Member, National Academy of Sciences (1997 - Present)
Investigator, HHMI (1988 - 2021)
Honors & Awards
Director’s Award, National Institutes of Health (1984)
Investigator, Howard Hughes Medical Institute (1988 to present)
Member, National Academy of Sciences (1997)
Merit Award, National Institutes of Health (2000)
Outstanding Inventor, Stanford University (2004)
Laureate in Chemistry with Stuart Schreiber, Thomas Scientific (2006)
David Korn Professorship, Stanford University (2008)
Faculty Mentor of the Year, Stanford University (2008)
Javits Neuroscience Investigator Award, National Institutes of Health (2013)
Fellow, American Association for the Advancement of Science (2015)
Current Research and Scholarly Interests
We are interested in the role of chromatin regulation in development and human cancer. Recent studies have show that over 20% of all human cancers have mutations in the subunits of an ATP-dependent chromatin regulatory complex we discovered several years ago. The genes behave as tumor suppressors and sometimes as oncogenes. We hope to understand the fundamental mechanisms used by these complex to prevent cancer.
These same chromatin remodeling complexes are frequently mutated in a variety of human neurologic diseases, reflecting their roles in the development of the nervous system. It appears that these specialized roles in the nervous system are due to the use of unique neural specific assemblies in the developing human and mouse brain. We hope to understand their fundamental mechanism of action through biochemical and genetic approaches in combination with genome-wide analysis and genome sequencing studies.
Finally, we are developing new ways of making conditional alleles of mammalian genes using synthetic ligands that we hope will bring about a new fusion of biochemical and genetic approaches to understanding and controlling fundamental biologic processes. Recently we have developed an effective way of both assaying and modifying chromatin regulation in living cells.
Independent Studies (21)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Developmental Biology
DBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Immunology
IMMUNOL 299 (Aut, Win, Spr, Sum)
- Directed Reading in Pathology
PATH 299 (Aut, Win, Spr, Sum)
- Directed Reading in Stem Cell Biology and Regenerative Medicine
STEMREM 299 (Aut, Win, Spr)
- Early Clinical Experience in Immunology
IMMUNOL 280 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Pathology
PATH 280 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
DBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
IMMUNOL 399 (Aut, Win, Spr, Sum)
- Graduate Research
PATH 399 (Aut, Win, Spr, Sum)
- Graduate Research
STEMREM 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
DBIO 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
PATH 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
STEMREM 370 (Aut, Win, Spr)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Teaching in Immunology
IMMUNOL 290 (Aut, Win, Spr, Sum)
- Undergraduate Research
DBIO 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
IMMUNOL 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
PATH 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
STEMREM 199 (Aut, Win, Spr)
- Directed Reading in Cancer Biology
- Prior Year Courses
A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation.
Mutations in ARID1A rank amongst the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediated ARID1A knockout in primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity and mucinous differentiation. Genetic Wnt/B-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation. ARID1A mutation induced transcriptional regulatory modules characteristic of MSI and EBV subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with non-essential Wnt-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells.
View details for DOI 10.1158/2159-8290.CD-20-1109
View details for PubMedID 33451982
BAF subunit switching regulates chromatin accessibility to control cell cycle exit in the developing mammalian cortex.
Genes & development
mSWI/SNF or BAF chromatin regulatory complexes are dosage-sensitive regulators of human neural development frequently mutated in autism spectrum disorders and intellectual disability. Cell cycle exit and differentiation of neural stem/progenitor cells is accompanied by BAF subunit switching to generate neuron-specific nBAF complexes. We manipulated the timing of BAF subunit exchange in vivo and found that early loss of the npBAF subunit BAF53a stalls the cell cycle to disrupt neurogenesis. Loss of BAF53a results in decreased chromatin accessibility at specific neural transcription factor binding sites, including the pioneer factors SOX2 and ASCL1, due to Polycomb accumulation. This results in repression of cell cycle genes, thereby blocking cell cycle progression and differentiation. Cell cycle block upon Baf53a deletion could be rescued by premature expression of the nBAF subunit BAF53b but not by other major drivers of proliferation or differentiation. WNT, EGF, bFGF, SOX2, c-MYC, or PAX6 all fail to maintain proliferation in the absence of BAF53a, highlighting a novel mechanism underlying neural progenitor cell cycle exit in the continued presence of extrinsic proliferative cues.
View details for DOI 10.1101/gad.342345.120
View details for PubMedID 33602870
LSH mediates gene repression through macroH2A deposition.
2020; 11 (1): 5647
The human Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is a severe disease with increased mortality caused by mutation in the LSH gene. Although LSH belongs to a family of chromatin remodeling proteins, it remains unknown how LSH mediates its function on chromatin in vivo. Here, we use chemical-induced proximity to rapidly recruit LSH to an engineered locus and find that LSH specifically induces macroH2A1.2 and macroH2A2 deposition in an ATP-dependent manner. Tethering of LSH induces transcriptional repression and silencing is dependent on macroH2A deposition. Loss of LSH decreases macroH2A enrichment at repeat sequences and results in transcriptional reactivation. Likewise, reduction of macroH2A by siRNA interference mimicks transcriptional reactivation. ChIP-seq analysis confirmed that LSH is a major regulator of genome-wide macroH2A distribution. Tethering of ICF4 mutations fails to induce macroH2A deposition and ICF4 patient cells display reduced macroH2A deposition and transcriptional reactivation supporting a pathogenic role for altered marcoH2A deposition. We propose that LSH is a major chromatin modulator of the histone variant macroH2A and that its ability to insert marcoH2A into chromatin and transcriptionally silence is disturbed in the ICF4 syndrome.
View details for DOI 10.1038/s41467-020-19159-0
View details for PubMedID 33159050
CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation.
Proceedings of the National Academy of Sciences of the United States of America
The chromatin remodeler CHD8 is among the most frequently mutated genes in autism spectrum disorder (ASD). CHD8 has a dosage-sensitive role in ASD, but when and how it becomes critical to human social function is unclear. Here, we conducted genomic analyses of heterozygous and homozygous Chd8 mouse embryonic stem cells and differentiated neural progenitors. We identify dosage-sensitive CHD8 transcriptional targets, sites of regulated accessibility, and an unexpected cooperation with SOX transcription factors. Collectively, our findings reveal that CHD8 negatively regulates expression of neuronal genes to maintain pluripotency and also during differentiation. Thus, CHD8 is essential for both the maintenance of pluripotency and neural differentiation, providing mechanistic insight into its function with potential implications for ASD.
View details for DOI 10.1073/pnas.1921963117
View details for PubMedID 32839322
Loss of the neural-specific BAF subunit ACTL6B relieves repression of early response genes and causes recessive autism.
Proceedings of the National Academy of Sciences of the United States of America
Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such "early activation" genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors (Fos, Fosl2, Fosb, and Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism.
View details for DOI 10.1073/pnas.1908238117
View details for PubMedID 32312822
Chemical Inhibitors of a Selective SWI/SNF Function Synergize with ATR Inhibition in Cancer Cell Killing.
ACS chemical biology
SWI/SNF (BAF) complexes are a diverse family of ATP-dependent chromatin remodelers produced by combinatorial assembly that are mutated in and thought to contribute to 20% of human cancers and a large number of neurologic diseases. The gene-activating functions of BAF complexes are essential for viability of many cell types, limiting the development of small molecule inhibitors. To circumvent the potential toxicity of SWI/SNF inhibition, we identified small molecules that inhibit the specific repressive function of these complexes but are relatively nontoxic and importantly synergize with ATR inhibitors in killing cancer cells. Our studies suggest an avenue for therapeutic enhancement of ATR/ATM inhibition and provide evidence for chemical synthetic lethality of BAF complexes as a therapeutic strategy in cancer.
View details for DOI 10.1021/acschembio.0c00312
View details for PubMedID 32369697
- Chromatin state as a mechanism of anthracycline response in breast cancer AMER ASSOC CANCER RESEARCH. 2019
Chromatin regulators mediate anthracycline sensitivity in breast cancer.
Anthracyclines are a highly effective component of curative breast cancer chemotherapy but are associated with substantial morbidity1,2. Because anthracyclines work in part by inhibiting topoisomerase-II (TOP2) on accessible DNA3,4, we hypothesized that chromatin regulatory genes (CRGs) that mediate DNA accessibility might predict anthracycline response. We studied the role of CRGs in anthracycline sensitivity in breast cancer through integrative analysis of patient and cell line data. We identified a consensus set of 38 CRGs associated with anthracycline response across ten cell line datasets. By evaluating the interaction between expression and treatment in predicting survival in a metacohort of 1006 patients with early-stage breast cancer, we identified 54 CRGs whose expression levels dictate anthracycline benefit across the clinical subgroups; of these CRGs, 12 overlapped with those identified in vitro. CRGs that promote DNA accessibility, including Trithorax complex members, were associated with anthracycline sensitivity when highly expressed, whereas CRGs that reduce accessibility, such as Polycomb complex proteins, were associated with decreased anthracycline sensitivity. We show that KDM4B modulates TOP2 accessibility to chromatin, elucidating a mechanism of TOP2 inhibitor sensitivity. These findings indicate that CRGs mediate anthracycline benefit by altering DNA accessibility, with implications for the stratification of patients with breast cancer and treatment decision making.
View details for DOI 10.1038/s41591-019-0638-5
View details for PubMedID 31700186
- Cyclosporin A Specifically Inhibits Function of Nuclear Proteins Involved in T Cell Activation (Reprinted from Science vol 246, pg 1617, 1989) JOURNAL OF IMMUNOLOGY 2018; 201 (11): 3149–52
Nucleosome Turnover Regulates Histone Methylation Patterns over the Genome.
Recent studies have indicated that nucleosome turnover is rapid, occurring several times per cell cycle. To access the effect of nucleosome turnover on theepigenetic landscape, we investigated H3K79 methylation, which is produced by a single methyltransferase (Dot1l) with no known demethylase. Using chemical-induced proximity (CIP), we find that the valency of H3K79 methylation (mono-, di-, and tri-) is determined by nucleosome turnover rates. Furthermore, propagation of this mark is predicted by nucleosome turnover simulations over the genome and accounts for the asymmetric distribution of H3K79me toward the transcriptional unit. More broadly, a meta-analysis of other conserved histone modifications demonstrates that nucleosome turnover models predict both valency and chromosomal propagation of methylation marks. Based on data from worms, flies, and mice, we propose that the turnover of modified nucleosomes is a general means of propagation of epigenetic marks and a determinant of methylation valence.
View details for PubMedID 30472189
Rapid chromatin repression by Aire provides precise control of immune tolerance
2018; 19 (2): 162-+
Aire mediates the expression of tissue-specific antigens in thymic epithelial cells to promote tolerance against self-reactive T lymphocytes. However, the mechanism that allows expression of tissue-specific genes at levels that prevent harm is unknown. Here we show that Brg1 generates accessibility at tissue-specific loci to impose central tolerance. We found that Aire has an intrinsic repressive function that restricts chromatin accessibility and opposes Brg1 across the genome. Aire exerted this repressive influence within minutes after recruitment to chromatin and restrained the amplitude of active transcription. Disease-causing mutations that impair Aire-induced activation also impair the protein's repressive function, which indicates dual roles for Aire. Together, Brg1 and Aire fine-tune the expression of tissue-specific genes at levels that prevent toxicity yet promote immune tolerance.
View details for PubMedID 29335648
Chemically induced proximity in biology and medicine.
Science (New York, N.Y.)
2018; 359 (6380)
Proximity, or the physical closeness of molecules, is a pervasive regulatory mechanism in biology. For example, most posttranslational modifications such as phosphorylation, methylation, and acetylation promote proximity of molecules to play deterministic roles in cellular processes. To understand the role of proximity in biologic mechanisms, chemical inducers of proximity (CIPs) were developed to synthetically model biologically regulated recruitment. Chemically induced proximity allows for precise temporal control of transcription, signaling cascades, chromatin regulation, protein folding, localization, and degradation, as well as a host of other biologic processes. A systematic analysis of CIPs in basic research, coupled with recent technological advances utilizing CRISPR, distinguishes roles of causality from coincidence and allows for mathematical modeling in synthetic biology. Recently, induced proximity has provided new avenues of gene therapy and emerging advances in cancer treatment.
View details for PubMedID 29590011
Small Molecule Targeting of Specific BAF (mSWI/SNF) Complexes for HIV Latency Reversal.
Cell chemical biology
The persistence of a pool of latently HIV-1-infected cells despite combination anti-retroviral therapy treatment is the major roadblock for a cure. The BAF (mammalian SWI/SNF) chromatin remodeling complex is involved in establishing and maintaining viral latency, making it an attractive drug target for HIV-1 latency reversal. Here we report a high-throughput screen for inhibitors of BAF-mediated transcription in cells and the subsequent identification of a 12-membered macrolactam. This compound binds ARID1A-specific BAF complexes, prevents nucleosomal positioning, and relieves transcriptional repression of HIV-1. Through this mechanism, these compounds are able to reverse HIV-1 latency in an in vitro T cell line, an ex vivo primary cell model of HIV-1 latency, and in patient CD4+ T cells without toxicity or T cell activation. These macrolactams represent a class of latency reversal agents with unique mechanism of action, and can be combined with other latency reversal agents to improve reservoir targeting.
View details for PubMedID 30197195
Dominant-negative SMARCA4 mutants alter the accessibility landscape of tissue-unrestricted enhancers.
Nature structural & molecular biology
2018; 25 (1): 61–72
Mutation of SMARCA4 (BRG1), the ATPase of BAF (mSWI/SNF) and PBAF complexes, contributes to a range of malignancies and neurologic disorders. Unfortunately, the effects of SMARCA4 missense mutations have remained uncertain. Here we show that SMARCA4 cancer missense mutations target conserved ATPase surfaces and disrupt the mechanochemical cycle of remodeling. We find that heterozygous expression of mutants alters the open chromatin landscape at thousands of sites across the genome. Loss of DNA accessibility does not directly overlap with Polycomb accumulation, but is enriched in 'A compartments' at active enhancers, which lose H3K27ac but not H3K4me1. Affected positions include hundreds of sites identified as superenhancers in many tissues. Dominant-negative mutation induces pro-oncogenic expression changes, including increased expression of Myc and its target genes. Together, our data suggest that disruption of enhancer accessibility represents a key source of altered function in disorders with SMARCA4 mutations in a wide variety of tissues.
View details for PubMedID 29323272
Tethering of Lsh at the Oct4 locus promotes gene repression associated with epigenetic changes
2018; 13 (2): 173–81
Lsh is a chromatin remodeling factor that regulates DNA methylation and chromatin function in mammals. The dynamics of these chromatin changes and whether they are directly controlled by Lsh remain unclear. To understand the molecular mechanisms of Lsh chromatin controlled regulation of gene expression, we established a tethering system that recruits a Gal4-Lsh fusion protein to an engineered Oct4 locus through Gal4 binding sites in murine embryonic stem (ES) cells. We examined the molecular epigenetic events induced by Lsh binding including: histone modification, DNA methylation and chromatin accessibility to determine nucleosome occupancy before and after embryonic stem cell differentiation. Our results indicate that Lsh assists gene repression upon binding to the Oct4 promoter region. Furthermore, we detected less chromatin accessibility and reduced active histone modifications at the tethered site in undifferentiated ES, while GFP reporter gene expression and DNA methylation patterns remained unchanged at this stage. Upon differentiation, association of Lsh promotes transcriptional repression of the reporter gene accompanied by the increase of repressive histone marks and a gain of DNA methylation at distal and proximal Oct4 enhancer sites. Taken together, this approach allowed us to examine Lsh mediated epigenetic regulation as a dynamic process and revealed chromatin accessibility changes as the primary consequence of Lsh function.
View details for PubMedID 28621576
View details for PubMedCentralID PMC5873361
Rapid and reversible epigenome editing by endogenous chromatin regulators
2017; 8: 560
Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9-MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin regulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic information to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endogenous multi-subunit chromatin regulator complexes.Understanding the link between epigenetic marks and gene regulation requires the development of new tools to directly manipulate chromatin. Here the authors demonstrate a Cas9-based system to recruit chromatin remodelers to loci of interest, allowing rapid, reversible manipulation of epigenetic states.
View details for PubMedID 28916764
Chd8 Mutation Leads to Autistic-like Behaviors and Impaired Striatal Circuits
2017; 19 (2): 335-350
Autism spectrum disorder (ASD) is a heterogeneous disease, but genetically defined models can provide an entry point to studying the molecular underpinnings of this disorder. We generated germline mutant mice with loss-of-function mutations in Chd8, a de novo mutation strongly associated with ASD, and demonstrate that these mice display hallmark ASD behaviors, macrocephaly, and craniofacial abnormalities similar to patient phenotypes. Chd8(+/-) mice display a broad, brain-region-specific dysregulation of major regulatory and cellular processes, most notably histone and chromatin modification, mRNA and protein processing, Wnt signaling, and cell-cycle regulation. We also find altered synaptic physiology in medium spiny neurons of the nucleus accumbens. Perturbation of Chd8 in adult mice recapitulates improved acquired motor learning behavior found in Chd8(+/-) animals, suggesting a role for CHD8 in adult striatal circuits. These results support a mechanism linking chromatin modification to striatal dysfunction and the molecular pathology of ASD.
View details for DOI 10.1016/j.celrep.2017.03.052
View details for Web of Science ID 000401132600010
View details for PubMedID 28402856
- The BAF45a/PHF10 subunit of SWI/SNF-like chromatin remodeling complexes is essential for hematopoietic stem cell maintenance EXPERIMENTAL HEMATOLOGY 2017; 48: 58-71
A General Non-Radioactive ATPase Assay for Chromatin Remodeling Complexes.
Current protocols in chemical biology
2017; 9 (1): 1-10
Chromatin remodeling complexes couple the energy released from ATP hydrolysis to facilitate transcription, recombination, and repair mechanisms essential for a wide variety of biologic responses. While recombinant expression of the regulatory subunits of these enzymes is possible, measuring catalytic (ATPase) activity of the intact complexes recovered from normal or mutant cells is critical for understanding their mechanisms. SWI/SNF-like remodeling complexes can be megadaltons in size and include many regulatory subunits, making reconstitution of purified subunits challenging for recapitulating in vivo function. The protocol in this article defines the first highly quantitative ATPase assay for intact remodeling complexes that does not require radiation or reconstitution of recombinantly expressed subunits. This protocol is specifically useful for defining the catalytic role of active-site mutations in the context of other regulatory subunits and quantitatively rank-ordering inactivating catalytic-site mutations. © 2017 by John Wiley & Sons, Inc.
View details for DOI 10.1002/cpch.16
View details for PubMedID 28253434
TOP2 synergizes with BAF chromatin remodeling for both resolution and formation of facultative heterochromatin.
Nature structural & molecular biology
The resolution and formation of facultative heterochromatin are essential for development, reprogramming, and oncogenesis. The mechanisms underlying these changes are poorly understood owing to the difficulty of studying heterochromatin dynamics and structure in vivo. We devised an in vivo approach to investigate these mechanisms and found that topoisomerase II (TOP2), but not TOP1, synergizes with BAF (mSWI/SNF) ATP-dependent chromatin remodeling complexes genome-wide to resolve facultative heterochromatin to accessible chromatin independent of transcription. This indicates that changes in DNA topology that take place through (de-)catenation rather than the release of torsional stress through swiveling are necessary for heterochromatin resolution. TOP2 and BAF cooperate to recruit pluripotency factors, which explains some of the instructive roles of BAF complexes. Unexpectedly, we found that TOP2 also plays a role in the re-formation of facultative heterochromatin; this finding suggests that facultative heterochromatin and accessible chromatin exist at different states of catenation or other topologies, which might be critical to their structures.
View details for DOI 10.1038/nsmb.3384
View details for PubMedID 28250416
Dynamics of BAF-Polycomb complex opposition on heterochromatin in normal and oncogenic states
2017; 49 (2): 213-222
The opposition between Polycomb repressive complexes (PRCs) and BAF (mSWI/SNF) complexes has a critical role in both development and disease. Mutations in the genes encoding BAF subunits contribute to more than 20% of human malignancies, yet the underlying mechanisms remain unclear, owing largely to a lack of assays to assess BAF function in living cells. To address this, we have developed a widely applicable recruitment assay system through which we find that BAF opposes PRC by rapid, ATP-dependent eviction, leading to the formation of accessible chromatin. The reversal of this process results in reassembly of facultative heterochromatin. Surprisingly, BAF-mediated PRC eviction occurs in the absence of RNA polymerase II (Pol II) occupancy, transcription, and replication. Further, we find that tumor-suppressor and oncogenic mutant BAF complexes have different effects on PRC eviction. The results of these studies define a mechanistic sequence underlying the resolution and formation of facultative heterochromatin, and they demonstrate that BAF opposes PRC on a minute-by-minute basis to provide epigenetic plasticity.
View details for DOI 10.1038/ng.3734
View details for Web of Science ID 000393148600010
View details for PubMedID 27941796
Smarca4 ATPase mutations disrupt direct eviction of PRC1 from chromatin
2017; 49 (2): 282-288
Trithorax-group proteins and their mammalian homologs, including those in BAF (mSWI/SNF) complexes, are known to oppose the activity of Polycomb repressive complexes (PRCs). This opposition underlies the tumor-suppressive role of BAF subunits and is expected to contribute to neurodevelopmental disorders. However, the mechanisms underlying opposition to Polycomb silencing are poorly understood. Here we report that recurrent disease-associated mutations in BAF subunits induce genome-wide increases in PRC deposition and activity. We show that point mutations in SMARCA4 (also known as BRG1) mapping to the ATPase domain cause loss of direct binding between BAF and PRC1 that occurs independently of chromatin. Release of this direct interaction is ATP dependent, consistent with a transient eviction mechanism. Using a new chemical-induced proximity assay, we find that BAF directly evicts Polycomb factors within minutes of its occupancy, thereby establishing a new mechanism for the widespread BAF-PRC opposition underlying development and disease.
View details for DOI 10.1038/ng.3735
View details for Web of Science ID 000393148600018
View details for PubMedID 27941795
The BAF45a/PHF10 subunit of SWI/SNF-like chromatin remodeling complexes is essential for hematopoietic stem cell maintenance.
2017; 48: 58–71.e15
The ability of hemopoietic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that establish and maintain stage-specific patterns of gene expression. However, the epigenetic factors responsible for mediating these regulatory events remain poorly defined. Here we provide evidence that BAF45a/PHF10, a subunit of SWI/SNF-like chromatin remodeling complexes, is essential for adult hemopoietic stem cell maintenance and myeloid lineage development. Deletion of BAF45a in the mouse is embryonic lethal. Acute deletion of BAF45a in the adult hemopoietic system causes a dose-dependent decrease in the frequency of long-term repopulating hemopoietic stem cells and committed myeloid progenitors without affecting their rate of proliferation. BAF45a-deficient hemopoietic stem cells and myeloid progenitors are selectively lost from mixed bone marrow chimeras, indicating their impaired function even in an intact microenvironment. Together, these studies suggest that the BAF45a subunit of SWI/SNF-like chromatin remodeling complexes plays nonredundant and specialized roles within the developing hemopoietic tissue.
View details for PubMedID 27931852
DNA binding drives the association of BRG1/hBRM bromodomains with nucleosomes.
2017; 8: 16080
BRG1 and BRM, central components of the BAF (mSWI/SNF) chromatin remodelling complex, are critical in chromatin structure regulation. Here, we show that the human BRM (hBRM) bromodomain (BRD) has moderate specificity for H3K14ac. Surprisingly, we also find that both BRG1 and hBRM BRDs have DNA-binding activity. We demonstrate that the BRDs associate with DNA through a surface basic patch and that the BRD and an adjacent AT-hook make multivalent contacts with DNA, leading to robust affinity and moderate specificity for AT-rich elements. Although we show that the BRDs can bind to both DNA and H3K14ac simultaneously, the histone-binding activity does not contribute substantially to nucleosome targeting in vitro. In addition, we find that neither BRD histone nor DNA binding contribute to the global chromatin affinity of BRG1 in mouse embryonic stem cells. Together, our results suggest that association of the BRG1/hBRM BRD with nucleosomes plays a regulatory rather than targeting role in BAF activity.
View details for PubMedID 28706277
View details for PubMedCentralID PMC5519978
The BAF chromatin remodelling complex is an epigenetic regulator of lineage specification in the early mouse embryo.
2016; 143 (8): 1271-1283
Dynamic control of gene expression is essential for the development of a totipotent zygote into an embryo with defined cell lineages. The accessibility of genes responsible for cell specification to transcriptional machinery is dependent on chromatin remodelling complexes such as the SWI\SNF (BAF) complex. However, the role of the BAF complex in early mouse development has remained unclear. Here, we demonstrate that BAF155, a major BAF complex subunit, regulates the assembly of the BAF complex in vivo and regulates lineage specification of the mouse blastocyst. We find that associations of BAF155 with other BAF complex subunits become enriched in extra-embryonic lineages just prior to implantation. This enrichment is attributed to decreased mobility of BAF155 in extra-embryonic compared with embryonic lineages. Downregulation of BAF155 leads to increased expression of the pluripotency marker Nanog and its ectopic expression in extra-embryonic lineages, whereas upregulation of BAF155 leads to the upregulation of differentiation markers. Finally, we show that the arginine methyltransferase CARM1 methylates BAF155, which differentially influences assembly of the BAF complex between the lineages and the expression of pluripotency markers. Together, our results indicate a novel role of BAF-dependent chromatin remodelling in mouse development via regulation of lineage specification.
View details for DOI 10.1242/dev.131961
View details for PubMedID 26952987
View details for PubMedCentralID PMC4852518
The Many Roles of BAF (mSWI/SNF) and PBAF Complexes in Cancer.
Cold Spring Harbor perspectives in medicine
2016; 6 (8)
During the last decade, a host of epigenetic mechanisms were found to contribute to cancer and other human diseases. Several genomic studies have revealed that ∼20% of malignancies have alterations of the subunits of polymorphic BRG-/BRM-associated factor (BAF) and Polybromo-associated BAF (PBAF) complexes, making them among the most frequently mutated complexes in cancer. Recurrent mutations arise in genes encoding several BAF/PBAF subunits, including ARID1A, ARID2, PBRM1, SMARCA4, and SMARCB1 These subunits share some degree of conservation with subunits from related adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in model organisms, in which a large body of work provides insight into their roles in cancer. Here, we review the roles of BAF- and PBAF-like complexes in these organisms, and relate these findings to recent discoveries in cancer epigenomics. We review several roles of BAF and PBAF complexes in cancer, including transcriptional regulation, DNA repair, and regulation of chromatin architecture and topology. More recent results highlight the need for new techniques to study these complexes.
View details for DOI 10.1101/cshperspect.a026930
View details for PubMedID 27413115
- Generation of BAF53b-Cre transgenic mice with pan-neuronal Cre activities GENESIS 2015; 53 (7): 440-448
Mammalian SWI/SNF chromatin remodeling complexes and cancer: Mechanistic insights gained from human genomics.
2015; 1 (5)
Over the past 4 years, nearly 100 exome sequencing studies have revealed the high frequency of mutations in the genes encoding the subunits of ATP-dependent chromatin remodelers in human cancer. Most of these mutations are within the genes encoding subunits of the BAF (Brg/Brahma-associated factors) or mSWI/SNF complex, which is one of two dozen predicted ATP-dependent chromatin remodeling complexes in mammals. Considering BAF complexes as a single entity, the 15 subunits encoded by 29 genes are mutated in >20% of human cancer, across a broad range of tumor types. These observations demonstrate that there is little redundancy in the oncogenic function of BAF complexes with the other remodeling complexes, underscoring their unique roles. Several important conclusions emerge from these genomic data: specific subunits appear to be mutated in specific cancers, highlighting tissue-specific protective roles; mutations can function as tumor suppressors or oncogenes; mutations can be homozygous or, more commonly, heterozygous, implying their dosage-sensitive roles in an unknown yet fundamental process used to suppress the genesis of cancer. These new human genetic findings paired with biochemical studies are challenging old ideas on how chromatin remodeling complexes function, generating new hypotheses with respect to their normal and oncogenic mechanisms and highlighting potential avenues for therapeutic intervention in human cancer.
View details for DOI 10.1126/sciadv.1500447
View details for PubMedID 26601204
View details for PubMedCentralID PMC4640607
Generation of BAF53b-Cre transgenic mice with pan-neuronal Cre activities.
Genesis (New York, N.Y. : 2000)
2015; 53 (7): 440–48
Molecular and functional studies of genes in neurons in mouse models require neuron-specific Cre lines. The current available neuronal Cre transgenic or knock-in lines either result in expression in a subset of neurons or expression in both neuronal and non-neuronal tissues. Previously we identified BAF53b as a neuron-specific subunit of the chromatin remodeling BAF complexes. Using a bacteria artificial chromosome (BAC) construct containing the BAF53b gene, we generated a Cre transgenic mouse under the control of BAF53b regulatory elements. Like the endogenous BAF53b gene, we showed that BAF53b-Cre is largely neuron-specific. In both central and peripheral nervous systems, it was expressed in all developing neurons examined and was not observed in neural progenitors or glial cells. In addition, BAF53b-Cre functioned in primary cultures in a pan-neuron-specific manner. Thus, BAF53b-Cre mice will be a useful genetic tool to manipulate gene expression in developing neurons for molecular, biochemical, and functional studies.
View details for PubMedID 26077106
- The Role of BAF (mSWI/SNF) Complexes in Mammalian Neural Development AMERICAN JOURNAL OF MEDICAL GENETICS PART C-SEMINARS IN MEDICAL GENETICS 2014; 166 (3): 333-349
Essential role of BRG, the ATPase subunit of BAF chromatin remodeling complexes, in leukemia maintenance
2014; 123 (11): 1720-1728
In mammals, combinatorial assembly of alternative families of subunits confers functional specificity to adenosine triphosphate (ATP)-dependent SWI/SNF-like Brg/Brm-associated factor (BAF) chromatin remodeling complexes by creating distinct polymorphic surfaces for interaction with regulatory elements and DNA-binding factors. Although redundant in terms of biochemical activity, the core ATPase subunits, BRG/SMARCA4 and BRM/SMARCA2, are functionally distinct and may contribute to complex specificity. Here we show using quantitative proteomics that BAF complexes expressed in leukemia are specifically assembled around the BRG ATPase. Moreover, using a mouse model of acute myeloid leukemia, we demonstrate that BRG is essential for leukemia maintenance, as leukemic cells lacking BRG rapidly undergo cell-cycle arrest and apoptosis. Most importantly, we show that BRG is dispensable for the maintenance of immunophenotypic long-term repopulating hematopoietic stem cells, suggesting that adroit targeting of BRG in leukemia may have potent and specific therapeutic effects.
View details for DOI 10.1182/blood-2013-02-483495
View details for Web of Science ID 000335847700019
View details for PubMedID 24478402
View details for PubMedCentralID PMC3954053
Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains
EPIGENETICS & CHROMATIN
Cellular differentiation and reprogramming are accompanied by changes in replication timing and 3D organization of large-scale (400 to 800 Kb) chromosomal domains ('replication domains'), but few gene products have been identified whose disruption affects these properties.Here we show that deletion of esBAF chromatin-remodeling complex components BAF250a and Brg1, but not BAF53a, disrupts replication timing at specific replication domains. Also, BAF250a-deficient fibroblasts reprogrammed to a pluripotency-like state failed to reprogram replication timing in many of these same domains. About half of the replication domains affected by Brg1 loss were also affected by BAF250a loss, but a much larger set of domains was affected by BAF250a loss. esBAF binding in the affected replication domains was dependent upon BAF250a but, most affected domains did not contain genes whose transcription was affected by loss of esBAF.Loss of specific esBAF complex subunits alters replication timing of select replication domains in pluripotent cells.
View details for DOI 10.1186/1756-8935-6-42
View details for Web of Science ID 000330071200001
View details for PubMedID 24330833
View details for PubMedCentralID PMC3895691
Creating a neural specific chromatin landscape by npBAF and nBAF complexes.
Current opinion in neurobiology
2013; 23 (6): 903-913
Several features make the chromatin environment of neurons likely to be different than any other cell type. These include the fact that several hundred types of neurons exist, each requiring specialized patterns of gene expression and in turn specialized chromatin landscapes. In addition, neurons have the most stable morphology of any cell type, a unique feature essential for memory. Yet these stable morphologies must allow the emergence of new stable morphologies in response to environmental influences permitting learning to occur by altered morphology and new synapse formation. Several years ago we found that neurons have specific chromatin remodeling mechanisms not present in any other cell type that are produced by combinatorial assembly of ATP-dependent chromatin remodeling complexes. The neural specific subunits are essential for normal neural development, learning and memory. Remarkably, recreating these neural specific complexes in fibroblasts leads to their conversion to neurons. Recently, the subunits of these complexes have been found to have genetically dominant roles in several human neurologic diseases. The genetic dominance of these mutations suggests that less severe mutations will contribute to phenotypic variation in human neuronally derived traits.
View details for DOI 10.1016/j.conb.2013.09.003
View details for PubMedID 24090879
View details for PubMedCentralID PMC3878911
The BAF Complex Interacts with Pax6 in Adult Neural Progenitors to Establish a Neurogenic Cross-Regulatory Transcriptional Network
CELL STEM CELL
2013; 13 (4): 403-418
Numerous transcriptional regulators of neurogenesis have been identified in the developing and adult brain, but how neurogenic fate is programmed at the epigenetic level remains poorly defined. Here, we report that the transcription factor Pax6 directly interacts with the Brg1-containing BAF complex in adult neural progenitors. Deletion of either Brg1 or Pax6 in the subependymal zone (SEZ) causes the progeny of adult neural stem cells to convert to the ependymal lineage within the SEZ while migrating neuroblasts convert to different glial lineages en route to or in the olfactory bulb (OB). Genome-wide analyses reveal that the majority of genes downregulated in the Brg1 null SEZ and OB contain Pax6 binding sites and are also downregulated in Pax6 null SEZ and OB. Downstream of the Pax6-BAF complex, we find that Sox11, Nfib, and Pou3f4 form a transcriptional cross-regulatory network that drives neurogenesis and can convert postnatal glia into neurons. Taken together, elements of our work identify a tripartite effector network activated by Pax6-BAF that programs neuronal fate.
View details for DOI 10.1016/j.stem.2013.07.002
View details for Web of Science ID 000329571100010
View details for PubMedID 23933087
Reprogramming human fibroblasts to neurons by recapitulating an essential microRNA-chromatin switch
CURRENT OPINION IN GENETICS & DEVELOPMENT
2013; 23 (5): 591-598
The development of the vertebrate nervous system requires a switch of ATP-dependent chromatin remodeling mechanisms, which occurs by substituting subunits within these complexes near cell cycle exit. This switching involves a triple negative genetic circuitry in which REST represses miR-9 and miR-124, which in turn repress BAF53a, which in turn repress the homologous neuron-specific BAF53b. Recapitulation of this microRNA/chromatin switch in human fibroblasts converts them to neurons. The genes involved in this fate-determining chromatin switch play genetically dominant roles in several human neurologic diseases suggesting that they are rate-limiting for aspects of human neural development. We review how this switch in ATP-dependent chromatin complexes might interface with traditional ideas about neural determination and reprogramming.
View details for DOI 10.1016/j.gde.2013.07.001
View details for Web of Science ID 000324970600015
View details for PubMedID 24035011
View details for PubMedCentralID PMC3951511
Exome sequencing to identify de novo mutations in sporadic ALS trios.
2013; 16 (7): 851-855
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease whose causes are still poorly understood. To identify additional genetic risk factors, we assessed the role of de novo mutations in ALS by sequencing the exomes of 47 ALS patients and both of their unaffected parents (n = 141 exomes). We found that amino acid-altering de novo mutations were enriched in genes encoding chromatin regulators, including the neuronal chromatin remodeling complex (nBAF) component SS18L1 (also known as CREST). CREST mutations inhibited activity-dependent neurite outgrowth in primary neurons, and CREST associated with the ALS protein FUS. These findings expand our understanding of the ALS genetic landscape and provide a resource for future studies into the pathogenic mechanisms contributing to sporadic ALS.
View details for DOI 10.1038/nn.3412
View details for PubMedID 23708140
Kinetic Analysis of npBAF to nBAF Switching Reveals Exchange of SS18 with CREST and Integration with Neural Developmental Pathways.
journal of neuroscience
2013; 33 (25): 10348-10361
During the development of the vertebrate nervous system, neural progenitors divide, generate progeny that exit mitosis, and then migrate to sites where they elaborate specific morphologies and synaptic connections. Mitotic exit in neurons is accompanied by an essential switch in ATP-dependent chromatin regulatory complexes from the neural progenitor Brg/Brm-associated factor (npBAF) to neuron-specific nBAF complexes that is in part driven by miR-9/9* and miR-124. Recapitulating this microRNA/chromatin switch in fibroblasts leads to their direct conversion to neurons. We have defined the kinetics of neuron-specific BAF complex assembly in the formation of induced neurons from mouse embryonic stem cells, human fibroblasts, and normal mouse neural differentiation and, using proteomic analysis, found that this switch also includes the removal of SS18 and its replacement by CREST at mitotic exit. We found that switching of chromatin remodeling mechanisms is highly correlated with a broad switch in the use of neurogenic transcription factors. Knock-down of SS18 in neural stem cells causes cell-cycle exit and failure to self-renew, whereas continued expression of SS18 in neurons blocks dendritic outgrowth, underlining the importance of subunit switching. Because dominant mutations in BAF subunits underlie widely different human neurologic diseases arising in different neuronal types, our studies suggest that the characteristics of these diseases must be interpreted in the context of the different BAF assemblies in neurons rather than a singular mammalian SWItch/sucrose nonfermentable (mSWI/SNF) complex.
View details for DOI 10.1523/JNEUROSCI.1258-13.2013
View details for PubMedID 23785148
BAF complexes facilitate decatenation of DNA by topoisomerase IIa.
2013; 497 (7451): 624-627
Recent exon-sequencing studies of human tumours have revealed that subunits of BAF (mammalian SWI/SNF) complexes are mutated in more than 20% of all human malignancies, but the mechanisms involved in tumour suppression are unclear. BAF chromatin-remodelling complexes are polymorphic assemblies that use energy provided by ATP hydrolysis to regulate transcription through the control of chromatin structure and the placement of Polycomb repressive complex 2 (PRC2) across the genome. Several proteins dedicated to this multisubunit complex, including BRG1 (also known as SMARCA4) and BAF250a (also known as ARID1A), are mutated at frequencies similar to those of recognized tumour suppressors. In particular, the core ATPase BRG1 is mutated in 5-10% of childhood medulloblastomas and more than 15% of Burkitt's lymphomas. Here we show a previously unknown function of BAF complexes in decatenating newly replicated sister chromatids, a requirement for proper chromosome segregation during mitosis. We find that deletion of Brg1 in mouse cells, as well as the expression of BRG1 point mutants identified in human tumours, leads to anaphase bridge formation (in which sister chromatids are linked by catenated strands of DNA) and a G2/M-phase block characteristic of the decatenation checkpoint. Endogenous BAF complexes interact directly with endogenous topoisomerase IIα (TOP2A) through BAF250a and are required for the binding of TOP2A to approximately 12,000 sites across the genome. Our results demonstrate that TOP2A chromatin binding is dependent on the ATPase activity of BRG1, which is compromised in oncogenic BRG1 mutants. These studies indicate that the ability of TOP2A to prevent DNA entanglement at mitosis requires BAF complexes and suggest that this activity contributes to the role of BAF subunits as tumour suppressors.
View details for DOI 10.1038/nature12146
View details for PubMedID 23698369
View details for PubMedCentralID PMC3668793
- BAF complexes facilitate decatenation of DNA by topoisomerase IIa. Nature 2013; 497 (7451): 624-627
- Exome sequencing to identify de novo mutations in sporadic ALS trios. Nature neuroscience 2013; 16 (7): 851-855
Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy.
2013; 45 (6): 592-601
Subunits of mammalian SWI/SNF (mSWI/SNF or BAF) complexes have recently been implicated as tumor suppressors in human malignancies. To understand the full extent of their involvement, we conducted a proteomic analysis of endogenous mSWI/SNF complexes, which identified several new dedicated, stable subunits not found in yeast SWI/SNF complexes, including BCL7A, BCL7B and BCL7C, BCL11A and BCL11B, BRD9 and SS18. Incorporating these new members, we determined mSWI/SNF subunit mutation frequency in exome and whole-genome sequencing studies of primary human tumors. Notably, mSWI/SNF subunits are mutated in 19.6% of all human tumors reported in 44 studies. Our analysis suggests that specific subunits protect against cancer in specific tissues. In addition, mutations affecting more than one subunit, defined here as compound heterozygosity, are prevalent in certain cancers. Our studies demonstrate that mSWI/SNF is the most frequently mutated chromatin-regulatory complex (CRC) in human cancer, exhibiting a broad mutation pattern, similar to that of TP53. Thus, proper functioning of polymorphic BAF complexes may constitute a major mechanism of tumor suppression.
View details for DOI 10.1038/ng.2628
View details for PubMedID 23644491
From neural development to cognition: unexpected roles for chromatin
NATURE REVIEWS GENETICS
2013; 14 (5): 347-359
Recent genome-sequencing studies in human neurodevelopmental and psychiatric disorders have uncovered mutations in many chromatin regulators. These human genetic studies, along with studies in model organisms, are providing insight into chromatin regulatory mechanisms in neural development and how alterations to these mechanisms can cause cognitive deficits, such as intellectual disability. We discuss several implicated chromatin regulators, including BAF (also known as SWI/SNF) and CHD8 chromatin remodellers, HDAC4 and the Polycomb component EZH2. Interestingly, mutations in EZH2 and certain BAF complex components have roles in both neurodevelopmental disorders and cancer, and overlapping point mutations are suggesting functionally important residues and domains. We speculate on the contribution of these similar mutations to disparate disorders.
View details for DOI 10.1038/nrg3413
View details for Web of Science ID 000317732600012
View details for PubMedID 23568486
The neuron-specific chromatin regulatory subunit BAF53b is necessary for synaptic plasticity and memory
2013; 16 (5): 552-?
Recent exome sequencing studies have implicated polymorphic Brg1-associated factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several human intellectual disabilities and cognitive disorders. However, it is currently unknown how mutations in BAF complexes result in impaired cognitive function. Postmitotic neurons express a neuron-specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Mice harboring selective genetic manipulations of BAF53b have severe defects in long-term memory and long-lasting forms of hippocampal synaptic plasticity. We rescued memory impairments in BAF53b mutant mice by reintroducing BAF53b in the adult hippocampus, which suggests a role for BAF53b beyond neuronal development. The defects in BAF53b mutant mice appeared to derive from alterations in gene expression that produce abnormal postsynaptic components, such as spine structure and function, and ultimately lead to deficits in synaptic plasticity. Our results provide new insight into the role of dominant mutations in subunits of BAF complexes in human intellectual and cognitive disorders.
View details for DOI 10.1038/nn.3359
View details for Web of Science ID 000318029300011
View details for PubMedID 23525042
MicroRNAs: regulators of neuronal fate
CURRENT OPINION IN CELL BIOLOGY
2013; 25 (2): 215-221
Mammalian neural development has been traditionally studied in the context of evolutionarily conserved signaling pathways and neurogenic transcription factors. Recent studies suggest that microRNAs, a group of highly conserved noncoding regulatory small RNAs also play essential roles in neural development and neuronal function. A part of their action in the developing nervous system is to regulate subunit compositions of BAF complexes (ATP-dependent chromatin remodeling complexes), which appear to have dedicated functions during neural development. Intriguingly, ectopic expression of a set of brain-enriched microRNAs, miR-9/9* and miR-124 that promote the assembly of neuron-specific BAF complexes, converts the nonneuronal fate of human dermal fibroblasts towards postmitotic neurons, thereby revealing a previously unappreciated instructive role of these microRNAs. In addition to these global effects, accumulating evidence indicates that many microRNAs could also function locally, such as at the growth cone or at synapses modulating synaptic activity and neuronal connectivity. Here we discuss some of the recent findings about microRNAs' activity in regulating various developmental stages of neurons.
View details for DOI 10.1016/j.ceb.2012.12.007
View details for Web of Science ID 000317886100011
View details for PubMedID 23374323
Reversible Disruption of mSWI/SNF (BAF) Complexes by the SS18-SSX Oncogenic Fusion in Synovial Sarcoma
2013; 153 (1): 71-85
Recent exon sequencing studies have revealed that over 20% of human tumors have mutations in subunits of mSWI/SNF (BAF) complexes. To investigate the underlying mechanism, we studied human synovial sarcoma (SS), in which transformation results from the translocation of exactly 78 amino acids of SSX to the SS18 subunit of BAF complexes. We demonstrate that the SS18-SSX fusion protein competes for assembly with wild-type SS18, forming an altered complex lacking the tumor suppressor BAF47 (hSNF5). The altered complex binds the Sox2 locus and reverses polycomb-mediated repression, resulting in Sox2 activation. Sox2 is uniformly expressed in SS tumors and is essential for proliferation. Increasing the concentration of wild-type SS18 leads to reassembly of wild-type complexes retargeted away from the Sox2 locus, polycomb-mediated repression of Sox2, and cessation of proliferation. This mechanism of transformation depends on only two amino acids of SSX, providing a potential foundation for therapeutic intervention.
View details for DOI 10.1016/j.cell.2013.02.036
View details for Web of Science ID 000316853700007
View details for PubMedID 23540691
View details for PubMedCentralID PMC3655887
- Rethinking our intellectual origins: response to Kalinka et al. TRENDS IN GENETICS 2013; 29 (3): 127-129
ACTL6a enforces the epidermal progenitor state by suppressing SWI/SNF-dependent induction of KLF4.
Cell stem cell
2013; 12 (2): 193-203
Somatic progenitors suppress differentiation to maintain tissue self-renewal. The mammalian SWI/SNF chromatin-remodeling complex regulates nucleosome packaging to control differentiation in embryonic and adult stem cells. Catalytic Brg1 and Brm subunits are required for these processes; however, the roles of SWI/SNF regulatory subunits are not fully understood. Here, we show that ACTL6a/BAF53A modulates the SWI/SNF complex to suppress differentiation in epidermis. Conditional loss of ACTL6a resulted in terminal differentiation, cell-cycle exit, and hypoplasia, whereas ectopic expression of ACTL6a promoted the progenitor state. A significant portion of genes regulated by ACTL6a were found to also be targets of KLF4, a known activator of epidermal differentiation. Mechanistically, we show that ACTL6a prevents SWI/SNF complex binding to promoters of KLF4 and other differentiation genes and that SWI/SNF catalytic subunits are required for full induction of KLF4 targets. Thus, ACTL6a controls the epidermal progenitor state by sequestering SWI/SNF to prevent activation of differentiation programs.
View details for DOI 10.1016/j.stem.2012.12.014
View details for PubMedID 23395444
- Our fragile intellect: response to Dr Mitchell TRENDS IN GENETICS 2013; 29 (2): 60-62
Our fragile intellect. Part II
TRENDS IN GENETICS
2013; 29 (1): 3-5
Analysis of human mutation rates and the number of genes required for human intellectual and emotional fitness indicates that we are almost certainly losing these abilities. If so, how did we get them in the first place, and when did things begin to change?
View details for DOI 10.1016/j.tig.2012.10.003
View details for Web of Science ID 000314014700002
View details for PubMedID 23153597
Our fragile intellect. Part I
TRENDS IN GENETICS
2013; 29 (1): 1-3
New developments in genetics, anthropology, and neurobiology predict that a very large number of genes underlie our intellectual and emotional abilities, making these abilities genetically surprisingly fragile.
View details for DOI 10.1016/j.tig.2012.10.002
View details for Web of Science ID 000314014700001
View details for PubMedID 23153596
The BAF53a subunit of SWI/SNF-like BAF complexes is essential for hemopoietic stem cell function
2012; 120 (24): 4720-4732
ATP-dependent SWI/SNF-like BAF chromatin remodeling complexes are emerging as key regulators of embryonic and adult stem cell function. Particularly intriguing are the findings that specialized assemblies of BAF complexes are required for establishing and maintaining pluripotent and multipotent states in cells. However, little is known on the importance of these complexes in normal and leukemic hemopoiesis. Here we provide the first evidence that the actin-related protein BAF53a, a subunit of BAF complexes preferentially expressed in long-term repopulating stem cells, is essential for adult hemopoiesis. Conditional deletion of BAF53a resulted in multilineage BM failure, aplastic anemia, and rapid lethality. These severe hemopoietic defects originate from a proliferative impairment of BM HSCs and progenitors and decreased progenitor survival. Using hemopoietic chimeras, we show that the impaired function of BAF53a-deficient HSCs is cell-autonomous and independent of the BM microenvironment. Altogether, our studies highlight an unsuspected role for BAF chromatin remodeling complexes in the maintenance of HSC and progenitor cell properties.
View details for DOI 10.1182/blood-2012-04-427047
View details for Web of Science ID 000313115300014
View details for PubMedID 23018638
Screening for Inhibitors of an Essential Chromatin Remodeler in Mouse Embryonic Stem Cells by Monitoring Transcriptional Regulation
JOURNAL OF BIOMOLECULAR SCREENING
2012; 17 (9): 1221-1230
The SWI/SNF-like adenosine triphosphate (ATP)-dependent chromatin remodeling complex, esBAF, is both necessary and, in some contexts, sufficient to induce the pluripotent state. Furthermore, mutations in various BAF subunits are associated with cancer. Little is known regarding the precise mechanism(s) by which this complex exerts its activities. Thus, it is unclear which protein interactions would be important to disrupt to isolate a relevant readout of mechanism. To address this, we developed a gene expression-based assay to identify inhibitors of the native esBAF complex. Specifically, a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay was developed in mouse embryonic stem (ES) cells to monitor expression of Bmi1, a developmentally important gene repressed by the esBAF complex. The assay was miniaturized to a 384-well format and used to screen a diverse collection of compounds, including novel products of diversity-oriented synthesis (DOS). Confirmed hits were validated using a knock-in ES cell reporter line in which luciferase is inserted into the Bmi1 locus. Several of the validated hits regulate a panel of target genes in a manner similar to the BAF chromatin-remodeling complex. Together these data indicate that expression-based screening using qRT-PCR is a successful approach to identify compounds targeting the regulation of key developmental genes in ES cells.
View details for DOI 10.1177/1087057112455060
View details for Web of Science ID 000309352100012
View details for PubMedID 22853929
Dynamics of inherently bounded histone modification domains
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (33): 13296-13301
A central goal of chromatin biology is to reveal how posttranslational histone marks modulate gene expression; however, relatively little is known about the spatial or temporal dynamics of these marks. We previously showed that a dynamic model of histone mark nucleation, propagation, and turnover fits the mean enrichment profiles from 99% of noncentromeric histone H3 lysine 9 trimethylation (H3K9me3) domains in mouse embryonic stem cells without the need for boundary or insulator elements. Here we report the full details of this "inherently bounded" model of histone modification dynamics and describe several dynamic features of the model using H3K9me3 as a paradigm. By analyzing the kinetic and structural constraints that drive formation of inherently bounded domains, we find that such domains are optimized when the rates of marking and turnover are comparable. Additionally, we find that to establish such domains, propagation of the histone marks must occur primarily through local contacts.
View details for DOI 10.1073/pnas.1211172109
View details for Web of Science ID 000307807000039
View details for PubMedID 22847427
View details for PubMedCentralID PMC3421184
Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations
2012; 488 (7409): 106-110
Medulloblastomas are the most common malignant brain tumours in children. Identifying and understanding the genetic events that drive these tumours is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma on the basis of transcriptional and copy number profiles. Here we use whole-exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas have low mutation rates consistent with other paediatric tumours, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were newly identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR and LDB1. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant, but not wild-type, β-catenin. Together, our study reveals the alteration of WNT, hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic β-catenin signalling in medulloblastoma.
View details for DOI 10.1038/nature11329
View details for Web of Science ID 000307010700042
View details for PubMedID 22820256
View details for PubMedCentralID PMC3413789
Neonatal beta Cell Development in Mice and Humans Is Regulated by Calcineurin/NFAT
2012; 23 (1): 21-34
Little is known about the mechanisms governing neonatal growth and maturation of organs. Here we demonstrate that calcineurin/Nuclear Factor of Activated T cells (Cn/NFAT) signaling regulates neonatal pancreatic development in mouse and human islets. Inactivation of calcineurin b1 (Cnb1) in mouse islets impaired dense core granule biogenesis, decreased insulin secretion, and reduced cell proliferation and mass, culminating in lethal diabetes. Pancreatic β cells lacking Cnb1 failed to express genes revealed to be direct NFAT targets required for replication, insulin storage, and secretion. In contrast, glucokinase activation stimulated Cn-dependent expression of these genes. Calcineurin inhibitors, such as tacrolimus, used for human immunosuppression, induce diabetes. Tacrolimus exposure reduced Cn/NFAT-dependent expression of factors essential for insulin dense core granule formation and secretion and neonatal β cell proliferation, consistent with our genetic studies. Discovery of conserved pathways regulating β cell maturation and proliferation suggests new strategies for controlling β cell growth or replacement in human islet diseases.
View details for DOI 10.1016/j.devcel.2012.05.014
View details for Web of Science ID 000306583800007
View details for PubMedID 22814600
View details for PubMedCentralID PMC3587727
Dynamics and Memory of Heterochromatin in Living Cells
2012; 149 (7): 1447-1460
Posttranslational histone modifications are important for gene regulation, yet the mode of propagation and the contribution to heritable gene expression states remains controversial. To address these questions, we developed a chromatin in vivo assay (CiA) system employing chemically induced proximity to initiate and terminate chromatin modifications in living cells. We selectively recruited HP1α to induce H3K9me3-dependent gene silencing and describe the kinetics and extent of chromatin modifications at the Oct4 locus in fibroblasts and pluripotent cells. H3K9me3 propagated symmetrically and continuously at average rates of ~0.18 nucleosomes/hr to produce domains of up to 10 kb. After removal of the HP1α stimulus, heterochromatic domains were heritably transmitted, undiminished through multiple cell generations. Our data enabled quantitative modeling of reaction kinetics, which revealed that dynamic competition between histone marking and turnover, determines the boundaries and stability of H3K9me3 domains. This framework predicts the steady-state dynamics and spatial features of the majority of euchromatic H3K9me3 domains over the genome.
View details for DOI 10.1016/j.cell.2012.03.052
View details for Web of Science ID 000305753800012
View details for PubMedID 22704655
View details for PubMedCentralID PMC3422694
BAF53A enforces the epidermal progenitor state by re-targeting the SWI/SNF/BAF chromatin remodeling complex away from differentiation gene promoters
75th Annual Meeting of the Society-for-Investigative-Dermatology
NATURE PUBLISHING GROUP. 2012: S63–S63
View details for Web of Science ID 000302866900372
MicroRNA-mediated conversion of human fibroblasts to neurons
2011; 476 (7359): 228-U123
Neurogenic transcription factors and evolutionarily conserved signalling pathways have been found to be instrumental in the formation of neurons. However, the instructive role of microRNAs (miRNAs) in neurogenesis remains unexplored. We recently discovered that miR-9* and miR-124 instruct compositional changes of SWI/SNF-like BAF chromatin-remodelling complexes, a process important for neuronal differentiation and function. Nearing mitotic exit of neural progenitors, miR-9* and miR-124 repress the BAF53a subunit of the neural-progenitor (np)BAF chromatin-remodelling complex. After mitotic exit, BAF53a is replaced by BAF53b, and BAF45a by BAF45b and BAF45c, which are then incorporated into neuron-specific (n)BAF complexes essential for post-mitotic functions. Because miR-9/9* and miR-124 also control multiple genes regulating neuronal differentiation and function, we proposed that these miRNAs might contribute to neuronal fates. Here we show that expression of miR-9/9* and miR-124 (miR-9/9*-124) in human fibroblasts induces their conversion into neurons, a process facilitated by NEUROD2. Further addition of neurogenic transcription factors ASCL1 and MYT1L enhances the rate of conversion and the maturation of the converted neurons, whereas expression of these transcription factors alone without miR-9/9*-124 was ineffective. These studies indicate that the genetic circuitry involving miR-9/9*-124 can have an instructive role in neural fate determination.
View details for DOI 10.1038/nature10323
View details for Web of Science ID 000293731900041
View details for PubMedID 21753754
View details for PubMedCentralID PMC3348862
esBAF facilitates pluripotency by conditioning the genome for LIF/STAT3 signalling and by regulating polycomb function
NATURE CELL BIOLOGY
2011; 13 (8): 903-U334
Signalling by the cytokine LIF and its downstream transcription factor, STAT3, prevents differentiation of pluripotent embryonic stem cells (ESCs). This contrasts with most cell types where STAT3 signalling induces differentiation. We find that STAT3 binding across the pluripotent genome is dependent on Brg1, the ATPase subunit of a specialized chromatin remodelling complex (esBAF) found in ESCs. Brg1 is required to establish chromatin accessibility at STAT3 binding targets, preparing these sites to respond to LIF signalling. Brg1 deletion leads to rapid polycomb (PcG) binding and H3K27me3-mediated silencing of many Brg1-activated targets genome wide, including the target genes of the LIF signalling pathway. Hence, one crucial role of Brg1 in ESCs involves its ability to potentiate LIF signalling by opposing PcG. Contrary to expectations, Brg1 also facilitates PcG function at classical PcG targets, including all four Hox loci, reinforcing their repression in ESCs. Therefore, esBAF does not simply antagonize PcG. Rather, the two chromatin regulators act both antagonistically and synergistically with the common goal of supporting pluripotency.
View details for DOI 10.1038/ncb2285
View details for Web of Science ID 000293373700008
View details for PubMedID 21785422
View details for PubMedCentralID PMC3155811
Engineering the ABA Plant Stress Pathway for Regulation of Induced Proximity
2011; 4 (164)
Chemically induced proximity (CIP) systems use small molecules and engineered proteins to control and study biological processes. However, small molecule-based systems for controlling protein abundance or activities have been limited by toxicity, instability, cost, and slow clearance of the small molecules in vivo. To address these problems, we modified proteins of the plant abscisic acid (ABA) stress response pathway to control the proximity of cellular proteins and showed that the system could be used to regulate transcription, signal transduction, and subcellular localization of proteins in response to exogenously applied ABA. We also showed that the ABA CIP system can be combined with other CIP systems to simultaneously control multiple processes. We found that, when given to mice, ABA was orally available and had a 4-hour half-life. These properties, along with its lack of toxicity and low cost, suggest that ABA may be well suited for therapeutic applications and as an experimental tool to control diverse cellular activities in vivo.
View details for DOI 10.1126/scisignal.2001449
View details for Web of Science ID 000288366500004
View details for PubMedID 21406691
View details for PubMedCentralID PMC3110149
ATP-dependent chromatin remodeling: genetics, genomics and mechanisms
2011; 21 (3): 396-420
Macromolecular assemblies that regulate chromatin structure using the energy of ATP hydrolysis have critical roles in development, cancer, and stem cell biology. The ATPases of this family are encoded by 27 human genes and are usually associated with several other proteins that are stable, non-exchangeable subunits. One fundamental mechanism used by these complexes is thought to be the movement or exchange of nucleosomes to regulate transcription. However, recent genetic studies indicate that chromatin remodelers may also be involved in regulating other aspects of chromatin structure during many cellular processes. The SWI/SNF family in particular appears to have undergone a substantial change in subunit composition and mechanism coincident with the evolutionary advent of multicellularity and the appearance of linking histones. The differential usage of this greater diversity of mammalian BAF subunits is essential for the development of specific cell fates, including the progression from pluripotency to multipotency to committed neurons. Recent human genetic screens have revealed that BRG1, ARID1A, BAF155, and hSNF5 are frequently mutated in tumors, indicating that BAF complexes also play a critical role in the initiation or progression of cancer. The mechanistic bases underlying the genetic requirements for BAF and other chromatin remodelers in development and cancer are relatively unexplored and will be a focus of this review.
View details for DOI 10.1038/cr.2011.32
View details for Web of Science ID 000288064900004
View details for PubMedID 21358755
View details for PubMedCentralID PMC3110148
Chromatin remodelling during development
2010; 463 (7280): 474-484
New methods for the genome-wide analysis of chromatin are providing insight into its roles in development and their underlying mechanisms. Current studies indicate that chromatin is dynamic, with its structure and its histone modifications undergoing global changes during transitions in development and in response to extracellular cues. In addition to DNA methylation and histone modification, ATP-dependent enzymes that remodel chromatin are important controllers of chromatin structure and assembly, and are major contributors to the dynamic nature of chromatin. Evidence is emerging that these chromatin-remodelling enzymes have instructive and programmatic roles during development. Particularly intriguing are the findings that specialized assemblies of ATP-dependent remodellers are essential for establishing and maintaining pluripotent and multipotent states in cells.
View details for DOI 10.1038/nature08911
View details for Web of Science ID 000273981100039
View details for PubMedID 20110991
View details for PubMedCentralID PMC3060774
Chromatin Regulatory Mechanisms in Pluripotency
ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, VOL 26
2010; 26: 503-532
Stem cells of all types are characterized by a stable, heritable state permissive of multiple developmental pathways. The past five years have seen remarkable advances in understanding these heritable states and the ways that they are initiated or terminated. Transcription factors that bind directly to DNA and have sufficiency roles have been most easy to investigate and, perhaps for this reason, are most solidly implicated in pluripotency. In addition, large complexes of ATP-dependent chromatin-remodeling and histone-modification enzymes that have specialized functions have also been implicated by genetic studies in initiating and/or maintaining pluripotency or multipotency. Several of these ATP-dependent remodeling complexes play non-redundant roles, and the esBAF complex facilitates reprogramming of induced pluripotent stem cells. The recent finding that virtually all histone modifications can be rapidly reversed and are often highly dynamic has raised new questions about how histone modifications come to play a role in the steady state of pluripotency. Another surprise from genetic studies has been the frequency with which the global effects of mutations in chromatin regulators can be largely reversed by a single target gene. These genetic studies help define the arena for future mechanistic studies that might be helpful to harness pluripotency for therapeutic goals.
View details for DOI 10.1146/annurev-cellbio-051809-102012
View details for Web of Science ID 000284856700020
View details for PubMedID 20624054
View details for PubMedCentralID PMC3085914
MicroRNA-mediated switching of chromatin-remodelling complexes in neural development
2009; 460 (7255): 642-U112
One of the most distinctive steps in the development of the vertebrate nervous system occurs at mitotic exit when cells lose multipotency and begin to develop stable connections that will persist for a lifetime. This transition is accompanied by a switch in ATP-dependent chromatin-remodelling mechanisms that appears to coincide with the final mitotic division of neurons. This switch involves the exchange of the BAF53a (also known as ACTL6a) and BAF45a (PHF10) subunits within Swi/Snf-like neural-progenitor-specific BAF (npBAF) complexes for the homologous BAF53b (ACTL6b) and BAF45b (DPF1) subunits within neuron-specific BAF (nBAF) complexes in post-mitotic neurons. The subunits of the npBAF complex are essential for neural-progenitor proliferation, and mice with reduced dosage for the genes encoding its subunits have defects in neural-tube closure similar to those in human spina bifida, one of the most serious congenital birth defects. In contrast, BAF53b and the nBAF complex are essential for an evolutionarily conserved program of post-mitotic neural development and dendritic morphogenesis. Here we show that this essential transition is mediated by repression of BAF53a by miR-9* and miR-124. We find that BAF53a repression is mediated by sequences in the 3' untranslated region corresponding to the recognition sites for miR-9* and miR-124, which are selectively expressed in post-mitotic neurons. Mutation of these sites led to persistent expression of BAF53a and defective activity-dependent dendritic outgrowth in neurons. In addition, overexpression of miR-9* and miR-124 in neural progenitors caused reduced proliferation. Previous studies have indicated that miR-9* and miR-124 are repressed by the repressor-element-1-silencing transcription factor (REST, also known as NRSF). Indeed, expression of REST in post-mitotic neurons led to derepression of BAF53a, indicating that REST-mediated repression of microRNAs directs the essential switch of chromatin regulatory complexes.
View details for DOI 10.1038/nature08139
View details for Web of Science ID 000268454300053
View details for PubMedID 19561591
View details for PubMedCentralID PMC2921580
- SnapShot: Ca2+-Calcineurin-NFAT Signaling CELL 2009; 138 (1)
ATP-dependent chromatin remodeling in neural development
CURRENT OPINION IN NEUROBIOLOGY
2009; 19 (2): 120-126
Recent advances have revealed that modification of chromatin structure is an important determinant of cell fate and function. DNA methylation and covalent modifications of histone tails contribute to changes in chromatin architectures, either enhancing or repressing gene expression. Another mechanism underlying the modification of chromatin structure relies on the activity of the SWI/SNF-related ATP-dependent chromatin remodeling complexes that control the accessibility of DNA sequences to transcription factors. There is increasing evidence that ATP-dependent chromatin remodeling complexes based on the alternative DNA-dependent ATPases, Brg1 and Brm, plays essential roles during neural development in both vertebrates and invertebrates. This remodeling complex has dedicated functions at different stages of neural development that appear to arise by combinatorial assembly of its subunits.
View details for DOI 10.1016/j.conb.2009.04.006
View details for Web of Science ID 000269108900003
View details for PubMedID 19442513
View details for PubMedCentralID PMC2756292
An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (13): 5181-5186
Mammalian SWI/SNF [also called BAF (Brg/Brahma-associated factors)] ATP-dependent chromatin remodeling complexes are essential for formation of the totipotent and pluripotent cells of the early embryo. In addition, subunits of this complex have been recovered in screens for genes required for nuclear reprogramming in Xenopus and mouse embryonic stem cell (ES) morphology. However, the mechanism underlying the roles of these complexes is unclear. Here, we show that BAF complexes are required for the self-renewal and pluripotency of mouse ES cells but not for the proliferation of fibroblasts or other cells. Proteomic studies reveal that ES cells express distinctive complexes (esBAF) defined by the presence of Brg (Brahma-related gene), BAF155, and BAF60A, and the absence of Brm (Brahma), BAF170, and BAF60C. We show that this specialized subunit composition is required for ES cell maintenance and pluripotency. Our proteomic analysis also reveals that esBAF complexes interact directly with key regulators of pluripotency, suggesting that esBAF complexes are specialized to interact with ES cell-specific regulators, providing a potential explanation for the requirement of BAF complexes in pluripotency.
View details for DOI 10.1073/pnas.0812889106
View details for Web of Science ID 000264790600042
View details for PubMedID 19279220
View details for PubMedCentralID PMC2654396
An embryonic stem cell chromatin remodeling complex, esBAF, is an essential component of the core pluripotency transcriptional network
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (13): 5187-5191
Distinctive SWI/SNF-like ATP-dependent chromatin remodeling esBAF complexes are indispensable for the maintenance and pluripotency of mouse embryonic stem (ES) cells [Ho L, et al. (2009) Proc Natl Acad Sci USA 10.1073/pnas.0812889106]. To understand the mechanism underlying the roles of these complexes in ES cells, we performed high-resolution genome-wide mapping of the core ATPase subunit, Brg, using ChIP-Seq technology. We find that esBAF, as represented by Brg, binds to genes encoding components of the core ES transcriptional circuitry, including Polycomb group proteins. esBAF colocalizes extensively with transcription factors Oct4, Sox2 and Nanog genome-wide, and shows distinct functional interactions with Oct4 and Sox2 at its target genes. Surprisingly, no significant colocalization of esBAF with PRC2 complexes, represented by Suz12, is observed. Lastly, esBAF colocalizes with Stat3 and Smad1 genome-wide, consistent with a direct and critical role in LIF and BMP signaling for maintaining self-renewal. Taken together, our studies indicate that esBAF is an essential component of the core pluripotency transcriptional network, and might also be a critical component of the LIF and BMP signaling pathways essential for maintenance of self-renewal and pluripotency.
View details for DOI 10.1073/pnas.0812888106
View details for Web of Science ID 000264790600043
View details for PubMedID 19279218
View details for PubMedCentralID PMC2654397
The gene encoding early growth response 2, a target of the transcription factor NFAT, is required for the development and maturation of natural killer T cells
2009; 10 (3): 306-313
The influence of signals transmitted by the phosphatase calcineurin and the transcription factor NFAT on the development and function of natural killer T (NKT) cells is unclear. In this report, we demonstrate that the transcription factor early growth response 2 (Egr2), a target gene of NFAT, was specifically required for the ontogeny of NKT cells but not that of conventional CD4(+) or CD8(+) T cells. NKT cells developed normally in the absence of Egr1 or Egr3, which suggests that Egr2 is a specific regulator of NKT cell differentiation. We found that Egr2 was important in the selection, survival and maturation of NKT cells. Our findings emphasize the importance of the calcineurin-NFAT-Egr2 pathway in the development of the NKT lymphocyte lineage.
View details for DOI 10.1038/ni.1696
View details for Web of Science ID 000263456500015
View details for PubMedID 19169262
View details for PubMedCentralID PMC2728767
FK506-binding protein (FKBP) partitions a modified HIV protease inhibitor into blood cells and prolongs its lifetime in vivo
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (5): 1336-1341
HIV protease inhibitors are a key component of anti-retroviral therapy, but their susceptibility to cytochrome P(450) metabolism reduces their systemic availability and necessitates repetitive dosing. Importantly, failure to maintain adequate inhibitor levels is believed to provide an opportunity for resistance to emerge; thus, new strategies to prolong the lifetime of these drugs are needed. Toward this goal, numerous prodrug approaches have been developed, but these methods involve creating inactive precursors that require enzymatic processing. Using an alternative strategy inspired by the natural product FK506, we have synthetically modified an HIV protease inhibitor such that it acquires high affinity for the abundant, cytoplasmic chaperone, FK506-binding protein (FKBP). This modified protease inhibitor maintains activity against HIV-1 protease (IC(50) = 19 nM) and, additionally, it is partitioned into the cellular component of whole blood via binding to FKBP. Interestingly, redistribution into this protected niche reduces metabolism and improves its half-life in mice by almost 20-fold compared with the unmodified compound. Based on these findings, we propose that addition of FKBP-binding groups might partially overcome the poor pharmacokinetic properties of existing HIV protease inhibitors and, potentially, other drug classes.
View details for DOI 10.1073/pnas.0805375106
View details for Web of Science ID 000263074600011
View details for PubMedID 19164520
View details for PubMedCentralID PMC2635806
Calcineurin/NFAT Signaling Is Required for Neuregulin-Regulated Schwann Cell Differentiation
2009; 323 (5914): 651-654
Schwann cells develop from multipotent neural crest cells and form myelin sheaths around axons that allow rapid transmission of action potentials. Neuregulin signaling through the ErbB receptor regulates Schwann cell development; however, the downstream pathways are not fully defined. We find that mice lacking calcineurin B1 in the neural crest have defects in Schwann cell differentiation and myelination. Neuregulin addition to Schwann cell precursors initiates an increase in cytoplasmic Ca2+, which activates calcineurin and the downstream transcription factors NFATc3 and c4. Purification of NFAT protein complexes shows that Sox10 is an NFAT nuclear partner and synergizes with NFATc4 to activate Krox20, which regulates genes necessary for myelination. Our studies demonstrate that calcineurin and NFAT are essential for neuregulin and ErbB signaling, neural crest diversification, and differentiation of Schwann cells.
View details for DOI 10.1126/science.1166562
View details for Web of Science ID 000262862800048
View details for PubMedID 19179536
View details for PubMedCentralID PMC2790385
Understanding the Words of Chromatin Regulation
2009; 136 (2): 200-206
Recent studies indicate that chromatin regulatory complexes produce biological specificity in the way that letters produce meanings by combinations into words. Combinatorial assembly of chromatin regulatory complexes may be critical for maximizing the information content provided by arrays of histone modifications.
View details for DOI 10.1016/j.cell.2009.01.009
View details for Web of Science ID 000262648800007
View details for PubMedID 19167321
View details for PubMedCentralID PMC2770578
Chemical rescue of cleft palate and midline defects in conditional gsk-3beta mice
13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium
MEDIMOND S R L. 2009: 7–9
View details for Web of Science ID 000303383400003
- Developmental biology: The early heart remodelled. Nature 2009; 459 (7247): 654–55
Bursting into the Nucleus
2008; 1 (51)
An increase in extracellular Ca(2+) induces the nuclear localization of the Crz1 transcription factor and the activation of target genes in yeast. A recent study indicates that nuclear entry occurs in short stochastic bursts that are unsynchronized within the population of cells. The frequency but not the amplitude of the bursts is controlled by Ca(2+). Modulation of the frequency of the burst coordinates aspects of expression of Crz target genes.
View details for DOI 10.1126/scisignal.151pe54
View details for Web of Science ID 000207497900002
View details for PubMedID 19109237
View details for PubMedCentralID PMC2713346
An EZ Mark to Miss
CELL STEM CELL
2008; 3 (6): 577-578
Ezh2, the methyltransferase within Polycomb Repressive II Complexes, was thought to be essential for all H3K27me3 marks in embryonic stem cells (ESCs). Recently in Molecular Cell, Shen and colleagues (2008) revealed that EZH2 is dispensable for ESC derivation and self-renewal, and that EZH1 may unexpectedly compensate for its loss.
View details for DOI 10.1016/j.stem.2008.11.007
View details for Web of Science ID 000261670900002
View details for PubMedID 19041770
View details for PubMedCentralID PMC2704610
Selective role of calcineurin in haematopoiesis and lymphopoiesis
2008; 9 (11): 1141-1148
The calcineurin/NFAT (nuclear factor of activated T-cells) signalling pathway is essential for many aspects of vertebrate development and is the target of the widely used immunosuppressive drugs FK506 and cyclosporine A. The basis for the therapeutic specificity of these drugs has remained unclear, as calcineurin is expressed ubiquitously. By inactivating calcineurin during haematopoietic development, we found that although this signalling pathway has an important, non-redundant role in the regulation of lymphocyte developmental checkpoints, it is not essential for the development of blood myeloid lineages. These studies have shown that the specificity of calcineurin inhibitors arises from the selective use of calcineurin at distinct developmental stages. The requirement for calcineurin/NFAT in the development of the adaptive but not of the innate immune system is consistent with the idea that the evolutionary appearance of this pathway was involved in the emergence of vertebrates.
View details for DOI 10.1038/embor.2008.174
View details for Web of Science ID 000260586800017
View details for PubMedID 18818667
View details for PubMedCentralID PMC2581854
Modulation of Peripheral B Cell Tolerance by CD72 in a Murine Model
ARTHRITIS AND RHEUMATISM
2008; 58 (10): 3192-3204
B cells play a dominant role in the pathogenesis of several autoimmune diseases, including systemic lupus erythematosus. It is not well understood how B cell signaling contributes to autoantibody production. The goal of this study was to elucidate the role of CD72 in modulating B cell receptor (BCR)-mediated tolerogenic signaling and peripheral B cell tolerance.A mouse model utilizing hen egg lysozyme (HEL) "anergic" B cells was studied. CD72-deficient mice carrying the BCR-specific IgHEL and/or soluble HEL (sHEL) transgenes were generated by breeding IgHEL-transgenic MD4 mice and/or sHEL-transgenic ML5 mice with congenic, CD72-deficient C57BL/6J mice. Normal and anergic B cells were isolated for analyses of B cell signaling. Aged wild-type and CD72-deficient mice were also examined for autoimmune phenomena.In the absence of CD72, anergic B cells inappropriately proliferated and survived in response to stimulation with self antigen. Biochemical analyses indicated that in anergic B cells, CD72 dominantly down-regulated BCR signaling to limit the antigen-induced elevation in [Ca2+]i and the activation of NFATc1, NF-kappaB, MAPK, and Akt. Mechanistically, CD72 was associated with, and regulated, the molecular adaptor Cbl-b in anergic B cells, suggesting that Cbl-b may play a role in mediating the negative effects of CD72 on BCR signaling. Moreover, in aged CD72-deficient mice, spontaneous production of antinuclear and anti-double-stranded DNA autoantibodies and features of lupus-like autoimmune disease were observed.CD72 is required to maintain B cell anergy and functions as a regulator of peripheral B cell tolerance. Thus, altered CD72 expression may play a role during the development of systemic lupus erythematosus.
View details for DOI 10.1002/art.23812
View details for Web of Science ID 000260024400029
View details for PubMedID 18821699
View details for PubMedCentralID PMC2790383
Monster protein controls calcium entry and fights infection
2008; 28 (1): 13-14
Sustained calcium signaling in T cells is critical for development and activation. In this issue of Immunity, Matza et al. (2007) demonstrate that the huge scaffold protein, AHNAK1, interacts with L-type calcium channels, regulates Ca2+ influx, and defends against Leishmania major infection.
View details for DOI 10.1016/j.immuni.2007.12.009
View details for Web of Science ID 000252627300005
View details for PubMedID 18199413
Calcineurin sets the bandwidth for discrimination of signals during thymocyte development
2007; 450 (7170): 731-U11
At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the alphabeta T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection). Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.
View details for DOI 10.1038/nature06305
View details for Web of Science ID 000251209700056
View details for PubMedID 18046413
Down syndrome critical region-1 is a transcriptional target of nuclear factor of activated T cells-c1 within the endocardium during heart development
JOURNAL OF BIOLOGICAL CHEMISTRY
2007; 282 (42): 30673-30679
Patients with Down syndrome have characteristic heart valve lesions resulting from endocardial cushion defects. The Down syndrome critical region 1 (DSCR1) gene, identified at the conserved trisomic 21 region in those patients, encodes a calcineurin inhibitor that inactivates nuclear factor of activated T cells (NFATc) activity. Here, we identify a regulatory sequence in the promoter region of human DSCR1 that dictates specific expression of a reporter gene in the endocardium, defined by the temporal and spatial expression of Nfatc1 during heart valve development. Activation of this evolutionally conserved DSCR1 regulatory sequence requires calcineurin and NFATc1 signaling in the endocardium. NFATc1 proteins bind to the regulatory sequence and trigger its enhancer activity. NFATc1 is sufficient to induce the expression of Dscr1 in cells that normally have undetectable or minimal NFATc1 or DSCR1. Pharmacologic inhibition of calcineurin or genetic Nfatc1 null mutation in mice abolishes the endocardial activity of this DSCR1 enhancer. Furthermore, in mice lacking endocardial NFATc1, the endogenous Dscr1 expression is specifically inhibited in the endocardium but not in the myocardium. Thus, our studies indicate that the DSCR1 gene is a direct transcriptional target of NFATc1 proteins within the endocardium during a critical window of heart valve formation.
View details for DOI 10.1074/jbc.M703622200
View details for Web of Science ID 000250136300035
View details for PubMedID 17693409
View details for PubMedCentralID PMC2366997
Regulation of dendritic development by NeuronSpecific chromatin remodeling complexes
2007; 56 (1): 94-108
The diversity of dendritic patterns is one of the fundamental characteristics of neurons and is in part regulated by transcriptional programs initiated by electrical activity. We show that dendritic outgrowth requires a family of combinatorially assembled, neuron-specific chromatin remodeling complexes (nBAF complexes) distinguished by the actin-related protein BAF53b and based on the Brg/Brm ATPases. nBAF complexes bind tightly to the Ca(2+)-responsive dendritic regulator CREST and directly regulate genes essential for dendritic outgrowth. BAF53b is not required for nBAF complex assembly or the interaction with CREST, yet is required for their recruitment to the promoters of specific target genes. The highly homologous BAF53a protein, which is a component of neural progenitor and nonneural BAF complexes, cannot replace BAF53b's role in dendritic development. Remarkably, we find that this functional specificity is conferred by the actin fold subdomain 2 of BAF53b. These studies suggest that the genes encoding the individual subunits of BAF complexes function like letters in a ten-letter word to produce biologically specific meanings (in this case dendritic outgrowth) by combinatorial assembly of their products.
View details for DOI 10.1016/j.neuron.2007.08.021
View details for Web of Science ID 000250289800013
View details for PubMedID 17920018
- Renaming the DSCR1/Adapt78 gene family as RCAN: regulators of calcineurin FASEB JOURNAL 2007; 21 (12): 3023-3028
An essential switch in subunit composition of a chromatin remodeling complex during neural development
2007; 55 (2): 201-215
Mammalian neural stem cells (NSCs) have the capacity to both self-renew and to generate all the neuronal and glial cell-types of the adult nervous system. Global chromatin changes accompany the transition from proliferating NSCs to committed neuronal lineages, but the mechanisms involved have been unclear. Using a proteomics approach, we show that a switch in subunit composition of neural, ATP-dependent SWI/SNF-like chromatin remodeling complexes accompanies this developmental transition. Proliferating neural stem and progenitor cells express complexes in which BAF45a, a Krüppel/PHD domain protein and the actin-related protein BAF53a are quantitatively associated with the SWI2/SNF2-like ATPases, Brg and Brm. As neural progenitors exit the cell cycle, these subunits are replaced by the homologous BAF45b, BAF45c, and BAF53b. BAF45a/53a subunits are necessary and sufficient for neural progenitor proliferation. Preventing the subunit switch impairs neuronal differentiation, indicating that this molecular event is essential for the transition from neural stem/progenitors to postmitotic neurons. More broadly, these studies suggest that SWI/SNF-like complexes in vertebrates achieve biological specificity by combinatorial assembly of their subunits.
View details for DOI 10.1016/j.neuron.2007.06.019
View details for Web of Science ID 000248635800006
View details for PubMedID 17640523
View details for PubMedCentralID PMC2674110
- Exploiting protein destruction for constructive use PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2007; 104 (28): 11511-11512
Rescue of degradation-prone mutants of the FK506-rapamycin binding (FRB) protein with chemical ligands
2007; 8 (10): 1162-1169
We recently reported that certain mutations in the FK506-rapamycin binding (FRB) domain disrupt its stability in vitro and in vivo (Stankunas et al. Mol. Cell, 2003, 12, 1615). To determine the precise residues that cause instability, we calculated the folding free energy (Delta G) of a collection of FRB mutants by measuring their intrinsic tryptophan fluorescence during reversible chaotropic denaturation. Our results implicate the T2098L point mutation as a key determinant of instability. Further, we found that some of the mutants in this collection were destabilized by up to 6 kcal mol(-1) relative to the wild type. To investigate how these mutants behave in cells, we expressed firefly luciferase fused to FRB mutants in African green monkey kidney (COS) cell lines and mouse embryonic fibroblasts (MEFs). When unstable FRB mutants were used, we found that the protein levels and the luminescence intensities were low. However, addition of a chemical ligand for FRB, rapamycin, restored luciferase activity. Interestingly, we found a roughly linear relationship between the Delta G of the FRB mutants calculated in vitro and the relative chemical rescue in cells. Because rapamycin is capable of simultaneously binding both FRB and the chaperone, FK506-binding protein (FKBP), we next examined whether FKBP might contribute to the protection of FRB mutants. Using both in vitro experiments and a cell-based model, we found that FKBP stabilizes the mutants. These findings are consistent with recent models that suggest damage to intrinsic Delta G can be corrected by pharmacological chaperones. Further, these results provide a collection of conditionally stable fusion partners for use in controlling protein stability.
View details for DOI 10.1002/cbic.200700087
View details for Web of Science ID 000248067100014
View details for PubMedID 17525916
Selective role of NFATc3 in positive selection of thymocytes
JOURNAL OF IMMUNOLOGY
2007; 179 (1): 103-110
The four Ca(2+)-dependent NFATc proteins are both signal transducers and transcription factors that reside in the cytoplasm until dephosphorylation by calcineurin. Dephosphorylation exposes nuclear import sequences and sends NFATc proteins into the nucleus where they assemble with nuclear partners into NFAT transcription complexes. Recent genetic studies have indicated that calcineurin-NFAT signaling is a major determinant of vertebrate morphogenesis and development. Mice lacking calcineurin activity show a complete block in positive selection of CD4 and CD8 double-positive thymocytes, yet the role of the NFATc proteins in T cell development has been controversial. In this study, we address the requirement for NFATc3 in T cell development by generating NFATc3 conditional knockout mice. We show that specific deletion of NFATc3 in thymocytes causes a partial block at the double-negative stage 3 and also a partial block in positive selection. Furthermore, the defect does not become more pronounced when NFATc2 is also absent, consistent with the fact that NFATc2-null mice do not have a T cell developmental defect. Expression of a nuclear (and constitutively active) NFATc1 even at subphysiological levels can rescue the transition of double-negative to double-positive thymocytes in RAG-null mice, but is unable to rescue development of CD4 and CD8 single-positive cells. In addition to NFATc3, this suggests a role for NFATc1 in T cell development. Our studies indicate that the signals that direct positive selection likely use both NFATc1 and NFATc3 downstream of calcineurin.
View details for Web of Science ID 000247497600016
View details for PubMedID 17579027
Cell signaling. Nuclear actin as choreographer of cell morphology and transcription.
2007; 316 (5832): 1710-1711
View details for PubMedID 17588921
NFAT signaling and the invention of vertebrates
TRENDS IN CELL BIOLOGY
2007; 17 (6): 251-260
The calcium/calcineurin-dependent NFATc family is thought to have arisen following the recombination of an ancient precursor with a Rel domain about 500 million years ago, producing a new group of signaling and transcription factors (the NFATc genes) found only in the genomes of vertebrates. Cell biological, genetic and biochemical evidence indicates that the circuitry of this pathway is well suited for intercalation with older pathways. We propose that this recombination enabled Ca(2+) signals to be redirected to a new transcriptional program, which provided part of the groundwork for vertebrate morphogenesis and organogenesis. This notion predicts that calcineurin-NFAT signaling would be essential for much of vertebrate development. We review recent evidence supporting this prediction and propose a systematic approach to explore aspects of vertebrate morphogenesis.
View details for DOI 10.1016/j.tcb.2007.04.006
View details for Web of Science ID 000247508200001
View details for PubMedID 17493814
Engineering small molecule specificity in nearly identical cellular environments
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
2007; 17 (10): 2703-2705
Methotrexate (MTX), an inhibitor of dihydrofolate reductase, was tethered to an FKBP12 ligand (SLF), and the resulting bifunctional molecule (MTXSLF) potently inhibits either enzyme but not both simultaneously. MTXSLF is cytotoxic to fibroblasts derived from FKBP12-null mice but is detoxified 40-fold by FKBP12 in wild-type fibroblasts. These studies demonstrate that non-target proteins in an otherwise identical genetic background can be used to predictably regulate the biological activity of synthetic molecules.
View details for DOI 10.1016/j.bmcl.2007.03.012
View details for Web of Science ID 000246675700005
View details for PubMedID 17383876
View details for PubMedCentralID PMC1949043
Enhanced NFATc1 nuclear occupancy causes T cell activation independent of CD28 costimulation
JOURNAL OF IMMUNOLOGY
2007; 178 (7): 4315-4321
TCR signals induce the nuclear localization of NFATc proteins, which are removed from the nucleus after rephosphorylation by glycogen synthase kinase 3 and other kinases. Rapid nuclear export might allow continuous monitoring of receptor occupancy, making the transcriptional response proportional to the duration of TCR/CD28 signaling. To investigate this possibility, we analyzed mice in which T cells express a NFATc1 variant (NFATc1(nuc)) with serine-to-alanine changes at the glycogen synthase kinase 3 phosphorylation sites. NFATc1(nuc) T cells have constitutively nuclear NFATc1, enhanced T cell activation in vivo, and calcineurin-independent proliferation in vitro. NFATc1(nuc) T cells are hypersensitive to TCR/CD3 stimulation, resulting in enhanced proliferation and cytokine production that is independent of CD28 costimulation. These results support the notion that CD28 inhibits nuclear export of NFATc transcription factors. In addition, NFATc1(nuc) destabilizes a positive feedback loop in which NFATc1 activates its own transcription as well as its targets, such as CD40 ligand and Th1/Th2 cytokines.
View details for Web of Science ID 000245197300038
View details for PubMedID 17371988
Chemical rescue of cleft palate and midline defects in conditional GSK-3 beta mice
2007; 446 (7131): 79-82
Glycogen synthase kinase-3beta (GSK-3beta) has integral roles in a variety of biological processes, including development, diabetes, and the progression of Alzheimer's disease. As such, a thorough understanding of GSK-3beta function will have a broad impact on human biology and therapeutics. Because GSK-3beta interacts with many different pathways, its specific developmental roles remain unclear. We have discovered a genetic requirement for GSK-3beta in midline development. Homozygous null mice display cleft palate, incomplete fusion of the ribs at the midline and bifid sternum as well as delayed sternal ossification. Using a chemically regulated allele of GSK-3beta (ref. 6), we have defined requirements for GSK-3beta activity during discrete temporal windows in palatogenesis and skeletogenesis. The rapamycin-dependent allele of GSK-3beta produces GSK-3beta fused to a tag, FRB* (FKBP/rapamycin binding), resulting in a rapidly destabilized chimaeric protein. In the absence of drug, GSK-3beta(FRB)*(/FRB)* mutants appear phenotypically identical to GSK-3beta-/- mutants. In the presence of drug, GSK-3betaFRB* is rapidly stabilized, restoring protein levels and activity. Using this system, mutant phenotypes were rescued by restoring endogenous GSK-3beta activity during two distinct periods in gestation. This technology provides a powerful tool for defining windows of protein function during development.
View details for DOI 10.1038/nature05557
View details for Web of Science ID 000244525600041
View details for PubMedID 17293880
Stringent control of NFATc1 nuclear occupancy is critical for maintaining balanced immune response
GENE THERAPY AND MOLECULAR BIOLOGY
2007; 11B: 171-176
View details for Web of Science ID 000251610000008
Calcineurin/Nfat signaling is required for perinatal lung maturation and function
JOURNAL OF CLINICAL INVESTIGATION
2006; 116 (10): 2597-2609
Pulmonary surfactant proteins and lipids are required for lung function after birth. Lung immaturity and resultant surfactant deficiency cause respiratory distress syndrome, a common disorder contributing to morbidity and mortality in preterm infants. Surfactant synthesis increases prior to birth in association with formation of the alveoli that mediate efficient gas exchange. To identify mechanisms controlling perinatal lung maturation, the Calcineurin b1 (Cnb1) gene was deleted in the respiratory epithelium of the fetal mouse. Deletion of Cnb1 caused respiratory failure after birth and inhibited the structural maturation of the peripheral lung. Synthesis of surfactant and a lamellar body-associated protein, ABC transporter A3 (ABCA3), was decreased prior to birth. Nuclear factor of activated T cells (Nfat) calcineurin-dependent 3 (Nfatc3), a transcription factor modulated by calcineurin, was identified as a direct activator of Sftpa, Sftpb, Sftpc, Abca3, Foxa1, and Foxa2 genes. The calcineurin/Nfat pathway controls the morphologic maturation of lungs prior to birth and regulates expression of genes involved in surfactant homeostasis that are critical for adaptation to air breathing.
View details for DOI 10.1172/JCI27331
View details for Web of Science ID 000240965700010
View details for PubMedID 16998587
View details for PubMedCentralID PMC1570374
Role of transcription factor NFAT in glucose and insulin homeostasis
MOLECULAR AND CELLULAR BIOLOGY
2006; 26 (20): 7372-7387
Compromised immunoregulation contributes to obesity and complications in metabolic pathogenesis. Here, we demonstrate that the nuclear factor of activated T cell (NFAT) group of transcription factors contributes to glucose and insulin homeostasis. Expression of two members of the NFAT family (NFATc2 and NFATc4) is induced upon adipogenesis and in obese mice. Mice with the Nfatc2-/- Nfatc4-/- compound disruption exhibit defects in fat accumulation and are lean. Nfatc2-/- Nfatc4-/- mice are also protected from diet-induced obesity. Ablation of NFATc2 and NFATc4 increases insulin sensitivity, in part, by sustained activation of the insulin signaling pathway. Nfatc2-/- Nfatc4-/- mice also exhibit an altered adipokine profile, with reduced resistin and leptin levels. Mechanistically, NFAT is recruited to the transcription loci and regulates resistin gene expression upon insulin stimulation. Together, these results establish a role for NFAT in glucose/insulin homeostasis and expand the repertoire of NFAT function to metabolic pathogenesis and adipokine gene transcription.
View details for DOI 10.1128/MCB.00580-06
View details for Web of Science ID 000241252300003
View details for PubMedID 16908540
View details for PubMedCentralID PMC1636854
Calcineurin/NFAT signalling regulates pancreatic beta-cell growth and function
2006; 443 (7109): 345-349
The growth and function of organs such as pancreatic islets adapt to meet physiological challenges and maintain metabolic balance, but the mechanisms controlling these facultative responses are unclear. Diabetes in patients treated with calcineurin inhibitors such as cyclosporin A indicates that calcineurin/nuclear factor of activated T-cells (NFAT) signalling might control adaptive islet responses, but the roles of this pathway in beta-cells in vivo are not understood. Here we show that mice with a beta-cell-specific deletion of the calcineurin phosphatase regulatory subunit, calcineurin b1 (Cnb1), develop age-dependent diabetes characterized by decreased beta-cell proliferation and mass, reduced pancreatic insulin content and hypoinsulinaemia. Moreover, beta-cells lacking Cnb1 have a reduced expression of established regulators of beta-cell proliferation. Conditional expression of active NFATc1 in Cnb1-deficient beta-cells rescues these defects and prevents diabetes. In normal adult beta-cells, conditional NFAT activation promotes the expression of cell-cycle regulators and increases beta-cell proliferation and mass, resulting in hyperinsulinaemia. Conditional NFAT activation also induces the expression of genes critical for beta-cell endocrine function, including all six genes mutated in hereditary forms of monogenic type 2 diabetes. Thus, calcineurin/NFAT signalling regulates multiple factors that control growth and hallmark beta-cell functions, revealing unique models for the pathogenesis and therapy of diabetes.
View details for DOI 10.1038/nature05097
View details for PubMedID 16988714
- A Foxy tango with NFAT NATURE IMMUNOLOGY 2006; 7 (9): 906-908
Calcineurin/NFAT signaling in osteoblasts regulates bone mass
2006; 10 (6): 771-782
Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts.
View details for DOI 10.1016/j.devcel.2006.04.006
View details for Web of Science ID 000238244700011
View details for PubMedID 16740479
NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21
2006; 441 (7093): 595-600
Trisomy 21 results in Down's syndrome, but little is known about how a 1.5-fold increase in gene dosage produces the pleiotropic phenotypes of Down's syndrome. Here we report that two genes, DSCR1 and DYRK1A , lie within the critical region of human chromosome 21 and act synergistically to prevent nuclear occupancy of NFATc transcription factors, which are regulators of vertebrate development. We use mathematical modelling to predict that autoregulation within the pathway accentuates the effects of trisomy of DSCR1 and DYRK1A, leading to failure to activate NFATc target genes under specific conditions. Our observations of calcineurin-and Nfatc-deficient mice, Dscr1- and Dyrk1a-overexpressing mice, mouse models of Down's syndrome and human trisomy 21 are consistent with these predictions. We suggest that the 1.5-fold increase in dosage of DSCR1 and DYRK1A cooperatively destabilizes a regulatory circuit, leading to reduced NFATc activity and many of the features of Down's syndrome. More generally, these observations suggest that the destabilization of regulatory circuits can underlie human disease.
View details for DOI 10.1038/nature04678
View details for PubMedID 16554754
Thymocyte negative selection is mediated by protein kinase C- and Ca2+-dependent transcriptional induction of bim of cell death
JOURNAL OF IMMUNOLOGY
2006; 176 (4): 2299-2306
The processes of positive and negative selection in the thymus both determine the population of T cells that will enter the peripheral immune system and eliminate self-reactive T cells by apoptosis. Substantial evidence indicates that TCR signal intensity mediates this cell fate choice: low-intensity signals lead to survival and differentiation, whereas high-intensity signals generated by self-Ag lead to cell death. The molecular mechanism by which these graded signals are converted to discrete outcomes is not understood. Positive selection requires the Ca(2+)-dependent phosphatase calcineurin, whereas negative selection requires the proapoptotic Bcl-2 family member Bcl-2-interacting mediator of cell death (Bim). In this study, we investigated the regulation of Bim expression and the role of Ca(2+) in mediating negative selection. Our results show that transcription is necessary for both negative selection and Bim induction. Surprisingly, we also found that Ca(2+) is necessary for Bim induction. Induction of bim transcription appears to involve protein kinase C, but not calcineurin, JNK, p38 MAPK, or MEK. These results localize the decision point in positive vs negative selection to a step downstream of Ca(2+) signaling and suggest that negative selection signals induce Ca(2+)-dependent bim transcription through PKC.
View details for Web of Science ID 000235180900031
View details for PubMedID 16455986
The calcineurin phosphatase complex modulates immunogenic B cell responses
2006; 24 (2): 141-152
A series of signal-directed transitions regulates the development of distinct populations of self-tolerant B cells and ultimately the production of antibody-producing plasma cells. We studied the role of calcineurin/NFAT signaling in B cells by deleting the regulatory b1 subunit of calcineurin specifically in B cells. Follicular (FO) and marginal zone (MZ) B cells develop normally in these mice, but B1 cell numbers are reduced. In vitro, calcineurin b1-deficient B cells have a cell-intrinsic proliferation defect downstream of the B cell receptor. These mice have higher total serum IgM despite the absence of B1 cells and have enhanced T cell-independent-1 responses. Conversely, mice with calcineurin b1-deficient B cells develop larger germinal centers and have reduced plasma cell development and antigen-specific antibody production during T cell-dependent immune responses. By several different criteria, calcineurin is dispensable for B cell tolerance, indicating that this phosphatase complex modulates immunogenic, but not tolerogenic, responses in vivo.
View details for DOI 10.1016/j.immuni.2005.12.013
View details for Web of Science ID 000235479000006
View details for PubMedID 16473827
Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity
CHEMISTRY & BIOLOGY
2006; 13 (1): 99-107
Controlling protein dimerization with small molecules has broad application to the study of protein function. Rapamycin has two binding surfaces: one that binds to FKBP12 and the other to the Frb domain of mTor/FRAP, directing their dimerization. Rapamycin is a potent cell growth inhibitor, but chemical modification of the surface contacting Frb alleviates this effect. Productive interactions with Frb-fused proteins can be restored by mutation of Frb to accommodate the rapamycin analog (a rapalog). We have quantitatively assessed the interaction between rapalogs functionalized at C16 and C20 and a panel of Frb mutants. Several drug-Frb mutant combinations have different and nonoverlapping specificities. These Frb-rapalog partners permit the selective control of different Frb fusion proteins without crossreaction. The orthogonal control of multiple target proteins broadens the capabilities of chemical induction of dimerization to regulate biologic processes.
View details for DOI 10.1016/j.chembiol.2005.10.017
View details for Web of Science ID 000235090900014
View details for PubMedID 16426976
Lymphocyte calcium signaling from membrane to nucleus
2006; 7 (1): 25-32
Ca(2+) signals control a variety of lymphocyte responses, ranging from short-term cytoskeletal modifications to long-term changes in gene expression. The identification of molecules and channels that modulate Ca(2+) entry into T and B lymphocytes has both provided details of the molecular events leading to immune responses and raised controversy. Here we review studies of the pathways that allow Ca(2+) entry, the function of Ca(2+) in the regulation of cell polarity and motility and the principles by which Ca(2+)-dependent transcription regulates lymphocyte function.
View details for DOI 10.1038/ni1295
View details for Web of Science ID 000234139700015
View details for PubMedID 16357855
'Nature-inspired' drug-protein complexes as inhibitors of A beta aggregation
Conference on Molecular Mechanisms of Neurodegeneration
PORTLAND PRESS LTD. 2005: 543–547
Protein-protein interactions are a regulatory mechanism for a number of physiological and pathological cellular processes. Neurodegenerative diseases, such as AD (Alzheimer's disease), are associated with the accelerated production or delayed clearance of protein aggregates. Hence, inhibition of pathologic protein-protein interactions is a very attractive mechanism for drug development. This review focuses on a novel therapeutic strategy to inhibit the de novo formation of protein aggregates. Inspired by strategies used in Nature and optimized over millions of years of evolution, we have created a bifunctional molecule [SLF (synthetic ligand for FK506-binding protein)-CR (Congo Red)] that is able to block Abeta (amyloid beta) aggregation by borrowing the surface and steric bulk of a cellular chaperone.
View details for Web of Science ID 000231345700001
View details for PubMedID 16042540
Probing Wnt signaling using small molecule regulation of protein stability and localization.
64th Annual Meeting of the Society-for-Development-Biology
ACADEMIC PRESS INC ELSEVIER SCIENCE. 2005: 580–80
View details for Web of Science ID 000230683800049
Integration of Notch 1 and Calcineurin/NFAT signaling pathways in keratinocyte growth and differentiation control
2005; 8 (5): 665-676
The Notch and Calcineurin/NFAT pathways have both been implicated in control of keratinocyte differentiation. Induction of the p21(WAF1/Cip1) gene by Notch 1 activation in differentiating keratinocytes is associated with direct targeting of the RBP-Jkappa protein to the p21 promoter. We show here that Notch 1 activation functions also through a second Calcineurin-dependent mechanism acting on the p21 TATA box-proximal region. Increased Calcineurin/NFAT activity by Notch signaling involves downregulation of Calcipressin, an endogenous Calcineurin inhibitor, through a HES-1-dependent mechanism. Besides control of the p21 gene, Calcineurin contributes significantly to the transcriptional response of keratinocytes to Notch 1 activation, both in vitro and in vivo. In fact, deletion of the Calcineurin B1 gene in the skin results in a cyclic alopecia phenotype, associated with altered expression of Notch-responsive genes involved in hair follicle structure and/or adhesion to the surrounding mesenchyme. Thus, an important interconnection exists between Notch 1 and Calcineurin-NFAT pathways in keratinocyte growth/differentiation control.
View details for DOI 10.1016/j.devcel.2005.02.016
View details for Web of Science ID 000229103000007
View details for PubMedID 15866158
Recruitment of the extracellular signal-regulated kinase/ribosomal S6 kinase signaling pathway to the NFATc4 transcription activation complex
MOLECULAR AND CELLULAR BIOLOGY
2005; 25 (3): 907-920
Integration of protein kinases into transcription activation complexes influences the magnitude of gene expression. The nuclear factor of activated T cells (NFAT) group of proteins are critical transcription factors that direct gene expression in immune and nonimmune cells. A balance of phosphotransferase activity is necessary for optimal NFAT activation. Activation of NFAT requires dephosphorylation by the calcium-mediated calcineurin phosphatase to promote NFAT nuclear accumulation, and the Ras-activated extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase, which targets NFAT partners, to potentiate transcription. Whether protein kinases operate on NFAT and contribute positively to transcription activation is not clear. Here, we coupled DNA affinity isolation with in-gel kinase assays to avidly pull down the activated NFAT and identify its associated protein kinases. We demonstrate that p90 ribosomal S6 kinase (RSK) is recruited to the NFAT-DNA transcription complex upon activation. The formation of RSK-NFATc4-DNA transcription complex is also apparent upon adipogenesis. Bound RSK phosphorylates Ser(676) and potentiates NFATc4 DNA binding by escalating NFAT-DNA association. Ser(676) is also targeted by the ERK MAP kinase, which interacts with NFAT at a distinct region than RSK. Thus, integration of the ERK/RSK signaling pathway provides a mechanism to modulate NFATc4 transcription activity.
View details for DOI 10.1128/MCB.25.3.907-920.2005
View details for Web of Science ID 000226652900005
View details for PubMedID 15657420
View details for PubMedCentralID PMC544015
Immunology. Decoding calcium signaling.
2005; 307 (5706): 56-57
View details for PubMedID 15637261
Generalized resistance to thymic deletion in the NOD mouse: A polygenic trait characterized by defective induction of Bim
2004; 21 (6): 817-830
The cause of common polygenic autoimmune diseases is not understood because of genetic and cellular complexity. Here, we pinpoint the action of a subset of autoimmune susceptibility loci in the NOD mouse strain linked to D1mit181, D2mit490, D7mit101, and D15mit229, which cause a generalized resistance to thymic deletion in vivo that applies equally to Aire-induced organ-specific gene products in the thymic medulla and to systemic antigens expressed at high levels throughout the thymus and affects CD4(+), CD4(+)8(+), and CD4(+)25(+) thymocytes. Resistance to thymic deletion does not reflect a general deficit in TCR signaling to calcineurin- or ERK-induced genes, imbalance in constitutive regulators of apoptosis, nor excessive signaling to prosurvival genes but is distinguished by failure to induce the proapoptotic gene and protein, Bim, during in vivo encounter with high-avidity autoantigen. These findings establish defects in thymic deletion and Bim induction as a key mechanism in the pathogenesis of autoimmunity.
View details for Web of Science ID 000225901600009
View details for PubMedID 15589170
Harnessing chaperones to generate small-molecule inhibitors of amyloid beta aggregation
2004; 306 (5697): 865-869
Protein aggregation is involved in the pathogenesis of neurodegenerative diseases and hence is considered an attractive target for therapeutic intervention. However, protein-protein interactions are exceedingly difficult to inhibit. Small molecules lack sufficient steric bulk to prevent interactions between large peptide surfaces. To yield potent inhibitors of beta-amyloid (Abeta) aggregation, we synthesized small molecules that increase their steric bulk by binding to chaperones but also have a moiety available for interaction with Abeta. This strategy yields potent inhibitors of Abeta aggregation and could lead to therapeutics for Alzheimer's disease and other forms of neurodegeneration.
View details for Web of Science ID 000224969400045
View details for PubMedID 15514157
A field of myocardial-endocardial NFAT signaling underlies heart valve morphogenesis
2004; 118 (5): 649-663
The delicate leaflets that make up vertebrate heart valves are essential for our moment-to-moment existence. Abnormalities of valve formation are the most common serious human congenital defect. Despite their importance, relatively little is known about valve development. We show that the initiation of heart valve morphogenesis in mice requires calcineurin/NFAT to repress VEGF expression in the myocardium underlying the site of prospective valve formation. This repression of VEGF at E9 is essential for endocardial cells to transform into mesenchymal cells. Later, at E11, a second wave of calcineurin/NFAT signaling is required in the endocardium, adjacent to the earlier myocardial site of NFAT action, to direct valvular elongation and refinement. Thus, NFAT signaling functions sequentially from myocardium to endocardium within a valvular morphogenetic field to initiate and perpetuate embryonic valve formation. This mechanism also operates in zebrafish, indicating a conserved role for calcineurin/NFAT signaling in vertebrate heart valve morphogenesis.
View details for Web of Science ID 000223730300014
View details for PubMedID 15339668
Genetic loss of calcineurin blocks mechanical overload-induced skeletal muscle fiber type switching but not hypertrophy
JOURNAL OF BIOLOGICAL CHEMISTRY
2004; 279 (25): 26192-26200
The serine/threonine phosphatase calcineurin is an important regulator of calcium-activated intracellular responses in eukaryotic cells. In higher eukaryotes, calcium/calmodulin-mediated activation of calcineurin facilitates direct dephosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T-cells (NFAT). Recently, controversy has surrounded the role of calcineurin in mediating skeletal muscle cell hypertrophy. Here we examined the ability of calcineurin-deficient mice to undergo skeletal muscle hypertrophic growth following mechanical overload (MOV) stimulation or insulin-like growth factor-1 (IGF-1) stimulation. Two distinct models of calcineurin deficiency were employed: calcineurin Abeta gene-targeted mice, which show a approximately 50% reduction in total calcineurin, and calcineurin B1-LoxP-targeted mice crossed with a myosin light chain 1f cre knock-in allele, which show a greater than 80% loss of total calcineurin only in skeletal muscle. Calcineurin Abeta-/- and calcineurin B1-LoxP(fl/fl)-MLC-cre mice show essentially no defects in muscle growth in response to IGF-1 treatment or MOV stimulation, although calcineurin Abeta-/- mice show a basal defect in total fiber number in the plantaris and a mild secondary reduction in growth, consistent with a developmental defect in myogenesis. Both groups of gene-targeted mice show normal increases in Akt activation following MOV or IGF-1 stimulation. However, overload-mediated fiber-type switching was dramatically impaired in calcineurin B1-LoxP(fl/fl)-MLC-cre mice. NFAT-luciferase reporter transgenic mice failed to show a correlation between IGF-1- or MOV-induced hypertrophy and calcineurin-NFAT-dependent signaling in vivo. We conclude that calcineurin expression is important during myogenesis and fiber-type switching, but not for muscle growth in response to hypertrophic stimuli.
View details for DOI 10.1074/jbc.M313800200
View details for Web of Science ID 000222003000034
View details for PubMedID 15082723
Biochemical and structural basis for partially redundant enzymatic and ranscriptional functions of DCoH and DCoH2
2004; 43 (23): 7345-7355
An inherited form of diabetes, maturity-onset diabetes of the young type 3 (MODY3), results from mutations in the transcriptional activator, hepatocyte nuclear factor-1alpha (HNF1alpha). Transcription by HNF1alpha is stimulated by the bifunctional coactivator DCoH (dimerization cofactor of HNF1). Strikingly, an HNF1alpha deletion in mice causes more severe phenotypes than a DCoH deletion. It has been hypothesized that a DCoH homolog, DCoH2, partially complements the DCoH deletion. To test this idea, we determined the biochemical properties and the 1.6-A-resolution crystal structure of DCoH2. Like DCoH, DCoH2 forms a tetramer, displays pterin-4alpha-carbinolamine dehydratase activity, and binds HNF1alpha in vivo and in vitro. DCoH and DCoH2 adopt identical folds with structural differences confined largely to the protein surfaces and the tetramer interface. In contrast to the hyperstable DCoH tetramer, DCoH2 readily disproportionates and forms a 2:2 complex with HNF1 in vitro. Phylogenetic analysis reveals six major subfamilies of DCoH proteins, including unique DCoH and DCoH2 branches in metazoans. These results suggest distinct roles for DCoH and DCoH2. Differences in conserved surface residues could mediate binding to different effectors. We propose that HNF1alpha binding kinetics may distinguish regulation by DCoH2, under thermodynamic control, from regulation by DCoH, under kinetic control.
View details for DOI 10.1021/bi049620t
View details for Web of Science ID 000221915100011
View details for PubMedID 15182178
- Calcineurin: a central controller of signalling in eukaryotes. EMBO reports 2004; 5 (4): 343-348
Calcineurin is required in urinary tract mesenchyme for the development of the pyeloureteral peristaltic machinery
JOURNAL OF CLINICAL INVESTIGATION
2004; 113 (7): 1051-1058
Congenital obstructive nephropathy is the principal cause of renal failure in infants and children. The underlying molecular and cellular mechanisms of this disease, however, remain largely undetermined. We generated a mouse model of congenital obstructive nephropathy that resembles ureteropelvic junction obstruction in humans. In these mice, calcineurin function is removed by the selective deletion of Cnb1 in the mesenchyme of the developing urinary tract using the Cre/lox system. This deletion results in reduced proliferation in the smooth muscle cells and other mesenchymal cells in the developing urinary tract. Compromised cell proliferation causes abnormal development of the renal pelvis and ureter, leading to defective pyeloureteral peristalsis, progressive renal obstruction, and, eventually, fatal renal failure. Our study demonstrates that calcineurin is an essential signaling molecule in urinary tract development and is required for normal proliferation of the urinary tract mesenchymal cells in a cell-autonomous manner. These studies also emphasize the importance of functional obstruction, resulting from developmental abnormality, in causing congenital obstructive nephropathy.
View details for DOI 10.1172/JCI200420049
View details for Web of Science ID 000220651700018
View details for PubMedID 15057312
View details for PubMedCentralID PMC379320
Calcineurin B1 is essential for positive but not negative selection during thymocyte development
2004; 20 (3): 255-266
During development, discrete cell fates often result from variation in the intensity of a particular signal. The mechanisms underlying these seemingly analog-to-digital switches are not understood. In developing T lymphocytes, low-intensity signals through the antigen receptor result in positive selection while more intense signals give rise to negative selection. By deleting the genetic locus encoding the regulatory B1 subunit of calcineurin specifically in thymocytes, we found an absolute requirement for calcineurin in positive selection. In contrast, calcineurin activity was dispensable in several models of negative selection. Unexpectedly, we found that removal of calcineurin activity from thymocytes results in inefficient ERK activation at the double-positive stage of thymocyte development, when selection occurs. These studies clarify the mechanism by which graded signals are converted to discrete outcomes in T cell development and further indicate that the developmental roles of calcineurin likely contribute to immunosuppression by calcineurin inhibitors.
View details for Web of Science ID 000221442600004
View details for PubMedID 15030770
Dynamic changes in transcription factor complexes during erythroid differentiation revealed by quantitative proteomics
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2004; 11 (1): 73-80
During erythroid differentiation, beta-globin gene expression is regulated by the locus control region (LCR). The transcription factor NF-E2p18/MafK binds within this region and is essential for beta-globin expression in murine erythroleukemia (MEL) cells. Here we use the isotope-coded affinity tag (ICAT) technique of quantitative mass spectrometry to compare proteins interacting with NF-E2p18/MafK during differentiation. Our results define MafK as a 'dual-function' molecule that shifts from a repressive to an activating mode during erythroid differentiation. The exchange of MafK dimerization partner from Bach1 to NF-E2p45 is a key step in the switch from the repressed to the active state. This shift is associated with changes in the interaction of MafK with co-repressors and co-activators. Thus, our results suggest that in addition to its role as a cis-acting activator of beta-globin gene expression in differentiated erythroid cells, the LCR also promotes an active repression of beta-globin transcription in committed cells before terminal differentiation.
View details for DOI 10.1038/nsmb713
View details for Web of Science ID 000220143900016
View details for PubMedID 14718926
Conditional protein alleles using knockin mice and a chemical inducer of dimerization
2003; 12 (6): 1615-1624
We have developed a general method of making conditional alleles that allows the rapid and reversible regulation of specific proteins. A mouse line was produced in which proteins encoded by the endogenous glycogen synthase kinase-3 beta (GSK-3beta) gene are fused to an 89 amino acid tag, FRB*. FRB* causes the destabilization of GSK-3beta, producing a severe loss-of-function allele. In the presence of C20-MaRap, a highly specific, nontoxic, cell-permeable small molecule, GSK-3betaFRB* binds to the ubiquitously expressed FKBP12 protein. This interaction stabilizes GSK-3betaFRB* and restores both protein levels and activity. C20-MaRap-mediated stabilization is rapidly reversed by the addition of an FKBP12 binding competitor molecule. This technology may be applied to a wide range of FRB*-tagged mouse genes while retaining their native transcriptional control. Inducible stabilization could be valuable for many developmental and physiological studies and for drug target validation.
View details for Web of Science ID 000187511600028
View details for PubMedID 14690613
Sequential roles of Brg, the ATPase subunit of BAF chromatin remodeling complexes, in thymocyte development
2003; 19 (2): 169-182
T cells develop through distinct stages directed by a series of signals. We explored the roles of SWI/SNF-like BAF chromatin remodeling complexes in this process by progressive deletion of the ATPase subunit, Brg, through successive stages of early T cell development. Brg-deficient cells were blocked at each of the developmental transitions examined. Bcl-xL overexpression suppressed cell death without relieving the developmental blockades, leading to the accumulation of Brg-deleted cells that were unexpectedly cell cycle arrested. These defects resulted partly from the disruptions of pre-TCR and potentially Wnt signaling pathways controlling the expression of genes such as c-Kit and c-Myc critical for continued development. Our studies indicate that BAF complexes dynamically remodel chromatin to propel sequential developmental transitions in response to external signals.
View details for Web of Science ID 000184929000005
View details for PubMedID 12932351
Calcium signalling in lymphocytes
CURRENT OPINION IN IMMUNOLOGY
2003; 15 (3): 299-307
The modulation of intracellular calcium ion concentration, [Ca(2+)](i), is a common signalling mechanism used in many biological systems. B and T lymphocytes rely on Ca(2+) signalling to initiate both developmental and activation programs. Recent data has shed new light on the initiation of this signalling pathway, the connection between the release of intracellular Ca(2+) stores and the influx of extracellular Ca(2+), and the molecular identity of the elusive Ca(2+) release-activated Ca(2+) (CRAC) channel. In addition, recent gene profiling of T lymphocytes has identified the genes that are controlled by [Ca(2+)](i) and the Ca(2+)-dependent phosphatase calcineurin.
View details for DOI 10.1016/S0952-7915(03)00050-5
View details for Web of Science ID 000183483800010
View details for PubMedID 12787755
Neurotrophins and netrins require calcineurin/NFAT signaling to stimulate outgrowth of embryonic axons
2003; 113 (5): 657-670
Axon outgrowth is the first step in the formation of neuronal connections, but the pathways that regulate axon extension are still poorly understood. We find that mice deficient in calcineurin-NFAT signaling have dramatic defects in axonal outgrowth, yet have little or no defect in neuronal differentiation or survival. In vitro, sensory and commissural neurons lacking calcineurin function or NFATc2, c3, and c4 are unable to respond to neurotrophins or netrin-1 with efficient axonal outgrowth. Neurotrophins and netrins stimulate calcineurin-dependent nuclear localization of NFATc4 and activation of NFAT-mediated gene transcription in cultured primary neurons. These data indicate that the ability of these embryonic axons to respond to growth factors with rapid outgrowth requires activation of calcineurin/NFAT signaling by these factors. The precise parsing of signals for elongation turning and survival could allow independent control of these processes during development.
View details for Web of Science ID 000183397600015
View details for PubMedID 12787506
Sonographic staging of the developmental status of mouse embryos in utero
2003; 36 (1): 7-11
In mouse developmental studies it is frequently desirable to isolate embryos of a specific age. However, the traditional staging of embryonic development based on postcoital dates often erroneously predicts the embryonic age, resulting in unwarranted sacrifice of the pregnant mother. Here we report a noninvasive way of staging embryonic development in utero. A clinical 14 MHz ultrasound system was employed to assess the morphology and size of developing embryos from embryonic day 7.5 to 18.5. We demonstrate that the developmental age of the mouse embryos can be accurately determined based on the sonographic morphology and size of the embryos. This noninvasive ultrasound application requires no anesthesia of the mice and the entire process of staging can be completed within 5-10 min. Empirically, this approach is applicable to mice of various genetic backgrounds and significantly enhances the efficiency of studying murine embryogenesis.
View details for DOI 10.1002/gene.10186
View details for Web of Science ID 000183397100002
View details for PubMedID 12748962
Protein kinase A negatively modulates the nuclear accumulation of NF-ATc1 by priming for subsequent phosphorylation by glycogen synthase kinase-3
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (50): 48664-48676
The nuclear localization and transcriptional activity of the NF-ATc family of transcription factors, essential to many developmental, differentiation, and adaptation processes, are determined by the opposing activities of the phosphatase calcineurin, which promotes nuclear accumulation of NF-ATc, and several kinases, which promote cytoplasmic accumulation. Many reports suggest that protein kinase A (PKA) negatively modulates calcineurin-mediated NF-ATc activation. Here we show that overexpression of PKA causes phosphorylation and cytoplasmic accumulation of NF-ATc1 in direct opposition to calcineurin by phosphorylating Ser-245, Ser-269, and Ser-294 in the conserved serine-proline repeat domain, and that mutation of these serines blocks the effect of PKA. Activation of endogenous PKA is similarly able to promote phosphorylation of these sites on NF-ATc1 in two lymphoid cell lines. We further show that a complete block of NF-ATc1 nuclear localization by PKA requires a second kinase activity that can be supplied by glycogen synthase kinase-3 (GSK-3), and that mutation of either the PKA phosphorylation sites or the upstream GSK-3 sites prevents the effect of PKA. Thus, we propose that PKA functions cooperatively as a priming kinase for further phosphorylation by GSK-3 to oppose calcineurin-mediated nuclear accumulation and transcriptional activity of NF-ATc1 and that, through this mechanism, PKA may be an important modulator of many NF-ATc-dependent processes.
View details for DOI 10.1074/jbc.M207029200
View details for Web of Science ID 000179789600091
View details for PubMedID 12351631
Identification of a polymorphic, neuron-specific chromatin remodeling complex
GENES & DEVELOPMENT
2002; 16 (19): 2509-2517
A variety of chromatin remodeling complexes are thought to assist sequence-specific transcription factors. The complexes described to date are expressed ubiquitously, suggesting that they have general transcriptional functions. We show that vertebrate neurons have a specialized chromatin remodeling complex, bBAF, specifically containing the actin-related protein, BAF53b, which is first expressed in postmitotic neurons at about murine embryonic day 12.5 (E12.5). BAF53b is combinatorially assembled into polymorphic complexes with ubiquitous subunits including the two ATPases BRG1 and BRM. We speculate that bBAF complexes create neuronal-specific patterns of chromatin accessibility, thereby imparting new regulatory characteristics to ubiquitous sequence-specific transcription factors in neurons.
View details for DOI 10.1101/gad.992102
View details for Web of Science ID 000178468000007
View details for PubMedID 12368262
View details for PubMedCentralID PMC187451
Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2002; 99 (18): 11796-11801
Optimal activation of T cells requires effective occupancy of both the antigen-specific T cell receptor and a second coreceptor such as CD28. We used cDNA microarrays to characterize the genomic expression program in human peripheral T cells responding to stimulation of these receptors. We found that CD28 agonists alone elicited few, but reproducible, changes in gene expression, whereas CD3 agonists elicited a multifaceted temporally choreographed gene expression program. The principal effect of simultaneous engagement of CD28 was to increase the amplitude of the CD3 transcriptional response. The induced genes whose expression was most enhanced by costimulation were significantly enriched for known targets of nuclear factor of activated T cells (NFAT) transcription factors. This enhancement was nearly abolished by blocking the nuclear translocation of NFATc by using the calcineurin inhibitor FK506. CD28 signaling promoted phosphorylation, and thus inactivation, of the NFAT nuclear export kinase glycogen synthase kinase-3 (GSK3), coincident with enhanced dephosphorylation of NFATc proteins. These results provide a detailed picture of the transcriptional program of T cell activation and suggest that enhancement of transcriptional activation by NFAT, through inhibition of its nuclear export, plays a key role in mediating the CD28 costimulatory signal.
View details for DOI 10.1073/pnas.092284399
View details for PubMedID 12195013
Hyperphenylalaninemia and impaired glucose tolerance in mice lacking the bifunctional DCoH gene
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (32): 28884-28891
The bifunctional protein DCoH (Dimerizing Cofactor for HNF1) acts as an enzyme in intermediary metabolism and as a binding partner of the HNF1 family of transcriptional activators. HNF1 proteins direct the expression of a variety of genes in the liver, kidney, pancreas, and gut and are critical to the regulation of glucose homeostasis. Mutations of the HNF1alpha gene underlie maturity onset diabetes of the young (MODY3) in humans. DCoH acts as a cofactor for HNF1 that stabilizes the dimeric HNF1 complex. DCoH also catalyzes the recycling of tetrahydrobiopterin, a cofactor of aromatic amino acid hydroxylases. To examine the roles of DCoH, a targeted deletion allele of the murine DCoH gene was created. Mice lacking DCoH are viable and fertile but display hyperphenylalaninemia and a predisposition to cataract formation. Surprisingly, HNF1 function in DCoH null mice is only slightly impaired, and mice are mildly glucose-intolerant in contrast to HNF1alpha null mice, which are diabetic. DCoH function as it pertains to HNF1 activity appears to be partially complemented by a newly identified homolog, DCoH2.
View details for DOI 10.1074/jbc.M201983200
View details for Web of Science ID 000177342600072
View details for PubMedID 12011081
Reciprocal regulation of CD4/CD8 expression by SWI/SNF-like BAF complexes
2002; 418 (6894): 195-199
Thymic development produces two sub-lineages of T cells expressing either CD4 or CD8 co-receptors that assist antibody production and mediate cell killing, respectively. The mechanisms for mutually exclusive co-receptor expression remain poorly defined. We find that mutations in the high mobility group (HMG) domain of BAF57--a DNA-binding subunit of the mammalian SWI/SNF-like chromatin-remodelling BAF complexes--or in the BAF complex ATPase subunit Brg, impair both CD4 silencing and CD8 activation. Brg is haploinsufficient for CD8 activation, but not for CD4 silencing, whereas BAF57 mutations preferentially impair CD4 silencing, pointing to target- and subunit-specific mechanisms of chromatin remodelling. BAF complexes directly bind the CD4 silencer, but the BAF57 HMG domain is dispensable for tethering BAF complexes to the CD4 silencer or other chromatin loci in vivo, or for remodelling reconstituted templates in vitro, suggesting that chromatin remodelling in vivo requires HMG-dependent DNA bending. These results indicate that BAF complexes contribute to lineage bifurcation by reciprocally regulating lineage-specific genes, reminiscent of the role of the yeast SWI/SNF complex in mediating mating-type switching.
View details for DOI 10.1038/nature00876
View details for Web of Science ID 000176710400043
View details for PubMedID 12110891
NFAT signaling: Choreographing the social lives of cells
2002; 109: S67-S79
Calcium signaling activates the phosphatase calcineurin and induces movement of NFATc proteins into the nucleus, where they cooperate with other proteins to form complexes on DNA. Nuclear import is opposed by kinases such as GSK3, thereby rendering transcription continuously responsive to receptor occupancy. Disruptions of the genes involved in NFAT signaling are implicating this pathway as a regulator of developmental cell-cell interactions.
View details for Web of Science ID 000175181100007
View details for PubMedID 11983154
Phosphatidylinositol-dependent actin filament binding by the SWI/SNF-like BAF chromatin remodeling complex
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2002; 99 (5): 2824-2829
Recently, several chromatin remodeling complexes in yeast, Drosophila, and mammals have been shown to contain actin and actin-related proteins (arps). However, the function of actin in these complexes is unclear. Here, we show that the mammalian SWI/SNF-like BAF complex binds to phosphatidylinositol 4,5-bisphosphate (PIP2) micelles and PIP2-containing mixed lipid vesicles, and that PIP2 binding allows the complex to associate with actin pointed ends and branch points. Actin binds to at least two distinct domains in the C terminus of the Brg1 protein, and interaction with only one of these domains is sensitive to PIP2. Based on these findings, we propose a model for PIP2 activation of actin binding by relief of intramolecular capping of actin by Brg1.
View details for DOI 10.1073/pnas.032662899
View details for Web of Science ID 000174284600044
View details for PubMedID 11880634
View details for PubMedCentralID PMC122432
Nuclear actin and actin-related proteins in chromatin remodeling
ANNUAL REVIEW OF BIOCHEMISTRY
2002; 71: 755-781
The existence and function of actin in the nucleus has been hotly debated for forty years. Recently, beta-actin was found to be a component of mammalian SWI/SNF-like BAF chromatin remodeling complexes and still more recently other SWI/SNF-related chromatin remodeling complexes in yeast, flies, and man. Although the function of actin in these chromatin remodeling complexes is only starting to be explored, the fact that actin is one of the most regulated proteins in the cell suggests that control of nuclear actin may be a critical regulatory point in the control of chromatin remodeling. Actin rapidly shuttles between the nucleus and the cytoplasm offering additional sites and modes of regulation. In addition, actin-related proteins (Arps) are also components of these chromatin remodeling complexes and have been implicated in transcriptional control in yeast. The observation that the BAF chromatin remodeling complex in which actin was originally identified, is also a human tumor suppressor complex necessary for the actions of the retinoblastoma protein indicates that the study of nuclear actin is likely to contribute to understanding cell growth control.
View details for DOI 10.1146/annurev.biochem.71.110601.135507
View details for Web of Science ID 000177352600024
View details for PubMedID 12045110
NFAT signaling in vertebrate development
CURRENT OPINION IN GENETICS & DEVELOPMENT
2001; 11 (5): 505-512
NFATc proteins transduce Ca(2+) signals to the nucleus and then pair with other proteins on DNA to generate NFAT complexes that activate transcription in response to both electrical and tyrosine kinase signaling. The four NFATc genes arose at the origin of vertebrates, implying that they have evolved for the development of vertebrate-specific functions, such as a complex nervous system, a recombinational immune system, and a vascular system with a complex heart. These speculations are borne out by studies of mice with null mutations in the different family members.
View details for Web of Science ID 000171477500003
View details for PubMedID 11532391
Signals transduced by Ca2+/calcineurin and NFATc3/c4 pattern the developing vasculature
2001; 105 (7): 863-875
Vascular development requires an orderly exchange of signals between growing vessels and their supporting tissues, but little is known of the intracellular signaling pathways underlying this communication. We find that mice with disruptions of both NFATc4 and the related NFATc3 genes die around E11 with generalized defects in vessel assembly as well as excessive and disorganized growth of vessels into the neural tube and somites. Since calcineurin is thought to control nuclear localization of NFATc proteins, we introduced a mutation into the calcineurin B gene that prevents phosphatase activation by Ca(2+) signals. These CnB mutant mice exhibit vascular developmental abnormalities similar to the NFATc3/c4 null mice. We show that calcineurin function is transiently required between E7.5 and E8.5. Hence, early calcineurin/NFAT signaling initiates the later cross-talk between vessels and surrounding tissues that pattern the vasculature.
View details for Web of Science ID 000169664300007
View details for PubMedID 11439183
Cell signaling can direct either binary or graded transcriptional responses
2001; 20 (12): 3167-3176
Transcriptional control is generally thought to operate as a binary switch, a behavior that might explain observations such as monoallelic gene expression, stochastic phenotypic changes and bimodal gene activation kinetics. By measuring the activity of the single-copy GAL1 promoter in single cells, we found that changes in the activities of either the transcriptional activator, Gal4 (by simple recruitment with synthetic ligands), or the transcriptional repressor, Mig1, generated graded (non-binary) changes in gene expression that were proportional to signal intensity. However, in the context of the endogenous glucose-responsive signaling pathway, these transcription factors formed part of a binary transcriptional response. Genetic studies demonstrated that this binary response resulted from regulation of a second repressor, Gal80, whereas regulation of Mig1 by a distinct signaling pathway generated graded changes in GAL1 promoter activity. Surprisingly, isogenetic cells can respond to glucose with either binary or graded changes in gene expression, depending on growth conditions. Our studies demonstrate that a given promoter can adapt either binary or graded behavior, and identify the Mig1 and Gal80 genes as necessary for binary versus graded behavior of the Gal1 promoter.
View details for Web of Science ID 000169450000019
View details for PubMedID 11406593
View details for PubMedCentralID PMC150188
Monitoring the duration of antigen-receptor occupancy by calcineurin/glycogen-synthase-kinase-3 control of NF-AT nuclear shuttling
CURRENT OPINION IN IMMUNOLOGY
2001; 13 (3): 346-350
Recent structural studies have supported a kinetic model of TCR activation, raising the question of how the duration of receptor occupancy is translated into activation of immune response genes. We summarize evidence that the cytoplasmic-to-nuclear shuttling of NF-ATc family members monitors the duration of receptor occupancy.
View details for Web of Science ID 000168477300012
View details for PubMedID 11406367
Evolutionary relationships among Rel domains indicate functional diversification by recombination
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2001; 98 (10): 5740-5745
The recent sequencing of several complete genomes has made it possible to track the evolution of large gene families by their genomic structure. Following the large-scale association of exons encoding domains with well defined functions in invertebrates could be useful in predicting the function of complex multidomain proteins in mammals produced by accretion of domains. With this objective, we have determined the genomic structure of the 14 genes in invertebrates and vertebrates that contain rel domains. The sequence encoding the rel domain is defined by intronic boundaries and has been recombined with at least three structurally and functionally distinct genomic sequences to generate coding sequences for: (i) the rel/Dorsal/NFkappaB proteins that are retained in the cytoplasm by IkB-like proteins; (ii) the NFATc proteins that sense calcium signals and undergo cytoplasmic-to-nuclear translocation in response to dephosphorylation by calcineurin; and (iii) the TonEBP tonicity-responsive proteins. Remarkably, a single exon in each NFATc family member encodes the entire Ca(2+)/calcineurin sensing region, including nuclear import/export, calcineurin-binding, and substrate regions. The Rel/Dorsal proteins and the TonEBP proteins are present in Drosophila but not Caenorhabditis elegans. On the other hand, the calcium-responsive NFATc proteins are present only in vertebrates, suggesting that the NFATc family is dedicated to functions specific to vertebrates such as a recombinational immune response, cardiovascular development, and vertebrate-specific aspects of the development and function of the nervous system.
View details for Web of Science ID 000168623300067
View details for PubMedID 11344309
Jun N-terminal kinase 2 modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear factor of activated T cell (NF-AT)
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2001; 98 (4): 1769-1774
The Jun N-terminal kinases (JNKs) recently have been shown to be required for thymocyte apoptosis and T cell differentiation and/or proliferation. To investigate the molecular targets of JNK signaling in lymphoid cells, we used mice in which the serines phosphorylated by JNK in c-Jun were replaced by homologous recombination with alanines (junAA mice). Lymphocytes from these mice showed no phosphorylation of c-Jun in response to activation stimuli, whereas c-Jun was rapidly phosphorylated in wild-type cells. Despite the fact that c-jun is essential for early development, junAA mice develop normally; however, c-Jun N-terminal phosphorylation was required for efficient T cell receptor-induced and tumor necrosis factor-alpha-induced thymocyte apoptosis. In contrast, c-Jun phosphorylation by JNK is not required for T cell proliferation or differentiation. Because jnk2-/- T cells display a proliferation defect, we concluded that JNK2 must have other substrates required for lymphocyte function. Surprisingly, jnk2-/- T cells showed reduced NF-AT DNA-binding activity after activation. Furthermore, overexpression of JNK2 in Jurkat T cells strongly enhanced NF-AT-dependent transcription. These results demonstrate that JNK signaling differentially uses c-Jun and NF-AT as molecular effectors during thymocyte apoptosis and T cell proliferation.
View details for Web of Science ID 000166949200083
View details for PubMedID 11172026
View details for PubMedCentralID PMC29332
- Calcium, calcineurin, and the control of transcription JOURNAL OF BIOLOGICAL CHEMISTRY 2001; 276 (4): 2313-2316
- Calcineurin inhibitors and the generalization of the presenting protein strategy ADVANCES IN PROTEIN CHEMISTRY, VOL 56 2001; 56: 253-?
A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes
2000; 6 (6): 1287-1295
Mammalian SWI/SNF complexes utilize either brahma (Brm) or brahma-related gene 1 (Brg1) catalytic subunits to remodel nucleosomes in an ATP-dependent manner. Brm was previously shown to be dispensable, suggesting that Brm and Brg1 are functionally redundant. To test this hypothesis, we have generated a Brg1 null mutation by gene targeting, and, surprisingly, homozygotes die during the periimplantation stage. Furthermore, blastocyst outgrowth studies indicate that neither the inner cell mass nor trophectoderm survives. However, experiments with other cell types demonstrate that Brg1 is not a general cell survival factor. In addition, Brg1 heterozygotes are predisposed to exencephaly and tumors. These results provide evidence that biochemically similar chromatin-remodeling complexes have dramatically different functions during mammalian development.
View details for Web of Science ID 000166239500002
View details for PubMedID 11163203
- Transcription - Regulation of the regulators NATURE 2000; 408 (6808): 46-47
Structural basis of dimerization, coactivator recognition and MODY3 mutations in HNF-1 alpha
NATURE STRUCTURAL BIOLOGY
2000; 7 (9): 744-748
Maturity-onset diabetes of the young type 3 (MODY3) results from mutations in the transcriptional activator hepatocyte nuclear factor-1alpha (HNF-1alpha). Several MODY3 mutations target the HNF-1alpha dimerization domain (HNF-p1), which binds the coactivator, dimerization cofactor of HNF-1 (DCoH). To define the mechanism of coactivator recognition and the basis for the MODY3 phenotype, we determined the cocrystal structure of the DCoH-HNF-p1 complex and characterized biochemically the effects of MODY3 mutations in HNF-p1. The DCoH-HNF-p1 complex comprises a dimer of dimers in which HNF-p1 forms a unique four-helix bundle. Through rearrangements of interfacial side chains, a single, bifunctional interface in the DCoH dimer mediates both HNF-1alpha binding and formation of a competing, transcriptionally inactive DCoH homotetramer. Consistent with the structure, MODY3 mutations in HNF-p1 reduce activator function by two distinct mechanisms.
View details for Web of Science ID 000089078500014
View details for PubMedID 10966642
The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte-antigen-presenting cell interface
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2000; 97 (18): 10150-10155
During the interaction of a T cell with an antigen-presenting cell (APC), several receptor ligand pairs, including the T cell receptor (TCR)/major histocompatibility complex (MHC), accumulate at the T cell/APC interface in defined geometrical patterns. This accumulation depends on a movement of the T cell cortical actin cytoskeleton toward the interface. Here we study the involvement of the guanine nucleotide exchange factor vav in this process. We crossed 129 vav(-/-) mice with B10/BR 5C.C7 TCR transgenic mice and used peptide-loaded APCs to stimulate T cells from the offspring. We found that the accumulation of TCR/MHC at the T cell/APC interface and the T cell actin cytoskeleton rearrangement were clearly defective in these vav(+/-) mice. A comparable defect in superantigen-mediated T cell activation of T cells from non-TCR transgenic 129 mice was also observed, although in this case it was more apparent in vav(-/-) mice. These data indicate that vav is an essential regulator of cytoskeletal rearrangements during T cell activation.
View details for Web of Science ID 000089067500061
View details for PubMedID 10963677
Chemically regulated transcription factors reveal the persistence of repressor-resistant transcription after disrupting activator function
JOURNAL OF BIOLOGICAL CHEMISTRY
2000; 275 (33): 25381-25390
Control of gene expression often requires that transcription terminates rapidly after destruction, inactivation, or nuclear export of transcription factors. However, the role of transcription factor inactivation in terminating transcription is unclear. We have developed a means of conducting order of addition and co-occupancy experiments in living cells by rapidly exchanging proteins bound to promoters. Using this approach, we found that, following specific disruption of activator function, transcription from active promoters decayed slowly, persisting through multiple cell divisions. This persistent transcriptional activity raised the question of what mechanisms return promoters to inactive states. By exchanging or directing co-occupancy of protein complexes bound to a promoter, we found that the transcriptional inhibitor, Ssn6-Tup1, lost its effectiveness as a repressor following activator dissociation. Similar experiments with another repressor, the histone deacetylase Sin3-Rpd3, reinforced this distinction between repression in the presence and absence of an activator. These results suggest that although repressors such as Ssn6-Tup1 and Sin3-Rpd3 prevent activation of gene expression, other mechanisms of repression return promoters to inactive states following the dissociation or inactivation of a transcriptional activator.
View details for Web of Science ID 000088849400046
View details for PubMedID 10801867
Perspectives: signal transduction. Inositol phosphates in the nucleus.
2000; 287 (5460): 1937-1939
View details for PubMedID 10755944
Searching for a function for nuclear actin
TRENDS IN CELL BIOLOGY
2000; 10 (3): 92-97
The abundant cytoskeletal protein actin has numerous cytoplasmic roles. Although there are many reports of the presence of actin in the nucleus, in general they have been discounted as artifactual. However, recent work has begun to provide evidence for important roles for actin in nuclear processes ranging from chromatin remodelling to splicing. In addition, several regulators of actin polymerization are localized to the nucleus or translocate to the nucleus in a regulated manner, suggesting that there is some function of actin in the nucleus that is subject to regulation. This review discusses the evidence for actin in the nucleus and summarizes recent work suggesting that actin or actin-related proteins are involved in the regulation of nuclear processes such as chromatin remodelling.
View details for Web of Science ID 000085420200004
View details for PubMedID 10675902
- The cytoskeleton in lymphocyte signaling ADVANCES IN IMMUNOLOGY, VOL 75 2000; 75: 89-114
L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons
1999; 401 (6754): 703-708
The molecular basis of learning and memory has been the object of several recent advances, which have focused attention on calcium-regulated pathways controlling transcription. One of the molecules implicated by pharmacological, biochemical and genetic approaches is the calcium/calmodulin-regulated phosphatase, calcineurin. In lymphocytes, calcineurin responds to specific calcium signals and regulates expression of several immediate early genes by controlling the nuclear import of the NF-ATc family of transcription factors. Here we show that NF-ATc4/NF-AT3 in hippocampal neurons can rapidly translocate from cytoplasm to nucleus and activate NF-AT-dependent transcription in response to electrical activity or potassium depolarization. The calcineurin-mediated translocation is critically dependent on calcium entry through L-type voltage-gated calcium channels. GSK-3 can phosphorylate NF-ATc4, promoting its export from the nucleus and antagonizing NF-ATc4-dependent transcription. Furthermore, we show that induction of the inositol 1,4,5-trisphosphate receptor type 1 is controlled by the calcium/calcineurin/NF-ATc pathway. This provides a new perspective on the function of calcineurin in the central nervous system and indicates that NF-AT-mediated gene expression may be involved in the induction of hippocampal synaptic plasticity and memory formation.
View details for Web of Science ID 000083207400058
View details for PubMedID 10537109
Continuous and widespread roles for the Swi-Snf complex in transcription
1999; 18 (8): 2254-2264
Chromatin presents a significant obstacle to transcription, but two means of overcoming its repressive effects, histone acetylation and the activities of the Swi-Snf complex, have been proposed. Histone acetylation and Swi-Snf activity have been shown to be crucial for transcriptional induction and to facilitate binding of transcription factors to DNA. By regulating the activity of the Swi-Snf complex in vivo, we found that active transcription requires continuous Swi-Snf function, demonstrating a role for this complex beyond the induction of transcription. Despite the presumably generalized packaging of genes into chromatin, previous studies have indicated that the transcriptional requirements for the histone acetyltransferase, Gcn5, and the Swi-Snf complex are limited to a handful of genes. However, inactivating Swi-Snf function in cells also lacking GCN5 revealed defects in transcription of several genes previously thought to be SWI-SNF- and GCN5-independent. These findings suggest that chromatin remodeling plays a widespread role in gene expression and that these two chromatin remodeling activities perform independent and overlapping functions during transcriptional activation.
View details for Web of Science ID 000079959200023
View details for PubMedID 10205178
View details for PubMedCentralID PMC1171308
- Generic signals and specific outcomes: Signaling through Ca2+, calcineurin, and NF-AT CELL 1999; 96 (5): 611-614
Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1999; 96 (5): 1953-1958
A general strategy is described for improving the binding properties of small-molecule ligands to protein targets. A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein. When the ligand of interest is presented to the target protein by the second protein, additional protein-protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event. We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide. This approach provides a way to modulate the potency and specificity of biologically active compounds.
View details for Web of Science ID 000078956600029
View details for PubMedID 10051576
View details for PubMedCentralID PMC26718
- The actin cytoskeleton and lymphocyte activation CELL 1999; 96 (1): 9-12
- Signaling through calcium, calcineurin, and NF-AT in lymphocyte activation and development 64th Symposia: Signaling and Gene Expression in the Immune System COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1999: 505–516
- The Actin Cytoskeleton and Lymphocyte Activation Cell -- 1999
Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling
1998; 95 (5): 625-636
Lymphocyte activation is accompanied by visible changes in chromatin structure. We find that antigen receptor signaling induces the rapid association of the BAF complex with chromatin. PIP2, which is regulated by activation stimuli, is sufficient in vitro to target the BAF complex to chromatin, but it has no effect on related chromatin remodeling complexes containing SNF2L or hISWI. Purification and peptide sequencing of the subunits of the complex revealed beta-actin as well as a novel actin-related protein, BAF53. beta-actin and BAF53 are required for maximal ATPase activity of BRG1 and are also required with BRG1 for association of the complex with chromatin/matrix. This work indicates that membrane signals control the activity of the mammalian SWI/SNF or BAF complex and demonstrates a direct interface between signaling and chromatin regulation.
View details for Web of Science ID 000077253700010
View details for PubMedID 9845365
Five SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin (SMARC) genes are dispersed in the human genome
1998; 51 (1): 140-143
The SWI/SNF-related, matrix-associated, actin-dependent regulators of chromatin (SMARC), also called BRG1-associated factors, are components of human SWI/SNF-like chromatin-remodeling protein complexes. We mapped five human SMARC genes toregions on four different human chromosomes, SMARCC1 to 3p23-p21, SMARCC2 to 12q13-q14, SMARCD1 to 12q13-q14, SMARCD2 to 17q23-q24, and SMARCD3 to 7q35-q36. SMARCC1, SMARCC2, and SMARCD1 are assigned to chromosomal regions that are frequently involved in somatic rearrangements in human cancers. SMARCD1 was mapped to the critical region of Allgrove syndrome; however, no mutation was identified in one Allgrove syndrome family studied.
View details for Web of Science ID 000075182000018
View details for PubMedID 9693044
Defects in actin-cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduction
1998; 8 (10): 563-572
Antigen-receptor interactions on lymphocytes result in local clustering of actin, receptors and signaling molecules into an asymmetric membrane structure termed a cap. Although actin polymerization is known to be required, the mechanisms underlying cap formation are unclear. We have studied the events underlying cap formation using mice bearing a null mutation in vav (vav-/-), a gene that encodes a guanine-nucleotide exchange factor for the GTPase Rac.Lymphocytes from vav-/- mice failed to form T-cell receptor caps following activation and had a defective actin cytoskeleton. The vav-/- T cells were deficient in interleukin-2 (IL-2) production and proliferation, and the peak of Ca2+ mobilization was reduced although of normal duration. Activation of Jun N-terminal kinase or stress-activated kinase (JNK or SAPK) and mitogen-activated protein kinase (MAPK) and the induction of the transcription factor NF-ATc1 and egr-1 genes was normal. Despite the reduced Ca2+ mobilization, translocation of cytoplasmic NF-ATc to the nucleus was normal, reflecting that the lower levels of Ca2+ in vav-/- cells were still sufficient to activate calcineurin. Treatment of lymphocytes with cytochalasin D, which blocks actin polymerization, inhibited cap formation and produced defects in signaling and IL-2 transcriptional induction in response to antigen-receptor signaling that were nearly identical to those seen in vav-/- cells. In transfection studies, either constitutively active Vav or Rac could complement constitutively active calcineurin to activate NF-AT-dependent transcription.These results indicate that Vav is required for cap formation in lymphocytes. Furthermore, the correlation between cap formation, IL-2 production and proliferation supports the hypothesis that an actin-dependent pathway is a source of specialized growth regulatory signals.
View details for Web of Science ID 000073590300014
View details for PubMedID 9601640
Role of the NF-ATc transcription factor in morphogenesis of cardiac valves and septum
1998; 392 (6672): 182-186
In lymphocytes, the expression of early immune response genes is regulated by NF-AT transcription factors which translocate to the nucleus after dephosphorylation by the Ca2+-dependent phosphatase, calcineurin. We report here that mice bearing a disruption in the NF-ATc gene fail to develop normal cardiac valves and septa and die of circulatory failure before day 14.5 of development. NF-ATc is first expressed in the heart at day 7.5, and is restricted to the endocardium, a specialized endothelium that gives rise to the valves and septum. Within the endocardium, specific inductive events appear to activate NF-ATc: it is localized to the nucleus only in endocardial cells that are adjacent to the interface with the cardiac jelly and myocardium, which are thought to give the inductive stimulus to the valve primordia. Treatment of wild-type embryos with FK506, a specific calcineurin inhibitor, prevents nuclear localization of NF-ATc. These data indicate that the Ca2+/calcineurin/NF-ATc signalling pathway is essential for normal cardiac valve and septum morphogenesis; hence, NF-ATc and its regulatory pathways are candidates for genetic defects underlying congenital human heart disease.
View details for Web of Science ID 000072462700063
View details for PubMedID 9515963
Architectural DNA binding by a high-mobility-group/kinesin-like subunit in mammalian SWI/SNF-related complexes
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1998; 95 (2): 492-498
The SWI/SNF complex in yeast and Drosophila is thought to facilitate transcriptional activation of specific genes by antagonizing chromatin-mediated transcriptional repression. The mechanism by which it is targeted to specific genes is poorly understood and may involve direct DNA binding and/or interactions with specific or general transcription factors. We have previously purified a mammalian complex by using antibodies against BRG1, a human homologue of SWI2/SNF2. This complex is likely functionally related to the yeast SWI/SNF complex because all five subunit identified so far (referred to as BAFs, for BRG1-associated factors) are homologues of the yeast SWI/SNF subunits. However, we now describe the cloning of the 57-kDa subunit (BAF57), which is present only in higher eukaryotes but not in yeast. BAF57 is shared by all mammalian complexes and contains a high-mobility-group (HMG) domain adjacent to a kinesin-like region. Both recombinant BAF57 and the whole complex bind four-way junction (4WJ) DNA, which is thought to mimic the topology of DNA as it enters or exits the nucleosome. Surprisingly, complexes with mutations in the HMG domain of BAF57 can still bind 4WJ DNA and mediate ATP-dependent nucleosome disruption. Our work describes the first DNA binding subunit for SWI/SNF-like complexes and suggest that the mechanism by which mammalian and Drosophila SWI/SNF-like complexes interact with chromatin may involve recognition of higher-order chromatin structure by two or more DNA binding domains.
View details for Web of Science ID 000071606000012
View details for PubMedID 9435219
View details for PubMedCentralID PMC18447
Dimerization as a regulatory mechanism in signal transduction
ANNUAL REVIEW OF IMMUNOLOGY
1998; 16: 569-592
Dynamic protein-protein interactions are a key component of biological regulatory networks. Dimerization events--physical interactions between related proteins--represent an important subset of protein-protein interactions and are frequently employed in transducing signals from the cell surface to the nucleus. Importantly, dimerization between different members of a protein family can generate considerable functional diversity when different protein combinations have distinct regulatory properties. A survey of processes known to be controlled by dimerization illustrates the diverse physical and biological outcomes achieved through this regulatory mechanism. These include: facilitated proximity and orientation; differential regulation by heterodimerization; generation of temporal and spatial boundaries; enhancement of specificity; and regulated monomer-to-dimer transitions. Elucidation of these mechanisms has led to the design of new approaches to study and to manipulate signal transduction pathways.
View details for Web of Science ID 000073129400021
View details for PubMedID 9597142
Characterization of Saccharomyces cerevisiae dna2 mutants suggests a role for the helicase late in S phase
MOLECULAR BIOLOGY OF THE CELL
1997; 8 (12): 2519-2537
The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like "lipid kinase" domain and are required for early G1 progression in eukaryotes. Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p). This gene is identical to DNA2, encoding a helicase required for DNA replication. As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants. Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions. Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and MEC1-dependent manner, suggesting that Dna2p has a role in S phase. Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis. Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2 mutants are not deficient in bulk DNA synthesis. These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation.
View details for Web of Science ID A1997YK93000013
View details for PubMedID 9398673
View details for PubMedCentralID PMC25725
Protein acetylation: more than chromatin modification to regulate transcription
CHEMISTRY & BIOLOGY
1997; 4 (12): 885-888
Histone acetyltransferases and deacetylases are involved in the regulation of gene transcription. Recently, tumor suppressor protein p53 has been shown to be a target for transcriptional coactivators that have histone acetyltransferase activity, suggesting acetylation is also involved in the regulation of cell proliferation and tumorigenesis.
View details for Web of Science ID 000071493800001
View details for PubMedID 9427654
Small molecule-dependent genetic selection in stochastic nanodroplets as a means of detecting protein-ligand interactions on a large scale
CHEMISTRY & BIOLOGY
1997; 4 (12): 961-968
Understanding the cellular role of a protein often requires a means of altering its function, most commonly by mutating the gene encoding the protein. Alternatively, protein function can be altered directly using a small molecule that binds to the protein, but no general method exists for the systematic discovery of small molecule ligands. Split-pool synthesis provides a means of synthesizing vast numbers of small molecules. Synthetic chemists will soon be able to synthesize natural product-like substances by this method, so compatible screening methods that detect the activity of minute quantities of molecules among many inactive ones will be in demand.We describe two advances towards achieving the above goals. First, a technique is described that uses a simple spray gun to create 5000-8000 droplets randomly, each having a volume of 50-200 nanoliters. The individual 'nanodroplets' contain a controlled number of cells and many also contain individual synthesis beads. As small molecules can be photochemically released from the beads in a time-dependent manner, the concentration of ligands that the cells are exposed to can be controlled. The spatial segregation of nanodroplets prevents the mixing of compounds from other beads so the effects of each molecule can be assayed individually. Second, a small molecule-dependent genetic selection involving engineered budding yeast cells was used to detect intracellular protein-ligand interactions in nanodroplets.The technique described here should facilitate the discovery of new cell-permeable ligands, especially when combined with a positive selection assay that detects intracellular binding of small molecules to proteins. Using 'anchored combinatorial libraries', it may be possible to screen entire libraries of natural product-like molecules against the entire collection of proteins encoded within cDNA libraries in a single experiment.
View details for Web of Science ID 000071493800009
View details for PubMedID 9427663
Proximity and orientation underlie signaling by the non-receptor tyrosine kinase ZAP70
1997; 16 (18): 5618-5628
Signaling by the antigen receptor of T lymphocytes initiates different developmental transitions, each of which require the tyrosine kinase ZAP70. Previous studies with agonist and antagonist peptides have indicated that ZAP70 might respond differently to different structures of the TCR-CD3 complex induced by bound peptides. The roles of membrane proximity and orientation in activation of ZAP70 signaling were explored using synthetic ligands and their binding proteins designed to produce different architectures of membrane-bound complexes composed of ZAP70 fusion proteins. Transient membrane recruitment of physiological levels of ZAP70 with the membrane-permeable synthetic ligand FK1012A leads to rapid phosphorylation of ZAP70 and activation of the ras/MAPK and Ca2+/calcineurin signaling pathways. ZAP70 SH2 domains are not required for signaling when the kinase is artifically recruited to the membrane, indicating that the SH2 domains function solely in recruitment and not in kinase activation. Using additional synthetic ligands and their binding proteins that recruit ZAP70 equally well but orient it at the cell membrane in different ways, we define a requirement for a specific presentation of ZAP70 to its downstream targets. These results provide a mechanism by which ZAP70, bound to the phosphorylated receptor, could discriminate between conformational changes induced by the binding of different MHC-peptide complexes to the antigen receptor and introduce an approach to exploring the role of spatial orientation of signaling complexes in living cells.
View details for Web of Science ID A1997XY11200015
View details for PubMedID 9312021
Redundant expression but selective utilization of nuclear factor of activated T cells family members
JOURNAL OF IMMUNOLOGY
1997; 159 (6): 2735-2740
Nuclear factor of activated T cells (NF-AT) complexes regulate the induction of many early T cell activation molecules. Four related proteins can function as the cytoplasmic subunit of NF-AT, and their overlapping expression patterns and the mild phenotype of the NF-ATp null mice suggest that they may be functionally redundant. We characterized the distribution and activation of cytoplasmic NF-AT proteins in mature lymphocytes and found that NF-ATc, NF-ATp, and NF-AT4/x/c3 are co-expressed and co-regulated in mature T and B cells. Each protein forms independent DNA binding complexes, and at physiologic concentrations, NF-ATc and NF-ATp complexes out-compete NF-AT4/x/c3 for occupancy of NF-AT sites from the IL-2, IL-3/granulocyte-macrophage CSF, IL-4, and CD40 ligand genes. This predicts heavily redundant immune regulatory functions of NF-ATp and NF-ATc, but distinct activities for NF-AT4/x/c3. Additionally, Ab interaction with NF-ATp induces high affinity NF-kappaB site interaction, suggesting that nuclear partners may dramatically vary the specificity of the NF-AT family.
View details for Web of Science ID A1997XV75000025
View details for PubMedID 9300694
Rapid targeting of nuclear proteins to the cytoplasm
1997; 7 (9): 638-644
The transcription factor NF-ATc plays a key role in the activation of many early immune response genes and is regulated by subcellular localization. NF-ATc translocates from the cytoplasm to the nucleus in response to a rise in intracellular calcium, and immediately returns to the cytoplasm when intracellular calcium levels fall. The rapid nuclear exit of NF-ATc is thought to be one mechanism by which cells distinguish between sustained and transient calcium signals.To study the nuclear export of NF-ATc, we have developed a general, non-invasive assay for the identification and study of nuclear export signals (NESs). The NES is defined by its ability to translocate a protein from the nucleus to the cytoplasm when the two are tethered by a membrane-permeable ligand. This procedure has allowed us to identify a NES within NF-ATc that functions in concert with a glycogen synthase kinase-regulated process to direct the rapid nuclear exit of NF-ATc.The rapid nuclear export of NF-ATc via its NES and a glycogen synthase kinase-regulated event may be an important mechanism for insulating cells from transient spikes in intracellular calcium which might otherwise lead to inappropriate activation. The assay we have developed allows the rapid identification of NESs and can be used as a general method for the inducible cytoplasmic export of nuclear proteins.
View details for Web of Science ID A1997XW88900025
View details for PubMedID 9285717
- The transcriptional paradox: Octamer factors and B and T cells SCIENCE 1997; 277 (5323): 193-194
Different nuclear signals are activated by the B cell receptor during positive versus negative signaling
1997; 6 (4): 419-428
It is not known how immunogenic versus tolerogenic cellular responses are signaled by receptors such as the B cell antigen receptor (BCR). Here we compare BCR signaling in naive cells that respond positively to foreign antigen and self-tolerant cells that respond negatively to self-antigen. In naive cells, foreign antigen triggered a large biphasic calcium response and activated nuclear signals through NF-AT, NF-kappa B, JNK, and ERK/pp90rsk. In tolerant B cells, self-antigen stimulated low calcium oscillations and activated NF-AT and ERK/pp90rsk but not NF-kappa B or JNK. Self-reactive B cells lacking the phosphatase CD45 did not exhibit calcium oscillations or ERK/pp90rsk activation, nor did they repond negatively to self-antigen. These data reveal striking biochemical differences in BCR signaling to the nucleus during positive selection by foreign antigens and negative selection by self-antigens.
View details for PubMedID 9133421
Nuclear localization of NF-ATc by a calcineurin-dependent, cyclosporin-sensitive intramolecular interaction
GENES & DEVELOPMENT
1997; 11 (7): 824-834
The NF-AT family of transcription factors participates in the regulation of early immune response genes such as IL-2, IL-4, CD40 ligand, and Fas ligand in response to Ca2+/calcineurin signals initiated at the antigen receptor. Calcineurin activation leads to the rapid translocation of NF-AT family members from cytoplasm to nucleus, an event that is blocked by the immunosuppressive drugs cyclosporin A and FK506. We show that translocation requires two redundant nuclear localization sequences and that one sequence is in an intramolecular association with phosphorserines in a conserved motif located at the amino terminus of each NF-AT protein. Mutation of serines in this motif in NF-ATc both disrupts this intramolecular interaction and leads to nuclear localization, suggesting a model of NF-AT nuclear import in which dephosphorylation by calcineurin causes exposure of two nuclear localization sequences.
View details for Web of Science ID A1997WU79400002
View details for PubMedID 9106655
Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3
1997; 275 (5308): 1930-1933
The transcription factor NF-AT responds to Ca2+-calcineurin signals by translocating to the nucleus, where it participates in the activation of early immune response genes. Calcineurin dephosphorylates conserved serine residues in the amino terminus of NF-AT, resulting in nuclear import. Purification of the NF-AT kinase revealed that it is composed of a priming kinase activity and glycogen synthase kinase-3 (GSK-3). GSK-3 phosphorylates conserved serines necessary for nuclear export, promotes nuclear exit, and thereby opposes Ca2+-calcineurin signaling. Because GSK-3 responds to signals initiated by Wnt and other ligands, NF-AT family members could be effectors of these pathways.
View details for Web of Science ID A1997WQ51300045
View details for PubMedID 9072970
Fas signal transduction triggers either proliferation or apoptosis in human fibroblasts
JOURNAL OF INVESTIGATIVE DERMATOLOGY
1997; 108 (2): 215-219
Although shown to be highly expressed by the epidermis in inflammatory skin disease, the ability of the Fas protein to trigger apoptosis in the distinct cell subpopulations of cutaneous tissue, particularly with regard to receptor density and the degree of crosslinking, has not been fully characterized. We therefore determined the effect of Fas cross-linking in primary human dermal fibroblasts at both high and low levels of Fas receptor expression. First, we examined the effects of the anti-Fas monoclonal antibody, CH-11, on fibroblasts expressing low basal levels of Fas. In these cells Fas aggregation stimulated proliferation by 160 +/- 10% over untreated controls. In contrast, the same concentration of CH-11 had an inhibitory effect on epidermal keratinocyte growth. Because Fas is upregulated in inflamed skin, we next examined the effects of Fas cross-linking on fibroblasts expressing augmented levels ofFas. Fibroblasts were either transfected with plasmids for overexpression of full length or bioengineered Fas receptors or were transduced with a retroviral Fas expression vector. In these cells Fas oligomerization triggered the morphologic changes indicative of apoptosis regardless of whether or not the Fas-signaling domain was tethered to the plasma membrane. These studies indicate that Fas oligomerization in dermal fibroblasts may initiate dual signaling programs, either proliferation or apoptosis, and that the chosen outcome may depend upon the magnitude of Fas aggregation.
View details for PubMedID 9008237
Specific triggering of the Fas signal transduction pathway in normal human keratinocytes
JOURNAL OF BIOLOGICAL CHEMISTRY
1996; 271 (49): 31666-31669
The epidermis is continually exposed to genotoxic injury and requires an efficient mechanism to eliminate genetically altered cells. The membrane receptor, Fas, initiates apoptosis in many cell types, including keratinocytes. Receptor cross-linking is the vital post-ligand binding step in Fas signal transduction, and we have utilized FK1012, capable of oligomerizing proteins engineered to contain the FK506 binding protein (FKBP), to trigger Fas via FKBP-linked receptor cytoplasmic domains in human keratinocytes. An FKBP chimera containing the Fas cytoplasmic domain targeted to the plasma membrane induced an up to 89% decrease in viability of keratinocytes, as reflected by the activity of constitutive promoters, in response to FK1012. Oligomerization of Fas, either with engineered Fas.FKBP by FK1012 or via antibody cross-linking of full-length Fas-induced cellular changes consistent with apoptosis. The lpr Fas point mutation abolished this effect. A Fas.FKBP construct unlinked to the membrane was fully active in this assay. Early developmental age or pre-treatment of cells with GM-CSF, TGF-beta, EGF, KGF, IFN-gamma, or phorbol ester failed to protect against Fas effects. These findings reveal that the Fas signal transduction pathway is active in keratinocytes, requires no induction, and dominantly overrides growth stimuli.
View details for Web of Science ID A1996VW68600098
View details for PubMedID 8940187
Three-part inventions: Intracellular signaling and induced proximity
TRENDS IN BIOCHEMICAL SCIENCES
1996; 21 (11): 418-422
Everyone has had the experience of stoking a fire; putting the logs close together causes flames to appear from previously dying embers. In a similar way, mere proximity might mediate qualitative biological responses. We discuss natural molecules that appear to have arisen to bring two proteins together and illustrate how this simple mechanism can be used to control a wide variety of biological processes.
View details for Web of Science ID A1996VV78600003
View details for PubMedID 8987395
Rapid shuttling of NF-AT in discrimination of Ca2+ signals and immunosuppression
1996; 383 (6603): 837-840
Cells need to distinguish between transient Ca2+ signals that induce events such as muscle contraction, secretion, adhesion and synaptic transmission, and sustained Ca2+ signals that are involved in cell proliferation and differentiation. The latter class of events is blocked in lymphocytes by the immunosuppressive drugs cyclosporin A and FK506, which inhibit calcineurin, a Ca2+-activated serine/threonine phosphatase necessary for the nuclear import of NF-AT transcription factors. Here we report that sustained high concentrations of Ca2+, but not transient pulses, are required to maintain NF-AT transcription factors in the nucleus, where they participate in Ca2+-dependent induction of genes required for lymphocyte activation and proliferation. Furthermore, overexpression and constitutive nuclear localization of NF-AT, but not Jun, Fos, NF-kappaB, Oct or Ets family members, renders the interleukin-2 enhancer in Jurkat T lymphocytes resistant to FK506 and cyclosporin A. Thus a primary effect of these immunosuppressive reagents is to control the subcellular localization of the NF-AT family of transcription factors.
View details for Web of Science ID A1996VQ14400067
View details for PubMedID 8893011
Purification and biochemical heterogeneity of the mammalian SWI-SNF complex
1996; 15 (19): 5370-5382
We have purified distinct complexes of nine to 12 proteins [referred to as BRG1-associated factors (BAFs)] from several mammalian cell lines using an antibody to the SWI2-SNF2 homolog BRG1. Microsequencing revealed that the 47 kDa BAF is identical to INI1. Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene. A group of BAF47-associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co-purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI-SNF complex. Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4-VP16 to a nucleosomal template similar to the yeast SWI-SNF complex. Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition. The two SWI-SNF2 homologs, BRG1 and hbrm, were found in separate complexes. Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI-SNF complex may be tailored to the needs of a differentiated cell type.
View details for PubMedID 8895581
Diversity and specialization of mammalian SWI/SNF complexes
GENES & DEVELOPMENT
1996; 10 (17): 2117-2130
The SWI/SNF complex in yeast facilitates the function of transcriptional activators by opposing chromatin-dependent repression of transcription. We demonstrate that in mammals SWI/SNF complexes are present in multiple forms made up of 9-12 proteins that we refer to as BRG1-associated factors (BAFs) ranging from 47 to 250 kD. We have isolated cDNAs for human BAF155, BAF170, and BAF60. BAF155 and BAF170 are encoded by separate genes that are both homologs of yeast SWI3. Both contain a region of similarity to the DNA binding domain of myb, but lack the basic residues known to be necessary for interaction with DNA. The two SWI3 homologs copurify on antibody columns specific for either BAF155 or BAF170, indicating that they are in the same complex. BAF60 is encoded by a novel gene family. An open reading frame from yeast, which is highly homologous, encodes the previously uncharacterized 73-kD subunit of the yeast SWI/SNF complex required for transcriptional activation by the glucocorticoid receptor (Cairns et al., this issue). BAF60a is expressed in all tissues examined, whereas BAF60b and BAF60c are expressed preferentially in muscle and pancreas, respectively. BAF60a is present within the 2000-kD BRG1 complex, whereas BAF60b is in a distinct complex that shares some but not all subunits with the BRG1 complex. The observed similarity between mammalian BAF190, BAF170, BAF155, BAF60, and BAF47 and yeast SNF2/SWI2, SWI3, SWI3, SWP73, and SNF5, respectively, underscores the similarity of the mammalian and yeast complexes. However, the complexes in mammals are more diverse than the SWI/SNF complex in yeast and are likely dedicated to developmentally distinct functions.
View details for Web of Science ID A1996VG10700002
View details for PubMedID 8804307
The mechanism of action of cyclosporin A and FK506
Symposium on New Frontiers in the Immunoregulation of Allergic and Autoimmune Diseases
ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS. 1996: S40–S45
The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin suppress the immune response by inhibiting evolutionary conserved signal transduction pathways. CsA, FK506, and rapamycin bind to their intracellular receptors, immunophilins, creating composite surfaces that block the activity of specific targets. For CsA/cyclophilin and FK506/FKBP the target is calcineurin. Because of the large surface area of interaction of the drug-immunophilin complex with calcineurin, FK506 and CsA have a specificity for their biologic targets that is equivalent to growth factor-receptor interactions. To date, all the therapeutic as well as toxic effects of these drugs have been shown to be due to inhibition of calcineurin. Inhibition of the action of calcineurin results in a complete block in the translocation of the cytosolic component of the nuclear factor of activated T cells (NF-AT), resulting in a failure to activate the genes regulated by the NF-AT transcription factor. These genes include those required for B-cell help such as interleukin (IL-4) and CD40 ligand as well as those necessary for T-cell proliferation such as IL-2. The purpose of this article is to illustrate the means by which these drugs produce immunosuppression.
View details for Web of Science ID A1996VG75000006
View details for PubMedID 8811062
Dimeric ligands define a role for transcriptional activation domains in reinitiation
1996; 382 (6594): 822-826
Eukaryotic transcriptional activators mediate transcriptional induction through stabilization of the preinitiation complex, probably through direct interactions with basal transcription factors. In vitro studies on the role of an activator in the maintenance of on-going transcription (reinitiation) have been contradictory, suggesting that, after formation of a preinitiation complex, an activator may or may not be necessary for transcription to be maintained. We have developed a means of regulating transcription in living cells through the use of both homodimeric and heterodimerizing synthetic ligands that allow the ligand-dependent association and disassociation of a transcriptional activation domain with a promoter. Here we report that maintaining the transcription of endogenous genes in vivo, in both yeast and human cells, requires the continuous presence of the activation domain. The use of synthetic ligands as a transcriptional on-off switch represents a powerful means of controlling the transcription in vitro and in vivo for both experimental and therapeutic purposes.
View details for Web of Science ID A1996VE34700051
View details for PubMedID 8752278
Functional analysis of Fas signaling in vivo using synthetic inducers of dimerization
1996; 6 (7): 839-847
Genetic abnormalities in the Fas receptor or its trimeric ligand, FasL, result in massive T-cell proliferation and a lupus-like autoimmune syndrome, which was initially attributed to excessive lymphoproliferation but is now ascribed to the absence of Fas-mediated cell death. Although Fas is normally expressed on most thymocytes, negative selection seems to be unperturbed in Fas-deficient (lpr) mice. This suggests that Fas has an important function in peripheral, but not thymic, T cells.To explore the Fas-mediated cell death pathway both in vitro and in vivo, we used conditional alleles of the Fas receptor that can be triggered by an intracellularly active chemical inducer of dimerization known as FK1012. We found that membrane attachment is important for Fas function and, unlike previous results with anti-Fas monoclonal antibodies, we show that dimerization is sufficient to trigger apoptosis. Finally, the administration of FK1012 in vivo to transgenic animals expressing the conditional FAS receptor in thymocytes demonstrates that sensitivity to FAS-mediated apoptosis is restricted to CD4+CD8+ thymocytes.Here, we describe the first in vivo application of non-toxic, cell-permeable synthetic ligands to regulate signal transduction in transgenic mice expressing a conditional receptor. Using this system, we show that the Fas pathway is restricted to double-positive thymocytes in vivo, consistent with recent in vitro findings with thymocytes. This method promises to be more useful not only for developmental studies involving cell ablation, but also for studies involving the regulation of a wide variety of signaling molecules.
View details for Web of Science ID A1996UY12800026
View details for PubMedID 8805308
CHARACTERIZATION OF T-CELL MUTANTS WITH DEFECTS IN CAPACITATIVE CALCIUM-ENTRY - GENETIC-EVIDENCE FOR THE PHYSIOLOGICAL ROLES OF CRAC CHANNELS
JOURNAL OF CELL BIOLOGY
1995; 131 (3): 655-667
Prolonged Ca2+ influx is an essential signal for the activation of T lymphocytes by antigen. This influx is thought to occur through highly selective Ca2+ release-activated Ca2+ (CRAC) channels that are activated by the depletion of intracellular Ca2+ stores. We have isolated mutants of the Jurkat human T cell line NZdipA to explore the molecular mechanisms that underlie capacitative Ca2+ entry and to allow a genetic test of the functions of CRAC channels in T cells. Five mutant cell lines (CJ-1 through CJ-5) were selected based on their failure to express a lethal diphtheria toxin A chain gene and a lacZ reporter gene driven by NF-AT, a Ca(2+)- and protein kinase C-dependent transcription factor. The rate of Ca2+ influx evoked by thapsigargin was reduced to varying degrees in the mutant cells whereas the dependence of NF-AT/lacZ gene transcription on [Ca2+]i was unaltered, suggesting that the transcriptional defect in these cells is caused by a reduced level of capacitative Ca2+ entry. We examined several factors that determine the rate of Ca2+ entry, including CRAC channel activity, K(+)-channel activity, and Ca2+ clearance mechanisms. The only parameter found to be dramatically altered in most of the mutant lines was the amplitude of the Ca2+ current (ICRAC), which ranged from 1 to 41% of that seen in parental control cells. In each case, the severity of the ICRAC defect was closely correlated with deficits in Ca2+ influx rate and Ca(2-)-dependent gene transcription. Behavior of the mutant cells provides genetic evidence for several roles of ICRAC in T cells. First, mitogenic doses of ionomycin appear to elevate [Ca2+]i primarily by activating CRAC channels. Second, ICRAC promotes the refilling of empty Ca2+ stores. Finally, CRAC channels are solely responsible for the Ca2+ influx that underlies antigen-mediated T cell activation. These mutant cell lines may provide a useful system for isolating, expressing, and exploring the functions of genes involved in capacitative Ca2+ entry.
View details for Web of Science ID A1995TC10100009
View details for PubMedID 7593187
View details for PubMedCentralID PMC2120614
A GENERAL STRATEGY FOR PRODUCING CONDITIONAL ALLELES OF SRC-LIKE TYROSINE KINASES
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1995; 92 (21): 9805-9809
The Src-like tyrosine kinases require membrane localization for transformation and probably for their normal role in signal transduction. We utilized this characteristic to prepare Src-like tyrosine kinases that can be readily activated with the rationally designed chemical inducer of dimerization FK1012. Dimerization of cytoplasmic Src-like tyrosine kinases was not sufficient for signaling, but their recruitment to the plasma membrane led to the rapid activation of transcription factors identical to those regulated by crosslinking the antigen receptor. Moreover, recruitment of activated Src-like kinases to the membrane replaced signaling by the T-lymphocyte antigen receptor complex, leading to the activation of both the Ras/protein kinase C and Ca2+/calcineurin pathways normally activated by antigen receptor signaling. Since these chemical inducers of dimerization are cell permeable, this approach permits the production of conditional alleles of any of the Src-like tyrosine kinases, thereby allowing a delineation of their developmental roles.
View details for PubMedID 7568222
SIGNAL-TRANSDUCTION IN T-LYMPHOCYTES USING A CONDITIONAL ALLELE OF SOS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1995; 92 (21): 9810-9814
While Ras activation has been shown to play an important role in signal transduction by the T-lymphocyte antigen receptor, the mechanism of its activation in T cells is unclear. Membrane localization of the guanine nucleotide exchange factor Sos, but not Vav or Dbl, was sufficient for Ras-mediated signaling in T lymphocytes. Activation of Sos appears to involve membrane recruitment and not allosteric changes, because interaction of Sos with the linking molecule Grb-2 was not required for Ras activation. To extend this analysis, we constructed a modified Sos that could be localized to the membrane inducibly by using a rationally designed chemical inducer of dimerization, FK1012. The role of Grb-2 in signaling was mimicked with this technique, which induced the association of a modified Sos with the membrane, resulting in rapid activation of Ras-induced signaling. In contrast, inducible localization of Grb-2 to the membrane did not activate signaling and suggests that the interaction of Grb-2 with Sos in T cells is subject to regulation. This conditional allele of Sos demonstrates that membrane localization of Sos is sufficient for Ras activation in T cells and indicates that the role of Grb-2 is to realize the biologic advantages of linker-mediated dimerization: enhanced specificity and favorable kinetics for signaling. This method of generating conditional alleles may also be useful in dissecting other signal transduction pathways regulated by protein localization or protein-protein interactions.
View details for PubMedID 7568223
NFATC3, A LYMPHOID-SPECIFIC NFATC FAMILY MEMBER THAT IS CALCIUM-REGULATED AND EXHIBITS DISTINCT DNA FINDING SPECIFICITY
JOURNAL OF BIOLOGICAL CHEMISTRY
1995; 270 (34): 19898-19907
Signals transduced by the T cell antigen receptor (TCR) regulate developmental transitions in the thymus and also mediate the immunologic activation of mature, peripheral T cells. In both cases TCR stimulation leads to the assembly of the NFAT transcription complex as a result of the calcium-dependent nuclear translocation of cytosolic subunits, NFATc, and the Ras/protein kinase C-dependent induction of a nuclear subunit, NFATn. To further understand the diverse roles of antigen receptor signaling throughout T cell development, we have identified a new NFATc family member, NFATc3, that is expressed at highest levels in the thymus. NFATc3 is the product of a gene on murine chromosome 8 that is not linked to the other NFATc genes. NFATc3, like other NFATc family members, contains a conserved rel similarity domain, and also defines a region conserved among NFATc family members, the SP repeat region, characterized by the repeated motif SPxxSPxxSPrxsxx (D/E)(D/E)swl. NFATc3 activates NFAT site-dependent transcription when overexpressed, yet exhibits a pattern of DNA site specificity distinct from other NFATc proteins. Additionally, thymic NFATc3 undergoes modifications in response to agents that mimic T cell receptor signaling, including a decrease in apparent molecular mass upon elevation of intracellular calcium that is inhibited by the immunosuppressant FK506. Given the preferential expression of NFATc3 in the thymus, NFATc family members may regulate distinct subsets of genes during T cell development.
View details for PubMedID 7650004
ISOLATION OF MUTANT T-LYMPHOCYTES WITH DEFECTS IN CAPACITATIVE CALCIUM-ENTRY
1995; 3 (2): 239-250
Calcium and calcium-binding proteins play important roles in the signaling cascade leading from the initial engagement of TCRs on T cells to the fully activated state. To undertake a molecular dissection of this cascade, we first isolated a Jurkat T cell line derivative containing the NF-AT promoter element driving transcription of the diphtheria toxin A chain gene (dipA), resulting in rapid cell death. Selecting viable cells that fail to activate NF-AT-dependent transcription, we isolated two independent cell lines possessing defects in capacitative Ca2+ entry. NF-AT-dependent transcription can be restored in these cells by expression of a constitutively active calcineurin, but not overexpression of the Ca2+ regulatory protein CAML, which can normally replace the Ca2+ signal. The defect in these cell lines probably lies between CAML and calcineurin in the T cell activation cascade.
View details for Web of Science ID A1995RR33900009
View details for PubMedID 7648396
CLONING AND CHROMOSOMAL LOCALIZATION OF THE HUMAN AND MURINE GENES FOR THE T-CELL TRANSCRIPTION FACTORS NFATC AND NFATP
CYTOGENETICS AND CELL GENETICS
1995; 68 (3-4): 185-191
The nuclear factor of activated T cells (NFAT) is a transcription factor complex involved in the activation of cytokines and cell surface molecules associated with coordinating the actions of different cells required for an immune response. Two different genes have recently been cloned that encode proteins capable of functioning as the pre-existing (p) and cytosolic (c) component of the NFAT transcription complex, NFATc of human and NFATp of murine origin (Northrop et al., 1994; McCaffrey et al., 1993b). We report here the partial cDNA cloning of the murine homolog of NFATc and the human homolog of NFATp, and the chromosomal localization of both genes in both species to conserved syntenic regions. Through the use of mapping panels of human x Chinese hamster and mouse x rodent cells hybrids, the NFATc genes were mapped to human and mouse chromosomes 18. By analyzing a chromosome 18 radiation hybrid panel, the human NFATc gene was localized to the q terminus, closely linked to STS marker D18S497. The murine Nfatc gene was sublocalized to chromosome band 18E4 by FISH. The NFATp genes were mapped by somatic cell hybrid analysis to human chromosome 20 and mouse chromosome 2. Human NFATp was assigned to chromosome region 20q13.2-->q13.3 by FISH. Based on the conserved syntenic region on human chromosome 20 and mouse chromosome 2, murine Nfatp is predicted to reside in the vicinity of a mutant locus wasted. Homozygous wst/wst mice display a phenotype reminiscent of severe combined immune deficiency or ataxia telangiectasia, disorders that could therefore be considered candidates for NFATp mutations.
View details for PubMedID 7842733
INTERLEUKIN-2-MEDIATED ELIMINATION OF THE P27(KIP1) CYCLIN-DEPENDENT KINASE INHIBITOR PREVENTED BY RAPAMYCIN
1994; 372 (6506): 570-573
The cyclin-dependent kinase (Cdk) enzymes, when associated with the G1 cyclins D and E, are rate-limiting for entry into the S phase of the cell cycle. During T-cell mitogenesis, antigen-receptor signalling promotes synthesis of cyclin E and its catalytic partner, Cdk2, and interleukin-2 (IL-2) signalling activates cyclin E/Cdk2 complexes. Rapamycin is a potent immunosuppressant which specifically inhibits G1-to-S-phase progression, leading to cell-cycle arrest in yeast and mammals. Here we report that IL-2 allows Cdk activation by causing the elimination of the Cdk inhibitor protein p27Kip1, and that this is prevented by rapamycin. By contrast, the Cdk inhibitor p21 is induced by IL-2 and this induction is blocked by rapamycin. Our results show that p27Kip1 governs Cdk activity during the transition from quiescence to S phase in T lymphocytes and that p21 function may be restricted to cycling cells.
View details for Web of Science ID A1994PW08200058
View details for PubMedID 7990932
MOLECULAR ANALYSIS OF THE INTERACTION OF CALCINEURIN WITH DRUG-IMMUNOPHILIN COMPLEXES
JOURNAL OF BIOLOGICAL CHEMISTRY
1994; 269 (42): 26431-26437
The calcium/calmodulin-regulated phosphatase calcineurin (CN) is the site of action of the immunosuppressive drugs cyclosporin A (CsA) and FK506. CN has recently been established as a key signaling enzyme in the T cell signal transduction cascade and an important regulator of transcription factors such as NF-AT and OAP/Oct-1, which are involved in the expression of a number of important T cell early genes. CsA and FK506 act by forming complexes with their respective intracellular receptors cyclophilin and FKBP (immunophilins), which can then bind to CN, inhibiting its enzymatic activity and thereby preventing early gene expression. CN is comprised of two subunits: a 59-kDa catalytic subunit (CNA), which contains a calmodulin binding domain and autoinhibitory region, and a 19-kDa intrinsic calcium binding regulatory subunit (CNB). In this study, we have utilized a series of deletion mutants of the CNA subunit to investigate the subunit and molecular requirements that govern the interaction of CN with drug-immunophilin complexes. The calmodulin binding and autoinhibitory domains of the CNA subunit were found to be dispensable for the binding of CN to drug-immunophilin complexes. In contrast, we found that the regulatory CNB subunit appears to play an obligatory role in this interaction and have defined an amino acid sequence of the CNA subunit which forms the binding site for CNB. Although necessary, the CNB subunit per se is not sufficient to mediate an interaction with drug-immunophilin complexes; amino acid residues of the CNA subunit, specifically a region located within the putative catalytic domain, are also required for the interaction of CN with both FKBP-FK506 and cyclophilin A-CsA.
View details for Web of Science ID A1994PQ93000075
View details for PubMedID 7523407
SPECIFIC-INHIBITION OF FORMATION OF TRANSCRIPTION COMPLEXES BY A CALICHEAMICIN OLIGOSACCHARIDE - A PARADIGM FOR THE DEVELOPMENT OF TRANSCRIPTIONAL ANTAGONISTS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1994; 91 (20): 9203-9207
Sequence-specific DNA ligands that antagonize DNA-protein interactions represent a potentially powerful means of modulating gene expression. Calicheamicin gamma 1I, a member of the DNA-cleaving enediyne class of anticancer antibiotics, binds to specific DNA sequences through an aryltetrasaccharide domain. To take advantage of this unique sequence-specific recognition capability, the methyl glycoside of the aryltetrasaccharide of calicheamicin gamma 1I (CLM-MG) was used to investigate the ability of glycoconjugate DNA ligands to inhibit DNA-protein interactions. CLM-MG inhibits the formation of DNA-protein complexes at micromolar concentrations in a sequence-specific manner and rapidly dissociates preformed complexes. CLM-MG also inhibits transcription in vivo with similar sequence specificity. These results suggest a strategy for the development of a class of novel biological probes and therapeutic agents.
View details for Web of Science ID A1994PJ42800003
View details for PubMedID 7937742
NF-AT COMPONENTS DEFINE A FAMILY OF TRANSCRIPTION FACTORS TARGETED IN T-CELL ACTIVATION
1994; 369 (6480): 497-502
The NF-AT transcription complex is required for the expression of a group of proteins that collectively coordinate the immune response. Here we purify two proteins encoded by separate genes that represent the pre-existing (p) and cytosolic (c) components of NF-AT. Expression of the full-length complementary DNA encoding NF-ATc activates the interleukin (IL-2) promoter in non-T lymphocytes, whereas a dominant negative of NF-ATc specifically blocks activation of the IL-2 promoter in T lymphocytes, indicating that NF-ATc is required for IL-2 gene expression. NF-ATc RNA expression is largely restricted to lymphoid tissues and is induced upon T-cell activation. The other protein, NF-ATp, is highly homologous to NF-ATc over a limited domain which shows similarity to the Dorsal/Rel family, but has a wider tissue distribution. Agents that increase intracellular Ca2+ or activate protein kinase C independently modify NF-ATc, indicating that distinct signalling pathways converge on NF-ATc to regulate its function.
View details for PubMedID 8202141
CLONING AND CHARACTERIZATION OF NF-AT(C) AND NF-AT(P) - THE CYTOPLASMIC COMPONENTS OF NF-AT
5th International Conference on Mechanisms of Lymphocyte Activation and Immune Regulation
PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1994: 167–173
Present evidence indicates a pathway of signal transmission in T cells that is outlined in figure 1. The elevation in intracellular calcium that is induced by interactions at the antigen receptor leads to the activation of the calcium-dependent phosphatase calcineurin. This in turn leads to the nuclear association of the cytosolic component of NF-ATc. The activation of calcineurin and the nuclear import of NF-ATc can both be blocked by cyclosporin A or FK506 in complex with their respective immunophilins. Once in the nucleus, NF-ATc interacts with NF-ATn to form an active transcriptional complex. NF-ATn is a ubiquitous protein, can be synthesized in response to PMA, and has many similarities to AP-1. The mechanism by which NF-ATc enters the nucleus is unknown, and although it appears to require calcineurin, NF-ATc has not yet been shown to be an in vivo substrate of calcineurin. Alternative mechanisms include the possibility that NF-ATc operates on some cytoplasmic anchor or that other proteins that are controlled by calcineurin carry out the nuclear import of NF-ATc. Although NF-ATp copurifies with NF-ATc, there is as yet no understanding of how NF-ATp is functioning in vivo. Now that these proteins are purified and cloned, the major goals will be to understand their role and the roles of other family members in thymic development.
View details for Web of Science ID A1994BB86U00017
View details for PubMedID 7887301
BRG1 - A PUTATIVE MURINE HOMOLOG OF THE DROSOPHILA-BRAHMA GENE, A HOMEOTIC GENE REGULATOR
1994; 161 (1): 229-242
To identify potential regulators of Hox gene expression in mice, we have screened for genes highly related to brahma (brm), an activator of homeotic gene expression in Drosophila. We have cloned a murine gene, brg1, which, like brm, encodes a member of the DEGH protein family, suggesting that brg1 may be a DNA-dependent ATPase or a helicase. brg1 also contains a bromodomain which may be involved in transactivation. Although the sequences of a number of mammalian genes similar to Drosophila brm have been reported, they are related to brm only within specific portions of the putative helicase region, while brg1 is highly similar to brm throughout and outside of this region. A 5.8-kb brg1 transcript was detected throughout embryogenesis and in numerous adult tissues. RNA in situ hybridization revealed widespread expression of brg1 in embryonic tissues. At later stages of embryogenesis, differences in levels of brg1 expression were seen among different tissues. brg1 expression was highest in the spinal cord, the brain, parts of the peripheral nervous system, and the vertebral column. These expression domains within the spinal cord and vertebral column encompass major regions of Hox gene expression. Within the spinal cord, brain, and retina, mRNA levels were higher in regions consisting of differentiated cells than in regions consisting of undifferentiated, proliferating cells. These patterns of brg1 expression are consistent with a possible role for brg1 in Hox gene regulation as well as in other regulatory pathways.
View details for Web of Science ID A1994MQ97400023
View details for PubMedID 7904967
- SIGNAL TRANSMISSION BETWEEN THE PLASMA-MEMBRANE AND NUCLEUS OF T-LYMPHOCYTES ANNUAL REVIEW OF BIOCHEMISTRY 1994; 63: 1045-1083
CONTROLLING SIGNAL-TRANSDUCTION WITH SYNTHETIC LIGANDS
1993; 262 (5136): 1019-1024
Dimerization and oligomerization are general biological control mechanisms contributing to the activation of cell membrane receptors, transcription factors, vesicle fusion proteins, and other classes of intra- and extracellular proteins. Cell permeable, synthetic ligands were devised that can be used to control the intracellular oligomerization of specific proteins. To demonstrate their utility, these ligands were used to induce intracellular oligomerization of cell surface receptors that lacked their transmembrane and extracellular regions but contained intracellular signaling domains. Addition of these ligands to cells in culture resulted in signal transmission and specific target gene activation. Monomeric forms of the ligands blocked the pathway. This method of ligand-regulated activation and termination of signaling pathways has the potential to be applied wherever precise control of a signal transduction pathway is desired.
View details for Web of Science ID A1993MG18700025
View details for PubMedID 7694365
BRG1 CONTAINS A CONSERVED DOMAIN OF THE SWI2/SNF2 FAMILY NECESSARY FOR NORMAL MITOTIC GROWTH AND TRANSCRIPTION
1993; 366 (6451): 170-174
Sequence-specific DNA binding activators of gene transcription may be assisted by SWI2 (SNF2), which contains a DNA-dependent ATPase domain. We have isolated a human complementary DNA encoding a 205K nuclear protein, BRG1, that contains extensive homology to SWI2 and Drosophila brahma. We report here that a SWI2/BRG1 chimera with the DNA-dependent ATPase domain replaced by corresponding human sequence restored normal mitotic growth and capacity for transcriptional activation to swi2- yeast cells. Point mutation of the conserved ATP binding site lysine abolished this complementation. This mutation in SWI2 exerted a dominant negative effect on transcription in yeast. A lysine to arginine substitution at the corresponding residue of BRG1 also generated a transcriptional dominant negative in human cells. BRG1 is exclusively nuclear and present in a high M(r) complex of about 2 x 10(6). These results show that the SWI2 family DNA-dependent ATPase domain has functional conservation between yeast and humans and suggest that a SWI/SNF protein complex is required for the activation of selective mammalian genes.
View details for PubMedID 8232556
PRIMARY EXTRAUTERINE ENDOMETRIAL STROMAL NEOPLASMS - A CLINICOPATHOLOGICAL STUDY OF 20 CASES AND A REVIEW OF THE LITERATURE
INTERNATIONAL JOURNAL OF GYNECOLOGICAL PATHOLOGY
1993; 12 (4): 282-296
We present the results of a clinicopathologic study of 20 patients with primary extrauterine endometrial stromal sarcoma (ESS). The sites of the primary neoplasm and the number of patients with sufficient follow-up for survival analysis are as follows: ovary (three of four), fallopian tube (one of one), pelvic cavity (six of eight), abdominal cavity (five of six), and retroperitoneum (one of one). Evaluation of all patients included the mitotic index (MI) and cytologic atypia. Thirteen of the sixteen patients eligible for survival analysis had tumors with an MI < 10 and would be classified as low-grade stromal sarcomas in the Norris and Taylor scheme. Eight (62%) of the 13 had one or more relapses; of these, three died of disease at 35, 108, and 120 months, respectively, and another patient was alive with disease at 96 months. The other four patients who were treated after a relapse showed no evidence of disease after relapse at 36, 57, 63, and 146 months, respectively. Two of the 13 patients had tumor considered unresectable at the time of diagnosis; both died of disease at 5 and 10 months, respectively. Neither MI nor cytologic atypia were predictive of tumor recurrence or death from tumor. We also extracted clinical and morphologic data from all previous reports of primary extrauterine ESS, combined them with our 20 patients, and then compared the combined group with 17 cases of primary high-stage uterine ESS we presented in an earlier report. Not surprisingly, the behavior of the primary extrauterine ESS was more reminiscent of high-stage primary uterine ESS than low-stage primary uterine ESS.
View details for PubMedID 8253545
IDENTIFICATION OF THE IMMUNOPHILINS CAPABLE OF MEDIATING INHIBITION OF SIGNAL-TRANSDUCTION BY CYCLOSPORINE-A AND FK506 - ROLES OF CALCINEURIN BINDING AND CELLULAR LOCATION
MOLECULAR AND CELLULAR BIOLOGY
1993; 13 (8): 4760-4769
The immunosuppressants cyclosporin A (CsA) and FK506 appear to block T-cell function by inhibiting the calcium-regulated phosphatase calcineurin. While multiple distinct intracellular receptors for these drugs (cyclophilins and FKBPs, collectively immunophilins) have been characterized, the functionally active ones have not been discerned. We found that overexpression of cyclophilin A or B or FKBP12 increased T-cell sensitivity to CsA or FK506, respectively, demonstrating that they are able to mediate the inhibitory effects of their respective immunosuppressants in vivo. In contrast, cyclophilin C, FKBP13, and FKBP25 had no effect. Direct comparison of the Ki of each drug-immunophilin complex for calcineurin in vitro revealed that although calcineurin binding was clearly necessary, it was not sufficient to explain the in vivo activity of the immunophilin. Subcellular localization was shown also to play a role, since gene deletions of cyclophilins B and C which changed their intracellular locations altered their activities significantly. Cyclophilin B has been shown previously to be located within calcium-containing intracellular vesicles; its ability to mediate CsA inhibition implies that certain components of the signal transduction machinery are also spatially restricted within the cell.
View details for Web of Science ID A1993LN40500032
View details for PubMedID 7687744
GENES FOR THE DIMERIZATION COFACTOR OF HEPATOCYTE NUCLEAR FACTOR-1-ALPHA (DCOH) ARE ON HUMAN AND MURINE CHROMOSOMES-10
1993; 16 (1): 292-295
Hepatocyte nuclear factor-1 alpha (HNF-1 alpha; gene symbol, TCF1) forms dimers with itself as well as with HNF-1 beta and regulates the expression of several liver-specific genes. Recently, a dimerization cofactor of hepatocyte nuclear factor-1 alpha, called DCOH, has been identified. Here, we report the chromosomal localization of the genes for this cofactor to chromosomes 10 in both humans and mice by Southern blot analyses of somatic cell hybrids.
View details for Web of Science ID A1993KW20500050
View details for PubMedID 8486378
Regulation of the HNF-1 homeodomain proteins by DCoH.
Current opinion in genetics & development
1993; 3 (2): 246-253
The pattern of expression of homeodomain proteins often exceeds their apparent domain of activity. Tissue-specific proteins that modulate the in vivo activity of homeodomain proteins have been proposed to account for this functional restriction. The first identified example of such an accessory protein is DCoH, which confers transcriptional activity to the hepatocyte nuclear factor 1 and provides a model of how other accessory factors might modulate the function of homeodomain proteins.
View details for PubMedID 8504250
CHARACTERIZATION OF THE NUCLEAR AND CYTOPLASMIC COMPONENTS OF THE LYMPHOID-SPECIFIC NUCLEAR FACTOR OF ACTIVATED T-CELLS (NF-AT) COMPLEX
JOURNAL OF BIOLOGICAL CHEMISTRY
1993; 268 (4): 2917-2923
The lymphoid-specific transcription complex, NF-AT, is involved in early gene activation in T cells and is assembled from a pre-existing, T cell restricted cytoplasmic factor and an inducible ubiquitous nuclear component within 30 min after activation through the antigen receptor. Recent studies have implicated the family of AP1 factors as components of the murine NF-AT complex. Evidence is provided here that the nuclear component of human NF-AT contains the phorbol ester-inducible transcription factor AP1 (Jun/Fos). We further characterize which AP1 family members can assume this role. Antisera to Fos inhibits NF-AT DNA binding as does an oligonucleotide containing a binding site for AP1. Constitutive expression in vivo of Fos, and to a lesser extent Fra-1, eliminates the requirement for phorbol 12-myristate 13-acetate (PMA) stimulation, leaving NF-AT-directed transcription responsive to calcium ionophore alone. Overexpression of cJun or JunD, but not JunB, also eliminates the requirement for PMA, indicating that many but not all Jun- and Fos-related proteins functionally activate NF-AT-dependent transcription in the presence of the cytoplasmic component. NF-AT DNA binding can be reconstituted in vitro using semi-purified AP1 proteins mixed with cytosol from T lymphocytes. Fos proteins are not needed for this reconstitution, and although JunB is not functional, it can participate in the NF-AT DNA binding complex. Finally, we have partially purified the cytoplasmic component of NF-AT and show by elution and renaturation from SDS-polyacrylamide gel electrophoresis gels that it has a molecular mass between 94 and 116 kDa and may have multiple differentially modified forms.
View details for Web of Science ID A1993KK81500097
View details for PubMedID 8428966
JUN FAMILY MEMBERS ARE CONTROLLED BY A CALCIUM-REGULATED, CYCLOSPORINE-A-SENSITIVE SIGNALING PATHWAY IN ACTIVATED LYMPHOCYTES-T
GENES & DEVELOPMENT
1993; 7 (2): 188-196
The octamer-binding transcription factor Oct-1 is involved in a wide variety of cellular processes but appears to lack a strong transcriptional activation domain, suggesting that it functions in the context of other proteins. We demonstrated previously that Oct-1, in association with a 40-kD protein, OAP40, contributes to the induction of interleukin-2 (IL-2), an early activation gene and major growth factor for T lymphocytes. Here we report that amino acid sequences obtained from purified OAP40 are identical to regions within JunD and c-Jun. We demonstrate that each of these Jun family members can participate in a complex that includes Oct-1 and a regulatory element in the IL-2 enhancer. In transient transfections, both JunD and c-Jun can contribute to activation-specific transcription mediated by this antigen receptor response element. These studies reveal a role, distinct from AP-1 activity, for Jun family members that is controlled by a calcium-triggered, cyclosporin A-sensitive mechanism.
View details for Web of Science ID A1993KM85900003
View details for PubMedID 8436291
- CALCINEURIN IS A KEY SIGNALING ENZYME IN T-LYMPHOCYTE ACTIVATION AND THE TARGET OF THE IMMUNOSUPPRESSIVE DRUGS CYCLOSPORINE-A AND FK506 Conference on Immunosuppressive and Antiinflammatory Drugs NEW YORK ACAD SCIENCES. 1993: 20–30
RAPAMYCIN INHIBITS P34(CDC2) EXPRESSION AND ARRESTS T-LYMPHOCYTE PROLIFERATION AT THE G1/S TRANSITION
Conference on Immunosuppressive and Antiinflammatory Drugs
NEW YORK ACAD SCIENCES. 1993: 31–37
Rapamycin, a potent immunosuppressant and antifungal agent, inhibits an evolutionarily conserved mechanism regulating cell cycle progression. In an interleukin-2 (IL-2) dependent murine T cell, we demonstrate that rapamycin arrested T cells prior to the entry into S-phase of the cell cycle and that rapamycin inhibited the IL-2-stimulated expression of p34cdc2, a serine/threonine kinase that is required for cells to progress through the cell cycle. The mechanism of action of rapamycin appeared specific since the structural analogue and immunosuppressant FK506 had no effect on the progression of the cells through S-phase or the expression of p34cdc2. These results demonstrate a rapamycin-sensitive IL-2-dependent signaling pathway in T cells and suggest that the immunosuppressive properties of rapamycin are mediated by impinging on the IL-2-induced T cell expression of p34cdc2.
View details for Web of Science ID A1993BZ60M00005
View details for PubMedID 8109837
RAPAMYCIN SELECTIVELY INHIBITS INTERLEUKIN-2 ACTIVATION OF P70 S6 KINASE
1992; 358 (6381): 70-73
The macrolide rapamycin induces cell cycle G1 arrest in yeast and in mammalian cells, which suggests that an evolutionarily conserved, rapamycin-sensitive pathway may regulate entry into S phase. In mammals, rapamycin inhibits interleukin-2 receptor-induced S phase entry and subsequent T-cell proliferation, resulting in immunosuppression. Here we show that interleukin-2 selectively stimulates the phosphorylation and activation of p70 S6 kinase but not the erk-encoded MAP kinases and rsk-encoded S6 kinases. Rapamycin completely and rapidly inhibits interleukin-2-induced phosphorylation and activation of p70 S6 kinase at concentrations comparable to those blocking S phase entry of T cells (0.05-0.2 nM). The structurally related macrolide FK506 competitively antagonizes the actions of rapamycin, indicating that these effects are mediated by FKBP, which binds the transition-state mimic structure common to both rapamycin and FK506 (refs 4, 6, 9-11). The selective blockade of the p70 S6 kinase activation cascade by the rapamycin-FKBP complex implicates this signalling pathway in the regulation of T cell entry into S phase.
View details for Web of Science ID A1992JB34100056
View details for PubMedID 1614535
IDENTIFICATION OF CALCINEURIN AS A KEY SIGNALING ENZYME IN LYMPHOCYTE-T ACTIVATION
1992; 357 (6380): 695-697
The immunosuppressive drugs cyclosporin A (CsA) and FK506 both interfere with a Ca(2+)-sensitive T-cell signal transduction pathway, thereby preventing the activation of specific transcription factors (such as NF-AT and NF-IL2A) involved in lymphokine gene expression. CsA and FK506 seem to act by interaction with their cognate intracellular receptors, cyclophilin and FKBP, respectively (see ref. 11 for review). The Ca2+/calmodulin-regulated phosphatase calcineurin is a major target of drug-isomerase complexes in vitro. We have therefore tested the hypothesis that this interaction is responsible for the in vivo effects of CsA/FK506. We report here that overexpression of calcineurin in Jurkat cells renders them more resistant to the effects of CsA and FK506 and augments both NFAT- and NFIL2A-dependent transcription. These results identify calcineurin as a key enzyme in the T-cell signal transduction cascade and provide biological evidence to support the notion that the interaction of drug-isomerase complexes with calcineurin underlies the molecular basis of CsA/FK506-mediated immunosuppression.
View details for Web of Science ID A1992JA43000073
View details for PubMedID 1377362
NEGATIVE REGULATION OF INTERLEUKIN-2 TRANSCRIPTION BY THE GLUCOCORTICOID RECEPTOR
JOURNAL OF EXPERIMENTAL MEDICINE
1992; 175 (5): 1235-1245
Glucocorticoid-dependent transcriptional enhancement is known to occur through the interaction of the glucocorticoid receptor (GR) with specific DNA response elements. In contrast, negative regulation of gene expression by this class of hormone is less well understood. Glucocorticoids are potent immunosuppressive agents acting primarily by inhibiting T lymphocyte activation and lymphokine production. Interleukin 2 (IL-2) gene expression, a critical early event during T lymphocyte activation, is inhibited in glucocorticoid-sensitive cells by hormone treatment. We have studied the mechanism of this inhibition. In transgenic mice carrying c-myc linked to the IL-2 enhancer, mitogen-induced expression of the transgene is inhibited by concurrent glucocorticoid treatment, while a similar transgene construct driven by three copies of the binding site for nuclear factor of activated T cells is not inhibited. Cotransfection experiments into glucocorticoid-insensitive jurkat cells show that the NH2 terminus of the glucocorticoid receptor is dispensable for inhibition of the IL-2 enhancer but that an intact DNA binding domain, although not necessarily binding to DNA, is required. Hybrid GRs containing the DNA binding domains of either the estrogen receptor (ER) or thyroid receptor, as well as the entire wild-type ER, all function as repressors of the IL-2 enhancer. We have localized the site of inhibition to two sequences located in the proximal half of the enhancer. These sequences bind a similar, if not identical, inducible nuclear factor that has biologic characteristics that distinguish it from AP-1. The mechanism of IL-2 inhibition likely involves direct interactions between the GR and this factor.
View details for Web of Science ID A1992HQ18400009
View details for PubMedID 1569395
A TRANSCRIPTIONAL HIERARCHY INVOLVED IN MAMMALIAN CELL-TYPE SPECIFICATION
1992; 355 (6359): 457-461
Although transcriptional hierarchies have been extensively studied in invertebrates, their involvement in mammalian cell-type specification is poorly understood. Here we report a hepatocyte transcriptional cascade suggested by the expression patterns of hepatic transcription factors in dedifferentiated hepatomas and hepatocyte: fibroblast hybrids in which the liver phenotype was extinguished. These results indicated that the homeoprotein hepatocyte nuclear factor-1 alpha (HNF-1 alpha), and HNF-4, a member of the steroid hormone receptor superfamily, were regulated coordinately or in a hierarchy by a higher-order locus, independently of other hepatic transactivators. HNF-4 was implicated as an essential positive regulator of HNF-1 alpha, as deletion of an HNF-4 binding site in the HNF-1 alpha promoter abolished promoter activity, and HNF-4 potently transactivated the HNF-1 alpha promoter in cotransfection assays. Moreover, genetic complementation of dedifferentiated hepatomas with HNF-4 complementary DNA rescued expression of endogenous HNF-1 alpha messenger RNA and DNA-binding activity. Our studies therefore define an HNF-4----HNF-1 alpha (4----1 alpha) transcriptional hierarchy operative in differentiated hepatocytes but selectively inhibited by an extinguishing locus and somatic mutations which antagonize the liver phenotype.
View details for Web of Science ID A1992HB53000073
View details for PubMedID 1734282
INVITRO TRANSCRIPTION FAITHFULLY REFLECTING T-CELL ACTIVATION REQUIREMENTS
JOURNAL OF BIOLOGICAL CHEMISTRY
1992; 267 (1): 399-406
T-cell activation is a complex process mediated by cell membrane molecules including the T-cell antigen receptor (TCR), adhesive molecules, and cytokine receptors that collectively produce an increase in intracellular Ca2+, and activation of protein kinase C that initiate a genetic program resulting in immunologic function and irreversible differentiation. To understand how these cell membrane events are translated into a genetic regulatory cascade resulting in T-cell function, we have developed an in vitro transcription system, derived from Jurkat T-cells, which demonstrates inducible, cell-type-specific transcription following T-cell stimulation. Nuclear extracts from cells stimulated with phorbol 12-myristate 13-acetate and ionomycin, which activate protein kinase C and mimic physiological activation through the T-cell antigen receptor, transcribe an interleukin-2 (IL-2) enhancer (-326 to +24) template 5-fold more efficient than nuclear extracts from resting T-cells and severalfold more efficient than extracts from Jurkat cells treated with phorbol 12-myristate 13-acetate or ionomycin alone. Further results demonstrate that in vitro transcription of the IL-2 enhancer is T-cell specific since nuclear extracts from rat liver and stimulated HeLa cells are unable to induce IL-2 transcription. The activation-dependent, T-cell-specific in vitro transcription system described here should facilitate the dissection of signals that emanate from the T-cell surface resulting in IL-2 transcription and T-cell activation.
View details for Web of Science ID A1992GY43900065
View details for PubMedID 1730605
- REGULATION OF THE INTERLEUKIN-2 GENE CHEMICAL IMMUNOLOGY 1992; 51: 266-298
CHARACTERIZATION OF A COFACTOR THAT REGULATES DIMERIZATION OF A MAMMALIAN HOMEODOMAIN PROTEIN
1991; 254 (5039): 1762-1767
Dimerization among transcription factors has become a recurrent theme in the regulation of eukaryotic gene expression. Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) is a homeodomain-containing protein that functions as a dimer. A dimerization cofactor of HNF-1 alpha (DCoH) was identified that displayed a restricted tissue distribution and did not bind to DNA, but, rather, selectively stabilized HNF-1 alpha dimers. The formation of a stable tetrameric DCoH-HNF-1 alpha complex, which required the dimerization domain of HNF-1 alpha, did not change the DNA binding characteristics of HNF-1 alpha, but enhanced its transcriptional activity. However, DCoH did not confer transcriptional activation to the GAL4 DNA binding domain. These results indicate that DCoH regulates formation of transcriptionally active tetrameric complexes and may contribute to the developmental specificity of the complex.
View details for PubMedID 1763325
- SITE OF ACTION OF CYCLOSPORINE AND FK-506 IN THE PATHWAYS OF COMMUNICATION BETWEEN THE LYMPHOCYTE-T ANTIGEN RECEPTOR AND THE EARLY ACTIVATION GENES INTERNATIONAL CONGRESS ON FK 506 ELSEVIER SCIENCE INC. 1991: 2845–45
ACTIVATION OF EARLY GENE-EXPRESSION IN LYMPHOCYTES-T BY OCT-1 AND AN INDUCIBLE PROTEIN, OAP40
1991; 254 (5031): 558-562
After antigenic stimulation of T lymphocytes, genes essential for proliferation and immune function, such as the interleukin-2 (IL-2) gene, are transcriptionally activated. In both transient transfections and T lymphocyte-specific in vitro transcription, the homeodomain-containing protein Oct-1 participated in the inducible regulation of transcription of the IL-2 gene. Oct-1 functioned in this context with a 40-kilodalton protein called Oct-1-associated protein (OAP40). In addition to interacting specifically with DNA, OAP40 reduced the rate of dissociation of Oct-1 from its cognate DNA-binding site, suggesting that a direct interaction exists between Oct-1 and OAP40.
View details for Web of Science ID A1991GL79900045
View details for PubMedID 1683003
NUCLEAR-ASSOCIATION OF A T-CELL TRANSCRIPTION FACTOR BLOCKED BY FK-506 AND CYCLOSPORINE-A
1991; 352 (6338): 803-807
Cyclosporin A and FK506 inhibit T- and B-cell activation and other processes essential to an effective immune response. In T lymphocytes these drugs disrupt an unknown step in the transmission of signals from the T-cell antigen receptor to cytokine genes that coordinate the immune response. The putative intracellular receptors for FK506 and cyclosporin are cis-trans prolyl isomerases. Binding of the drug inhibits isomerase activity, but studies with other prolyl isomerase inhibitors and analysis of cyclosporin-resistant mutants in yeast suggest that the effects of the drug result from the formation of an inhibitory complex between the drug and isomerase, and not from inhibition of isomerase activity. A transcription factor, NF-AT, which is essential for early T-cell gene activation, seems to be a specific target of cyclosporin A and FK506 action because transcription directed by this protein is blocked in T cells treated with these drugs, with little or no effect on other transcription factors such as AP-1 and NF-kappa B. Here we demonstrate that NF-AT is formed when a signal from the antigen receptor induces a pre-existing cytoplasmic subunit to translocate to the nucleus and combine with a newly synthesized nuclear subunit of NF-AT. FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit.
View details for Web of Science ID A1991GC96400060
View details for PubMedID 1715516
INDEPENDENT REGULATION OF HNF-1-ALPHA AND HNF-1-BETA RETINOIC ACID IN F9-TERATOCARCINOMA CELLS
1991; 10 (8): 2231-2236
Hepatocyte Nuclear Factor-1 alpha (HNF-1 alpha) and HNF-1 beta are homeodomain-containing transcription factors which interact with the GTTAATNATTAAC motif essential to the function of more than 15 promoters selectively expressed in the liver. These homeoproteins can form homo- and heterodimers in solution and share identical DNA-binding domains but have different transcriptional activation properties. During retinoic acid (RA) induced differentiation of F9 embryonal carcinoma (EC) cells, which stimulates aspects of pre-implantation embryogenesis, both HNF-1 beta mRNA and immunoreactive DNA-binding activity are strongly induced approximately 24 h post RA-treatment. In contrast, HNF-1 alpha mRNA increases approximately 4-fold after 5 days, concomitant with elevation of HNF-1 alpha DNA-binding activity and expression of the HNF-1 target gene alpha-fetoprotein. These results indicate that HNF-1 alpha and -1 beta expression can be controlled by regulatory hierarchies downstream of primary RA-response genes, and suggest that independent regulatory mechanisms for these factors can confer distinct and interactive developmental functions.
View details for Web of Science ID A1991FW47300034
View details for PubMedID 2065662
HNF-1-ALPHA AND HNF-1-BETA (VHNF-1) SHARE DIMERIZATION AND HOMEO DOMAINS, BUT NOT ACTIVATION DOMAINS, AND FORM HETERODIMERS INVITRO
GENES & DEVELOPMENT
1991; 5 (6): 1042-1056
HNF-1 alpha (previously referred to as HNF-1, LPB1, and APF) is a vertebrate transcription factor that contains a divergent homeo domain and plays a prominent role in regulating genes that have the common characteristic of being expressed in hepatocytes and a complex group of endodermally and mesodermally derived tissues. HNF-1 alpha is unique among the vertebrate homeo domain-containing proteins in that it dimerizes in the absence of its DNA recognition sequence, suggesting the possibility that the function of HNF-1 alpha may be diversified by forming heterodimers with other related proteins. We report the initial characterization of HNF-1 beta, which is closely related to HNF-1 alpha and is able to form heterodimers with HNF-1 alpha in vitro. Although HNF-1 alpha, but not HNF-1 beta, is expressed in the liver, HNF-1 alpha and HNF-1 beta are coexpressed in the murine Hepa1A cell line and in the mammalian kidney where a subset of hepatocyte genes are expressed. In contrast, exclusive expression of HNF-1 beta is associated with repression of a subset of hepatocyte-specific genes in the dedifferentiated hepatocyte cell line C2, differentiated F9 cells, in somatic hybrids between hepatocytes and fibroblasts, and in the lung. The extent of heterodimerization may be regulated in a tissue-specific way because freely exchangeable heterodimers are formed in Jurkat T cells transfected with HNF-1 alpha and HNF-1 beta, whereas in liver cells stable homodimers are present. These studies define a pair of homeo domain proteins that have the potential to interact to produce an embryologically complex pattern of gene expression.
View details for Web of Science ID A1991FQ55400013
View details for PubMedID 2044952
- HNF-1, A MEMBER OF A NOVEL CLASS OF DIMERIZING HOMEODOMAIN PROTEINS JOURNAL OF BIOLOGICAL CHEMISTRY 1991; 266 (2): 677-680
THE ACTIONS OF CYCLOSPORINE-A AND FK506 SUGGEST A NOVEL STEP IN THE ACTIVATION OF LYMPHOCYTES-T
1990; 9 (13): 4425-4433
Cyclosporin A and FK506 are immunosuppressive compounds that have similar inhibitory effects on the expression of several lymphokines produced by T lymphocytes. Despite their similar effects the drugs bind to two different cytosolic protein, cyclophilin and FKBP respectively, which raises the possibility that they have different modes of action. Using constructs in which mRNA production controlled by a specific transcription factor could be readily measured we found that both cyclosporin A and FK506 completely inhibited transcription activated by NF-AT, NFIL2 A, NFIL2 B and partially inhibited transcription activated by NF kappa B. Cyclosporin A and FK506 inhibited only transcriptional activation that was dependent on Ca2+ mobilization. However, cyclosporin A and FK506 did not inhibit Ca2+ mobilization dependent expression of c-fos mRNA indicating that only a subset of signalling pathways regulated by Ca2+ is sensitive to these drugs. Furthermore, we did not observe any qualitative differences between the effect of cyclosporin A and FK506 on six different transcription factors which suggests that these drugs may interfere with the activity of a novel Ca2+ dependent step that regulates several transcription factors.
View details for Web of Science ID A1990EN92100026
View details for PubMedID 1702384
MOLECULAR-CLONING, FUNCTIONAL EXPRESSION, AND CHROMOSOMAL LOCALIZATION OF MOUSE HEPATOCYTE NUCLEAR FACTOR-I
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1990; 87 (24): 9838-9842
The homeodomain-containing transcription factor hepatocyte nuclear factor 1 (HNF-1) most likely plays an essential role during liver organogenesis by transactivating a family of greater than 15 predominantly hepatic genes. We have isolated cDNA clones encoding mouse HNF-1 and expressed them in monkey COS cells and in the human T-cell line Jurkat, producing HNF-1 DNA-binding activity as well as transactivation of reporter constructs containing multimerized HNF-1 binding sites. In addition, the HNF-1 gene was assigned by somatic cell hybrids and recombinant inbred strain mapping to mouse chromosome 5 near Bcd-1 and to human chromosome 12 region q22-qter, revealing a homologous chromosome region in these two species. The presence of HNF-1 mRNA in multiple endodermal tissues (liver, stomach, intestine) suggests that HNF-1 may constitute an early marker for endodermal, rather than hepatocyte, differentiation. Further, that HNF-1 DNA-binding and transcriptional activity can be conferred by transfecting the HNF-1 cDNA into several cell lines indicates that it is sufficient to activate transcription in the context of ubiquitously expressed factors.
View details for Web of Science ID A1990EN15900069
View details for PubMedID 2263635
SINGLE CELL ASSAY OF A TRANSCRIPTION FACTOR REVEALS A THRESHOLD IN TRANSCRIPTION ACTIVATED BY SIGNALS EMANATING FROM THE T-CELL ANTIGEN RECEPTOR
GENES & DEVELOPMENT
1990; 4 (10): 1823-1834
Stimulation of T lymphocytes through their antigen receptor leads to the appearance of several transcription factors, including NF-AT and NF-kappa B, which are involved in regulating genes required for immunologic activation. To investigate the activity of a single transcription factor in individual viable cells, we have applied an assay that uses the fluorescence-activated cell sorter to quantitate beta-galactosidase (beta-gal). We have analyzed the distribution of NF-AT transcriptional activity among T cells undergoing activation by using a construct in which three tandem copies of the NF-AT-binding site directs transcription of the lacZ gene. Unexpectedly, stimulation of cloned stably transfected Jurkat T cells leads to a bimodal pattern of beta-gal expression in which some cells express no beta-gal and others express high levels. This expression pattern cannot be accounted for by cell-cycle position or heritable variation. Further results, in which beta-gal activity is correlated with NF-AT-binding activity, indicate that the concentration of NF-AT must exceed a critical threshold before transcription initiates. This threshold likely reflects the NF-AT concentration-dependent assembly of transcription complexes at the promoter. Similar constructs controlled by NF-kappa B or the entire interleukin-2 enhancer show bimodal expression patterns during induction, suggesting that thresholds set by the concentration of transcription factors may be a common property of inducible genes.
View details for Web of Science ID A1990EC63000016
View details for PubMedID 2123468
CELL TYPE SPECIFICITY AND ACTIVATION REQUIREMENTS FOR NFAT-1 (NUCLEAR FACTOR OF ACTIVATED T-CELLS) TRANSCRIPTIONAL ACTIVITY DETERMINED BY A NEW METHOD USING TRANSGENIC MICE TO ASSAY TRANSCRIPTIONAL ACTIVITY OF AN INDIVIDUAL NUCLEAR FACTOR
JOURNAL OF BIOLOGICAL CHEMISTRY
1990; 265 (26): 15788-15795
Nuclear factor of activated T-cells (NFAT-1) is a transcription factor which is considered to be an important regulator in early T-cell activation. We have developed a system to monitor the transcriptional activity of NFAT-1 at the single cell level in whole animals. The system is based on the use of an oligomerized NFAT-1 binding motif that directs transcription of SV40 T-antigen in transgenic mice. This report represents the first demonstration that a multimerized short binding motif can function appropriately in transgenic mice. NFAT-1 activity had previously been thought to be confined to activated T-lymphocytes upon release of intracellular calcium. By targeting NFAT-1-dependent gene expression in transgenic mice we discovered new sites of NFAT-1 activity. Besides in T-lymphocytes NFAT-1 activity could also be induced in T-lymphocyte-depleted spleen cells and purified B-lymphocytes and requires agents that both release intracellular calcium and activate protein kinase C. A difference in the time course of appearance of NFAT-1 activity between T-lymphocytes and non-T-lymphocytes was revealed. Constitutive expression was observed in a small population of cells in the dermis and some mice have developed skin lesions. Interestingly, the tissue pattern of expression of the NFAT-1 activity resembles the expression pattern described for HIV-LTR/tat transgenic mice (Vogel, J., Hinrichs, S. H., Reynolds, R. K., Luciw, P. A., and Jay, G. (1988) Nature 335, 606-611). This similarity in expression and the fact that NFAT-1 has been shown to bind functional sequences in HIV-LTR suggest a role for NFAT-1 in dermal activation of the HIV-LTR.
View details for Web of Science ID A1990DY96400070
View details for PubMedID 2394747
HNF-1 SHARES 3 SEQUENCE MOTIFS WITH THE POU DOMAIN PROTEINS AND IS IDENTICAL TO LF-B1 AND APF
GENES & DEVELOPMENT
1990; 4 (3): 372-379
The coordinate expression of genes during development and differentiation is thought to be accomplished by common transcription factors operating on the promoters of families of coexpressed genes. HNF-1 is a transcriptional factor involved in the expression of genes in the liver and was originally defined as playing a major role in coordinating the expression of the linked fibrinogen genes. We have isolated cDNA clones for HNF-1 using oligonucleotides prepared to the sequence of the purified protein. The sequence of HNF-1 shares homeo domain, as well as short acidic and basic sequences with the POU family of transcriptional activators. Peptides from the protein interacting with the albumin proximal element, or B box (APF), and the factor interacting with the alpha 1-antitrypsin promoter (LF-B1) are found in the predicted sequence of HNF-1. HNF-1 mRNA is not present in the dedifferentiated hepatoma variant, C2, but reappears upon selection for gluconeogenesis coincident with the re-expression of liver-specific genes. Finally, the mRNA is not present in somatic cell hybrids in which liver-specific gene expression is extinguished. In contrast to earlier published results, we find that in addition to being present in the liver, HNF is expressed in the kidney, intestine, and spleen, but not in other tissues. This pattern of expression mirrors the complex pattern of expression of many genes, such as alpha-fetoprotein, alpha 1-antitrypsin, and fibrinogen, whose promoters contain HNF-1 sites. These data indicate that HNF-1 is a more broadly acting transcription factor than has been indicated by previous work.
View details for Web of Science ID A1990DD09600006
View details for PubMedID 1970973
- LYMPHOCYTE-T ANTIGEN INTERACTIONS IN TRANSPLANT REJECTION NEW ENGLAND JOURNAL OF MEDICINE 1990; 322 (8): 510-517
- TRANSMISSION OF SIGNALS FROM THE LYMPHOCYTE-T ANTIGEN RECEPTOR TO THE GENES RESPONSIBLE FOR CELL-PROLIFERATION AND IMMUNE FUNCTION - THE MISSING LINK ANNUAL REVIEW OF IMMUNOLOGY 1990; 8: 421-452
CYCLOSPORINE-A SPECIFICALLY INHIBITS FUNCTION OF NUCLEAR PROTEINS INVOLVED IN T-CELL ACTIVATION
1989; 246 (4937): 1617-1620
One action of cyclosporin A thought to be central to many of its immunosuppressive effects is its ability to inhibit the early events of T lymphocyte activation such as lymphokine gene transcription in response to signals initiated at the antigen receptor. Cyclosporin A was found to specifically inhibit the appearance of DNA binding activity of NF-AT, AP-3, and to a lesser extent NF-kappa B, nuclear proteins that appear to be important in the transcriptional activation of the genes for interleukin-2 and its receptor, as well as several other lymphokines. In addition, cyclosporin A abolished the ability of the NF-AT binding site to activate a linked promoter in transfected mitogen-stimulated T lymphocytes and in lymphocytes from transgenic mice. These results indicate that cyclosporin A either directly inhibits the function of nuclear proteins critical to T lymphocyte activation or inhibits the action of a more proximal member of the signal transmission cascade leading from the antigen receptor to the nucleus.
View details for Web of Science ID A1989CE81500052
View details for PubMedID 2595372
- THE ROLE OF HNF-1 IN LIVER-SPECIFIC GENE-EXPRESSION ANNALS OF THE NEW YORK ACADEMY OF SCIENCES 1989; 557: 272-279
CONTINGENT GENETIC REGULATORY EVENTS IN LYMPHOCYTE-T ACTIVATION
1989; 243 (4889): 355-361
Interaction of antigen in the proper histocompatibility context with the T lymphocyte antigen receptor leads to an orderly series of events resulting in morphologic change, proliferation, and the acquisition of immunologic function. In most T lymphocytes two signals are required to initiate this process, one supplied by the antigen receptor and the other by accessory cells or agents that activate protein kinase C. Recently, DNA sequences have been identified that act as response elements for one or the other of the two signals, but do not respond to both signals. The fact that these sequences lie within the control regions of the same genes suggests that signals originating from separate cell membrane receptors are integrated at the level of the responsive gene. The view is put forth that these signals initiate a contingent series of gene activations that bring about proliferation and impart immunologic function.
View details for Web of Science ID A1989R832400028
View details for PubMedID 2783497
THE ROLE OF HNF-1 IN LIVER-SPECIFIC GENE-EXPRESSION
CONF ON REGULATION OF THE ACUTE PHASE AND IMMUNE RESPONSES - A NEW CYTOKINE : INTERLEUKIN-6
NEW YORK ACAD SCIENCES. 1989: 272–279
View details for Web of Science ID A1989BQ78Y00027
PURIFIED HEPATOCYTE NUCLEAR FACTOR-I INTERACTS WITH A FAMILY OF HEPATOCYTE-SPECIFIC PROMOTERS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1988; 85 (21): 7937-7941
During development cell types arise through the activation or repression of classes of specific genes. One hypothesis is that this phenomenon is realized by tissue-specific factors playing a role at the transcription level. Recently we have described a liver-specific nuclear protein, hepatocyte nuclear factor 1, that appears to be involved in the transcription of the fibrinogen and alpha 1-antitrypsin genes. In this report we describe the purification of hepatocyte nuclear factor 1 and demonstrate that it interacts with essential promoter regions of many liver-specific genes, including albumin, alpha-fetoprotein, and transthyretin. This finding suggests that hepatocyte nuclear factor 1 could be one factor necessary for establishing the liver phenotype. We also show that this protein binds to the promoter of the surface-antigen gene of the hepatitis B virus, a virus characterized by a high degree of hepatotropism.
View details for Web of Science ID A1988Q834100023
View details for PubMedID 2460858
A VARIANT NUCLEAR-PROTEIN IN DEDIFFERENTIATED HEPATOMA-CELLS BINDS TO THE SAME FUNCTIONAL SEQUENCES IN THE BETA-FIBRINOGEN GENE PROMOTER AS HNF-1
1988; 7 (8): 2485-2493
Normal liver and differentiated hepatoma cell lines contain a nuclear factor, HNF-1, which binds functional sequences within the promoters of the alpha and beta chains of fibrinogen and alpha 1-antitrypsin. In UV cross-linking studies we find that HNF-1 has an apparent mol. wt of 92 kd in differentiated hepatocytes. Nuclear extracts from a dedifferentiated hepatoma cell line, Fao flC2 (C2), selected on the basis of morphological and biochemical dedifferentiation from Fao contains a protein, vHNF, which binds to the same DNA sequence motif as HNF-1 but has an apparent mol. wt of 72 rather than 92 kd. Mixing experiments indicate that this variant nuclear factor does not arise from HNF-1 by proteolysis. Reversion to the differentiated phenotype in C2-Rev7 (Rev7), selected by growth in glucose-free media, results in the re-expression of many liver-specific functions including the fibrinogen genes. In Rev7, HNF-1 is indistinguishable from that in the original differentiated cell line Fao. Transfection studies and nuclear run-on experiments indicate that reduced expression of fibrinogen RNA in C2 relative to Fao is related to reduced transcription. vHNF but not HNF-1 is present in somatic hybrids between fibroblasts and liver cells which show extinction of liver specific traits and it can also be detected in normal tissue, predominantly in lung nuclear extracts. Since vHNF and HNF-1 are not co-expressed yet correlate with the non-hepatic and hepatic phenotype, respectively, we suggest that the expression of these variant forms reflects determination events in establishing the hepatic phenotype.
View details for Web of Science ID A1988P464000026
View details for PubMedID 2847919
IDENTIFICATION OF A PUTATIVE REGULATOR OF EARLY T-CELL ACTIVATION GENES
1988; 241 (4862): 202-205
Molecules involved in the antigen receptor-dependent regulation of early T cell activation genes were investigated with the use of functional sequences of the T cell activation-specific enhancer of interleukin-2 (IL-2). One of these sequences forms a protein complex, NFAT-1, specifically with nuclear extracts of activated T cells. This complex appeared 10 to 25 minutes before the activation of the IL-2 gene. Studies with inhibitors of protein synthesis indicated that the time of synthesis of the activator of the IL-2 gene in Jurkat T cells corresponds to the time of appearance of NFAT-1. NFAT-1, or a very similar protein, bound functional sequences of the long terminal repeat (LTR) of the human immunodeficiency virus type 1; the LTR of this virus is known to be stimulated during early T cell activation. The binding site for this complex activated a linked promoter after transfection into antigen receptor-activated T cells but not other cell types. These characteristics suggest that NFAT-1 transmits signals initiated at the T cell antigen receptor.
View details for Web of Science ID A1988P120800034
View details for PubMedID 3260404
SP1, A CAAT-BINDING FACTOR, AND THE ADENOVIRUS MAJOR LATE PROMOTER TRANSCRIPTION FACTOR INTERACT WITH FUNCTIONAL REGIONS OF THE GAMMA-FIBRINOGEN PROMOTER
MOLECULAR AND CELLULAR BIOLOGY
1988; 8 (6): 2628-2637
To study the factors which influence the coordinately and developmentally regulated expression of the three adjacent fibrinogen genes, we have defined the functional regions of the gamma-fibrinogen promoter and the proteins which bind to them. Using a series of 5' and internal deletion mutations, we found that sequences between 88 and 43 base pairs (bp) upstream of the gamma-fibrinogen transcription initiation site functioned in cis to direct properly initiated mRNA accumulation in transfected hepatocytes. The efficient function of these sequences was highly distance dependent, since transcriptional activity decreased by 92% when they were moved 32 bp upstream of the TATA box. We demonstrated that two known and one putative transcriptional factors interacted with this 47-bp sequence. The transcription factor Sp1 interacted with sequences between -51 and -46 as demonstrated by protection from DNase I digestion with the purified protein. Directly adjacent to the Sp1 site, between nucleotides -66 and -53, there was a sequence which bound a CAAT-binding factor. Finally, sequences just 5' to the CAAT factor-binding site interacted with the adenovirus major late transcriptional factor as previously demonstrated. Internal deletion mutations which disrupt these interactions diminished the activity of the promoter in vivo. One consequence of the interaction of these proteins is that a bend is placed in the DNA at or near their sites of interaction.
View details for Web of Science ID A1988N633800044
View details for PubMedID 3043186
CHARACTERIZATION OF ANTIGEN RECEPTOR RESPONSE ELEMENTS WITHIN THE INTERLEUKIN-2 ENHANCER
MOLECULAR AND CELLULAR BIOLOGY
1988; 8 (4): 1715-1724
T-cell activation and induction of interleukin-2 (IL-2) expression in human T lymphocytes require both interaction of foreign antigen with the T-cell antigen receptor and protein kinase C (PKC) stimulation. Agents such as phorbol 12-myristate 13-acetate (PMA) that stimulate PKC augment the effects of antigen but are not sufficient for IL-2 activation. By analysis of deletion mutants, we identified three DNA sequences extending from -73 to -89, -217 to -255, and -263 to -279, designated IL-2 sites A, D, and E, respectively, that are required for maximal induction of IL-2 expression. One of these regions, site E, interacted with a protein (NF-IL-2E) present only in the nuclei of cells which have been stimulated. The other two sequences interacted with a protein (NF-IL-2A) that is constitutively expressed in T cells. When multiple tandem copies of either the E site or the A site were placed upstream of the gamma-fibrinogen promoter, they activated expression via this promoter in response to signals initiated at the antigen receptor but not following PMA stimulation. For this reason, we denoted them antigen receptor response elements. The uncoupling of antigen receptor and PKC requirements in these studies indicates that these signal pathways are, at least in part, distinct and integrated at the level of the gene.
View details for Web of Science ID A1988M717700038
View details for PubMedID 3260003
IDENTIFICATION AND CHARACTERIZATION OF NUCLEAR PROTEINS INVOLVED IN THE EXPRESSION OF FIBRINOGEN GENES
8TH INTERNATIONAL FIBRINOGEN WORKSHOP
ELSEVIER SCIENCE PUBL B V. 1988: 3–6
View details for Web of Science ID A1988BR83J00001
INTERACTION OF A LIVER-SPECIFIC NUCLEAR FACTOR WITH THE FIBRINOGEN AND ALPHA-1-ANTITRYPSIN PROMOTERS
1987; 238 (4827): 688-692
The orderly and sequential activation of genes during development is hypothesized to be related to the selective expression of groups of regulatory proteins acting primarily at the level of transcription. A nuclear protein was found in hepatocytes, but not other cell types, that binds to a sequence required for hepatocyte-specific transcription of the gene for the beta chain of fibrinogen. This protein, hepatocyte nuclear factor 1 (HNF1), also interacts with homologous sequences required for optimal promoter function of the genes for the alpha chain of fibrinogen and alpha 1-antitrypsin. The promoter or enhancer regions for several viral and cellular genes not expressed in the liver did not compete for this binding. The restricted expression of HNF1 and its selective interaction with the control regions of several liver-specific genes indicate that it is involved in developmentally regulated gene expression in the liver.
View details for Web of Science ID A1987K595500040
View details for PubMedID 3499668