Honors & Awards
ISSCR Outstanding Young Investigator Award, International Society for Stem Cell Research (2010)
Distinguished Young Scholar in Biomedical Research, W.M.Keck Foundation (2008-2013)
New Faculty Award, California Institute for Regenerative Medicine (2008-2013)
Searle Scholar, Chicago Community Trust (2007-2010)
Faculty Scholar, Baxter Foundation (2007)
Terman Fellowship, Fredrick E. Terman Foundation (2006-2009)
Postdoctoral Fellowship, Damon Runyon Cancer Research Foundation (2004-2006)
postdoctoral education, The Rockefeller University, Chromatin Biology (2006)
PhD, IBB Polish Academy of Science & Cold Spring Harbor Laboratory, Biochemistry (2003)
MSc, Warsaw University, Molecular Biology (1998)
Current Research and Scholarly Interests
EPIGENETIC REGULATION OF DIFFERENTIATION AND DEVELOPMENT
The biological question that is driving our research in the long-term is understanding the epigenetic basis of vertebrate development and differentiation. Although each cell of a multicellular organism is a progeny of a single zygote, and shares the same genetic information with every other cell, cells differentiate to specialized forms such as skin, muscle or nervous cells. Thus, new information emerges during development, and is inherited in a way that does not involve changes in DNA sequence. This fascinating process is called epigenesis. Epigenetic changes underlie not only normal, but also pathological development. Abnormal transmission of epigenetic information contributes to human pathology, such as aging, cancer, degenerative diseases, developmental defects and mental retardation.
In the last decade evidence emerged that a substantial portion of epigenetic information is transmitted in a form of chemical modifications of histones and associated DNA. Our research focuses on understanding the mechanistic basis by which covalent histone modifications regulate gene expression patterns during vertebrate development and differentiation. In particular, we are focusing on characterizing enzymatic activities responsible for "writing" the methyl mark on histones, called histone methyltransferases, as well as on downstream effectors, or "readers", which recognize the methyl marks and translate them into specific biological outcomes. The outstanding questions we are trying to address are: How are methylation patterns established? How do methyltransferases connect to the signaling pathways? What are their roles in regulating development and how did they functionally specialize during vertebrate evolution?
A second major area of our interest involves chromatin regulation in embryonic stem cells (ESCs), molecular basis of pluripotency and role of histone methyltransferases in cell fate decisions. ESCs share with the early embryo the potential to produce every type of cell in the human body. This rare biological property is known as pluripotency. Pluripotency is a unique epigenetic state, in that ESCs can self-renew, while retaining the potential for multilineage differentiation. We are investigating the role of writers and readers of histone methylation in human and mouse ESC self-renewal and commitment to different cell fates.
Independent Studies (11)
- Directed Reading in Chemical and Systems Biology
CSB 299 (Aut, Win, Spr, Sum)
- Directed Reading in Developmental Biology
DBIO 299 (Aut, Win, Spr, Sum)
- Graduate Research
CSB 399 (Aut, Win, Spr, Sum)
- Graduate Research
DBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
STEMREM 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
CSB 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
DBIO 370 (Aut, Win, Spr, Sum)
- TGR Dissertation
STEMREM 802 (Aut, Win, Spr, Sum)
- Undergraduate Research
CSB 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
DBIO 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
STEMREM 199 (Aut, Spr, Sum)
- Directed Reading in Chemical and Systems Biology
- Prior Year Courses
Graduate and Fellowship Programs
Epigenomic Annotation of Enhancers Predicts Transcriptional Regulators of Human Neural Crest
CELL STEM CELL
2012; 11 (5): 633-648
Neural crest cells (NCC) are a transient, embryonic cell population characterized by unusual migratory ability and developmental plasticity. To annotate and characterize cis-regulatory elements utilized by the human NCC, we coupled a hESC differentiation model with genome-wide profiling of histone modifications and of coactivator and transcription factor (TF) occupancy. Sequence analysis predicted major TFs binding at epigenomically annotated hNCC enhancers, including a master NC regulator, TFAP2A, and nuclear receptors NR2F1 and NR2F2. Although many TF binding events occur outside enhancers, sites coinciding with enhancer chromatin signatures show significantly higher sequence constraint, nucleosomal depletion, correlation with gene expression, and functional conservation in NCC isolated from chicken embryos. Simultaneous co-occupancy by TFAP2A and NR2F1/F2 is associated with permissive enhancer chromatin states, characterized by high levels of p300 and H3K27ac. Our results provide global insights into human NC chromatin landscapes and a rich resource for studies of craniofacial development and disease.
View details for DOI 10.1016/j.stem.2012.07.006
View details for Web of Science ID 000311471900010
View details for PubMedID 22981823
A unique chromatin signature uncovers early developmental enhancers in humans
2011; 470 (7333): 279-?
Cell-fate transitions involve the integration of genomic information encoded by regulatory elements, such as enhancers, with the cellular environment. However, identification of genomic sequences that control human embryonic development represents a formidable challenge. Here we show that in human embryonic stem cells (hESCs), unique chromatin signatures identify two distinct classes of genomic elements, both of which are marked by the presence of chromatin regulators p300 and BRG1, monomethylation of histone H3 at lysine 4 (H3K4me1), and low nucleosomal density. In addition, elements of the first class are distinguished by the acetylation of histone H3 at lysine 27 (H3K27ac), overlap with previously characterized hESC enhancers, and are located proximally to genes expressed in hESCs and the epiblast. In contrast, elements of the second class, which we term 'poised enhancers', are distinguished by the absence of H3K27ac, enrichment of histone H3 lysine 27 trimethylation (H3K27me3), and are linked to genes inactive in hESCs and instead are involved in orchestrating early steps in embryogenesis, such as gastrulation, mesoderm formation and neurulation. Consistent with the poised identity, during differentiation of hESCs to neuroepithelium, a neuroectoderm-specific subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos, poised enhancers are able to direct cell-type and stage-specific expression characteristic of their proximal developmental gene, even in the absence of sequence conservation in the fish genome. Our data demonstrate that early developmental enhancers are epigenetically pre-marked in hESCs and indicate an unappreciated role of H3K27me3 at distal regulatory elements. Moreover, the wealth of new regulatory sequences identified here provides an invaluable resource for studies and isolation of transient, rare cell populations representing early stages of human embryogenesis.
View details for DOI 10.1038/nature09692
View details for Web of Science ID 000287144200048
View details for PubMedID 21160473
Sequence-specific regulator Prdm14 safeguards mouse ESCs from entering extraembryonic endoderm fates
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2011; 18 (2): 120-U175
Prdm14 is a PR-domain and zinc-finger protein whose expression is restricted to the pluripotent cells of the early embryo, embryonic stem cells (ESCs), and germ cells. Here, we show that Prdm14 safeguards mouse ESC (mESC) maintenance by preventing induction of extraembryonic endoderm (ExEn) fates. Conversely, Prdm14 overexpression impairs ExEn differentiation during embryoid body formation. Prdm14 occupies and represses genomic loci encoding ExEn differentiation factors, while also binding to and promoting expression of genes associated with mESC self-renewal. Prdm14-associated genomic regions substantially overlap those occupied by Nanog and Oct4, are enriched in a chromatin signature associated with distal regulatory elements and contain a unique DNA-sequence motif recognized by Prdm14 in vitro. Our work identifies a new member of the mESC transcriptional network, Prdm14, which plays a dual role as a context-dependent transcriptional repressor or activator.
View details for DOI 10.1038/nsmb.2000
View details for Web of Science ID 000286969200003
View details for PubMedID 21183938
CHD7 cooperates with PBAF to control multipotent neural crest formation
2010; 463 (7283): 958-U135
Heterozygous mutations in the gene encoding the CHD (chromodomain helicase DNA-binding domain) member CHD7, an ATP-dependent chromatin remodeller homologous to the Drosophila trithorax-group protein Kismet, result in a complex constellation of congenital anomalies called CHARGE syndrome, which is a sporadic, autosomal dominant disorder characterized by malformations of the craniofacial structures, peripheral nervous system, ears, eyes and heart. Although it was postulated 25 years ago that CHARGE syndrome results from the abnormal development of the neural crest, this hypothesis remained untested. Here we show that, in both humans and Xenopus, CHD7 is essential for the formation of multipotent migratory neural crest (NC), a transient cell population that is ectodermal in origin but undergoes a major transcriptional reprogramming event to acquire a remarkably broad differentiation potential and ability to migrate throughout the body, giving rise to craniofacial bones and cartilages, the peripheral nervous system, pigmentation and cardiac structures. We demonstrate that CHD7 is essential for activation of the NC transcriptional circuitry, including Sox9, Twist and Slug. In Xenopus embryos, knockdown of Chd7 or overexpression of its catalytically inactive form recapitulates all major features of CHARGE syndrome. In human NC cells CHD7 associates with PBAF (polybromo- and BRG1-associated factor-containing complex) and both remodellers occupy a NC-specific distal SOX9 enhancer and a conserved genomic element located upstream of the TWIST1 gene. Consistently, during embryogenesis CHD7 and PBAF cooperate to promote NC gene expression and cell migration. Our work identifies an evolutionarily conserved role for CHD7 in orchestrating NC gene expression programs, provides insights into the synergistic control of distal elements by chromatin remodellers, illuminates the patho-embryology of CHARGE syndrome, and suggests a broader function for CHD7 in the regulation of cell motility.
View details for DOI 10.1038/nature08733
View details for Web of Science ID 000274582700048
View details for PubMedID 20130577
Jarid2/Jumonji Coordinates Control of PRC2 Enzymatic Activity and Target Gene Occupancy in Pluripotent Cells
2009; 139 (7): 1290-1302
Polycomb Repressive Complex 2 (PRC2) regulates key developmental genes in embryonic stem (ES) cells and during development. Here we show that Jarid2/Jumonji, a protein enriched in pluripotent cells and a founding member of the Jumonji C (JmjC) domain protein family, is a PRC2 subunit in ES cells. Genome-wide ChIP-seq analyses of Jarid2, Ezh2, and Suz12 binding reveal that Jarid2 and PRC2 occupy the same genomic regions. We further show that Jarid2 promotes PRC2 recruitment to the target genes while inhibiting PRC2 histone methyltransferase activity, suggesting that it acts as a "molecular rheostat" that finely calibrates PRC2 functions at developmental genes. Using Xenopus laevis as a model we demonstrate that Jarid2 knockdown impairs the induction of gastrulation genes in blastula embryos and results in failure of differentiation. Our findings illuminate a mechanism of histone methylation regulation in pluripotent cells and during early cell-fate transitions.
View details for DOI 10.1016/j.cell.2009.12.002
View details for Web of Science ID 000273048700017
View details for PubMedID 20064375
Chromatin remodelling factor Mll1 is essential for neurogenesis from postnatal neural stem cells
2009; 458 (7237): 529-U9
Epigenetic mechanisms that maintain neurogenesis throughout adult life remain poorly understood. Trithorax group (trxG) and Polycomb group (PcG) gene products are part of an evolutionarily conserved chromatin remodelling system that activate or silence gene expression, respectively. Although PcG member Bmi1 has been shown to be required for postnatal neural stem cell self-renewal, the role of trxG genes remains unknown. Here we show that the trxG member Mll1 (mixed-lineage leukaemia 1) is required for neurogenesis in the mouse postnatal brain. Mll1-deficient subventricular zone neural stem cells survive, proliferate and efficiently differentiate into glial lineages; however, neuronal differentiation is severely impaired. In Mll1-deficient cells, early proneural Mash1 (also known as Ascl1) and gliogenic Olig2 expression are preserved, but Dlx2, a key downstream regulator of subventricular zone neurogenesis, is not expressed. Overexpression of Dlx2 can rescue neurogenesis in Mll1-deficient cells. Chromatin immunoprecipitation demonstrates that Dlx2 is a direct target of MLL in subventricular zone cells. In differentiating wild-type subventricular zone cells, Mash1, Olig2 and Dlx2 loci have high levels of histone 3 trimethylated at lysine 4 (H3K4me3), consistent with their transcription. In contrast, in Mll1-deficient subventricular zone cells, chromatin at Dlx2 is bivalently marked by both H3K4me3 and histone 3 trimethylated at lysine 27 (H3K27me3), and the Dlx2 gene fails to properly activate. These data support a model in which Mll1 is required to resolve key silenced bivalent loci in postnatal neural precursors to the actively transcribed state for the induction of neurogenesis, but not for gliogenesis.
View details for DOI 10.1038/nature07726
View details for Web of Science ID 000264532400049
View details for PubMedID 19212323
H3K27 demethylases, at long last
2007; 131 (1): 29-32
Methylation of lysine 27 on histone H3 (H3K27me) by the Polycomb complex (PRC2) proteins is associated with gene silencing in many developmental processes. A cluster of recent papers (Agger et al., 2007; De Santa et al., 2007; Lan et al., 2007; Lee et al., 2007) identify the JmjC-domain proteins UTX and JMJD3 as H3K27-specific demethylases that remove this methyl mark, enabling the activation of genes involved in animal body patterning and the inflammatory response.
View details for DOI 10.1016/j.cell.2007.09.026
View details for Web of Science ID 000249934700008
View details for PubMedID 17923085
E2F activation of S phase promoters via association with HCF-1 and the MLL family of histone H3K4 methyltransferases
2007; 27 (1): 107-119
E2F transcriptional regulators control human-cell proliferation by repressing and activating the transcription of genes required for cell-cycle progression, particularly the S phase. E2F proteins repress transcription in association with retinoblastoma pocket proteins, but less is known about how they activate transcription. Here, we show that the human G1 phase regulator HCF-1 associates with both activator (E2F1 and E2F3a) and repressor (E2F4) E2F proteins, properties that are conserved in insect cells. Human HCF-1-E2F interactions are versatile: their associations and binding to E2F-responsive promoters are cell-cycle selective, and HCF-1 displays coactivator properties when bound to the E2F1 activator and corepressor properties when bound to the E2F4 repressor. During the G1-to-S phase transition, HCF-1 recruits the mixed-lineage leukemia (MLL) and Set-1 histone H3 lysine 4 methyltransferases to E2F-responsive promoters and induces histone methylation and transcriptional activation. These results suggest that HCF-1 induces cell-cycle-specific transcriptional activation by E2F proteins to promote cell proliferation.
View details for DOI 10.1016/j.molcel.2007.05.030
View details for Web of Science ID 000248088500010
View details for PubMedID 17612494
Methylation of lysine 4 on histone H3: Intricacy of writing and reading a single epigenetic mark
2007; 25 (1): 15-30
Cells employ elaborate mechanisms to introduce structural and chemical variation into chromatin. Here, we focus on one such element of variation: methylation of lysine 4 in histone H3 (H3K4). We assess a growing body of literature, including treatment of how the mark is established, the patterns of methylation, and the functional consequences of this epigenetic signature. We discuss structural aspects of the H3K4 methyl recognition by the downstream effectors and propose a distinction between sequence-specific recruitment mechanisms and stabilization on chromatin through methyl-lysine recognition. Finally, we hypothesize how the unique properties of the polyvalent chromatin fiber and associated effectors may amplify small differences in methyl-lysine recognition, simultaneously allowing for a dynamic chromatin architecture.
View details for DOI 10.1016/j.molcel.2006.12.014
View details for Web of Science ID 000243566700002
View details for PubMedID 17218268
A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling
2006; 442 (7098): 86-90
Lysine methylation of histones is recognized as an important component of an epigenetic indexing system demarcating transcriptionally active and inactive chromatin domains. Trimethylation of histone H3 lysine 4 (H3K4me3) marks transcription start sites of virtually all active genes. Recently, we reported that the WD40-repeat protein WDR5 is important for global levels of H3K4me3 and control of HOX gene expression. Here we show that a plant homeodomain (PHD) finger of nucleosome remodelling factor (NURF), an ISWI-containing ATP-dependent chromatin-remodelling complex, mediates a direct preferential association with H3K4me3 tails. Depletion of H3K4me3 causes partial release of the NURF subunit, BPTF (bromodomain and PHD finger transcription factor), from chromatin and defective recruitment of the associated ATPase, SNF2L (also known as ISWI and SMARCA1), to the HOXC8 promoter. Loss of BPTF in Xenopus embryos mimics WDR5 loss-of-function phenotypes, and compromises spatial control of Hox gene expression. These results strongly suggest that WDR5 and NURF function in a common biological pathway in vivo, and that NURF-mediated ATP-dependent chromatin remodelling is directly coupled to H3K4 trimethylation to maintain Hox gene expression patterns during development. We also identify a previously unknown function for the PHD finger as a highly specialized methyl-lysine-binding domain.
View details for DOI 10.1038/nature04815
View details for Web of Science ID 000238724500042
View details for PubMedID 16728976
WDR5 associates with histone H3 methylated at K4 and is essential for H3K4 methylation and vertebrate development
2005; 121 (6): 859-872
Histone H3 lysine 4 (K4) methylation has been linked to the transcriptional activation in a variety of eukaryotic species. Here we show that a common component of MLL1, MLL2, and hSet1 H3 K4 methyltransferase complexes, the WD40-repeat protein WDR5, directly associates with histone H3 di- and trimethylated at K4 and with H3-K4-dimethylated nucleosomes. WDR5 is required for binding of the methyltransferase complex to the K4-dimethylated H3 tail as well as for global H3 K4 trimethylation and HOX gene activation in human cells. WDR5 is essential for vertebrate development, in that WDR5-depleted X. laevis tadpoles exhibit a variety of developmental defects and abnormal spatial Hox gene expression. Our results are the first demonstration that a WD40-repeat protein acts as a module for recognition of a specific histone modification and suggest a mechanism for reading and writing an epigenetic mark for gene activation.
View details for DOI 10.1016/j.cell.2005.03.036
View details for Web of Science ID 000230011200009
View details for PubMedID 15960974
Enhancers as information integration hubs in development: lessons from genomics
TRENDS IN GENETICS
2012; 28 (6): 276-284
Transcriptional enhancers are the primary determinants of tissue-specific gene expression. Although the majority of our current knowledge of enhancer elements comes from detailed analyses of individual loci, recent progress in epigenomics has led to the development of methods for comprehensive and conservation-independent annotation of cell type-specific enhancers. Here, we discuss the advantages and limitations of different genomic approaches to enhancer mapping and summarize observations that have been afforded by the genome-wide views of enhancer landscapes, with a focus on development. We propose that enhancers serve as information integration hubs, at which instructions encoded by the genome are read in the context of a specific cellular state, signaling milieu and chromatin environment, allowing for exquisitely precise spatiotemporal control of gene expression during embryogenesis.
View details for DOI 10.1016/j.tig.2012.02.008
View details for Web of Science ID 000305094000004
View details for PubMedID 22487374
A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression
2011; 472 (7341): 120-U158
The genome is extensively transcribed into long intergenic noncoding RNAs (lincRNAs), many of which are implicated in gene silencing. Potential roles of lincRNAs in gene activation are much less understood. Development and homeostasis require coordinate regulation of neighbouring genes through a process termed locus control. Some locus control elements and enhancers transcribe lincRNAs, hinting at possible roles in long-range control. In vertebrates, 39 Hox genes, encoding homeodomain transcription factors critical for positional identity, are clustered in four chromosomal loci; the Hox genes are expressed in nested anterior-posterior and proximal-distal patterns colinear with their genomic position from 3' to 5'of the cluster. Here we identify HOTTIP, a lincRNA transcribed from the 5' tip of the HOXA locus that coordinates the activation of several 5' HOXA genes in vivo. Chromosomal looping brings HOTTIP into close proximity to its target genes. HOTTIP RNA binds the adaptor protein WDR5 directly and targets WDR5/MLL complexes across HOXA, driving histone H3 lysine 4 trimethylation and gene transcription. Induced proximity is necessary and sufficient for HOTTIP RNA activation of its target genes. Thus, by serving as key intermediates that transmit information from higher order chromosomal looping into chromatin modifications, lincRNAs may organize chromatin domains to coordinate long-range gene activation.
View details for DOI 10.1038/nature09819
View details for Web of Science ID 000289199400049
View details for PubMedID 21423168
Epigenomics of human embryonic stem cells and induced pluripotent stem cells: insights into pluripotency and implications for disease.
2011; 3 (6): 36-?
Human pluripotent cells such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) and their in vitro differentiation models hold great promise for regenerative medicine as they provide both a model for investigating mechanisms underlying human development and disease and a potential source of replacement cells in cellular transplantation approaches. The remarkable developmental plasticity of pluripotent cells is reflected in their unique chromatin marking and organization patterns, or epigenomes. Pluripotent cell epigenomes must organize genetic information in a way that is compatible with both the maintenance of self-renewal programs and the retention of multilineage differentiation potential. In this review, we give a brief overview of the recent technological advances in genomics that are allowing scientists to characterize and compare epigenomes of different cell types at an unprecedented scale and resolution. We then discuss how utilizing these technologies for studies of hESCs has demonstrated that certain chromatin features, including bivalent promoters, poised enhancers, and unique DNA modification patterns, are particularly pervasive in hESCs compared with differentiated cell types. We outline these unique characteristics and discuss the extent to which they are recapitulated in iPSCs. Finally, we envision broad applications of epigenomics in characterizing the quality and differentiation potential of individual pluripotent lines, and we discuss how epigenomic profiling of regulatory elements in hESCs, iPSCs and their derivatives can improve our understanding of complex human diseases and their underlying genetic variants.
View details for DOI 10.1186/gm252
View details for PubMedID 21658297
Flipping MLL1's Switch One Proline at a Time
2010; 141 (7): 1108-1110
The MLL1 (mixed lineage leukemia 1) protein, which is often disrupted in leukemia, both activates and represses Hox genes during hematopoiesis. Now, Wang et al. (2010) demonstrate that the cyclophilin CyP33 underpins this regulatory switch by altering the state of MLL1 through cis-trans proline isomerization in the linker region between MLL1's third PHD finger and bromodomain.
View details for DOI 10.1016/j.cell.2010.06.013
View details for Web of Science ID 000279148100006
View details for PubMedID 20602992
2009; 137 (2): 203-205
The memory of somatic cell gene expression is reset in the germline in a process that is accompanied by dramatic changes in chromatin modifications. In this issue, Katz et al. (2009) show that the histone demethylase Lsd1/Spr-5 may participate in this resetting process in the worm, thereby preventing a decline in germ cell epigenetic stability and viability over ensuing generations.
View details for DOI 10.1016/j.cell.2009.04.008
View details for Web of Science ID 000265456800010
View details for PubMedID 19379684
It takes a PHD to SUMO
TRENDS IN BIOCHEMICAL SCIENCES
2008; 33 (5): 191-194
PHD fingers and bromodomains are found in close proximity to each other in many chromatin-associated proteins and can functionally synergize. Recently, it has been demonstrated that the PHD finger of the KAP1 co-repressor functions as an E3 SUMO ligase for the adjacent bromodomain. This PHD-mediated SUMOylation stabilizes the association of the bromodomain with the chromatin modifiers SETDB1 and the nucleosome remodeling and deacetylation (NuRD) complex, thereby promoting establishment of the silent gene expression state.
View details for DOI 10.1016/j.tibs.2008.02.003
View details for Web of Science ID 000256750100001
View details for PubMedID 18406149
Epigenetic Regulation of Histone H3 Serine 10 Phosphorylation Status by HCF-1 Proteins in C. elegans and Mammalian Cells
2007; 2 (11)
The human herpes simplex virus (HSV) host cell factor HCF-1 is a transcriptional coregulator that associates with both histone methyl- and acetyltransferases, and a histone deacetylase and regulates cell proliferation and division. In HSV-infected cells, HCF-1 associates with the viral protein VP16 to promote formation of a multiprotein-DNA transcriptional activator complex. The ability of HCF proteins to stabilize this VP16-induced complex has been conserved in diverse animal species including Drosophila melanogaster and Caenorhabditis elegans suggesting that VP16 targets a conserved cellular function of HCF-1.To investigate the role of HCF proteins in animal development, we have characterized the effects of loss of the HCF-1 homolog in C. elegans, called Ce HCF-1. Two large hcf-1 deletion mutants (pk924 and ok559) are viable but display reduced fertility. Loss of Ce HCF-1 protein at reduced temperatures (e.g., 12 degrees C), however, leads to a high incidence of embryonic lethality and early embryonic mitotic and cytokinetic defects reminiscent of mammalian cell-division defects upon loss of HCF-1 function. Even when viable, however, at normal temperature, mutant embryos display reduced levels of phospho-histone H3 serine 10 (H3S10P), a modification implicated in both transcriptional and mitotic regulation. Mammalian cells with defective HCF-1 also display defects in mitotic H3S10P status.These results suggest that HCF-1 proteins possess conserved roles in the regulation of cell division and mitotic histone phosphorylation.
View details for DOI 10.1371/journal.pone.0001213
View details for Web of Science ID 000207459300003
View details for PubMedID 18043729
Identifying novel proteins recognizing histone modifications using peptide pull-down assay
2006; 40 (4): 339-343
Post-translational modifications of histones have been correlated with virtually all chromatin-templated processes, including gene expression regulation, DNA replication, mitosis and meiosis, and DNA repair. In order to better understand the mechanistic basis by which histone modifications participate in the control of cellular processes, it is essential to identify and characterize downstream effector proteins, or "readers", that are responsible for recognizing different marks and translating them into specific biological outcomes. Ideally, identification of potential histone-binding effectors should occur in an unbiased fashion. Although in the recent years much progress has been made in identifying readers of histone modifications, in particular methylation, recognition of the majority of known histone marks is still poorly understood. Here I describe a simple and unbiased biochemical pull-down assay that allows for the identification of novel histone effector proteins and utilizes biotinylated histone peptides modified at various residues. I provide detailed protocols and suggestions for troubleshooting.
View details for DOI 10.1016/j.ymeth.2006.05.028
View details for Web of Science ID 000242902600008
View details for PubMedID 17101446
Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF
2006; 442 (7098): 91-95
Mono-, di- and trimethylated states of particular histone lysine residues are selectively found in different regions of chromatin, thereby implying specialized biological functions for these marks ranging from heterochromatin formation to X-chromosome inactivation and transcriptional regulation. A major challenge in chromatin biology has centred on efforts to define the connection between specific methylation states and distinct biological read-outs impacting on function. For example, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with transcription start sites of active genes, but the molecular 'effectors' involved in specific recognition of H3K4me3 tails remain poorly understood. Here we demonstrate the molecular basis for specific recognition of H3(1-15)K4me3 (residues 1-15 of histone H3 trimethylated at K4) by a plant homeodomain (PHD) finger of human BPTF (bromodomain and PHD domain transcription factor), the largest subunit of the ATP-dependent chromatin-remodelling complex, NURF (nucleosome remodelling factor). We report on crystallographic and NMR structures of the bromodomain-proximal PHD finger of BPTF in free and H3(1-15)K4me3-bound states. H3(1-15)K4me3 interacts through anti-parallel beta-sheet formation on the surface of the PHD finger, with the long side chains of arginine 2 (R2) and K4me3 fitting snugly in adjacent pre-formed surface pockets, and bracketing an invariant tryptophan. The observed stapling role by non-adjacent R2 and K4me3 provides a molecular explanation for H3K4me3 site specificity. Binding studies establish that the BPTF PHD finger exhibits a modest preference for K4me3- over K4me2-containing H3 peptides, and discriminates against monomethylated and unmodified counterparts. Furthermore, we identified key specificity-determining residues from binding studies of H3(1-15)K4me3 with PHD finger point mutants. Our findings call attention to the PHD finger as a previously uncharacterized chromatin-binding module found in a large number of chromatin-associated proteins.
View details for DOI 10.1038/nature04802
View details for Web of Science ID 000238724500043
View details for PubMedID 16728978
Histone arginine methylation and its dynamic regulation
FRONTIERS IN BIOSCIENCE-LANDMARK
2006; 11: 344-355
Methylation of histones by protein arginine methyltransferases (PRMTs) is increasingly being found to play an important and dynamic role in gene regulation. In mammals, PRMT1- and CARM1-catalyzed histone asymmetric dimethyl-arginine is involved in gene activation while PRMT5-catalyzed histone symmetric dimethyl-arginine is associated with gene repression. Insight into mechanisms by which histone arginine methylation can be dynamically regulated comes from recent reports demonstrating that conversion of histone methylarginine residues to citrulline by peptidylarginine deiminase 4 (PADI4) leads to transcriptional repression. While the downstream cellular effects of histone arginine methylation remain poorly understood, recent findings indicate that protein arginine methylation, in general, is required for mammalian development and is also likely important for cellular proliferation and differentiation. Given the surge of interest in histone arginine methylation, this review article will focus on recent progress in this area.
View details for Web of Science ID 000232528000028
View details for PubMedID 16146736
Taking LSD1 to a new high
2005; 122 (5): 654-658
Histone modifications mediate changes in gene expression by altering the underlying chromatin structure or by serving as a binding platform to recruit other proteins. One such modification, histone methylation, was thought to be irreversible until last year when Shi and co-workers broke new ground with their discovery of a lysine-specific histone demethylase (LSD 1). They showed that LSD 1, a nuclear amine oxidase homolog, is a bona fide histone H3 lysine 4 demethylase (Shi et al., 2004). Now, a new study from published in a recent issue of Molecular Cell, together with two studies recently published by and in Nature, reveal that LSD 1's specificity and activity is in fact regulated by associated protein cofactors.
View details for DOI 10.1016/j.cell.2005.08.022
View details for Web of Science ID 000231844300006
View details for PubMedID 16143099
Physical association and coordinate function of the H3K4 methyltransferase MLL1 and the H4K16 acetyltransferase MOF
2005; 121 (6): 873-885
A stable complex containing MLL1 and MOF has been immunoaffinity purified from a human cell line that stably expresses an epitope-tagged WDR5 subunit. Stable interactions between MLL1 and MOF were confirmed by reciprocal immunoprecipitation, cosedimentation, and cotransfection analyses, and interaction sites were mapped to MLL1 C-terminal and MOF zinc finger domains. The purified complex has a robust MLL1-mediated histone methyltransferase activity that can effect mono-, di-, and trimethylation of H3 K4 and a MOF-mediated histone acetyltransferase activity that is specific for H4 K16. Importantly, both activities are required for optimal transcription activation on a chromatin template in vitro and on an endogenous MLL1 target gene, Hox a9, in vivo. These results indicate an activator-based mechanism for joint MLL1 and MOF recruitment and targeted methylation and acetylation and provide a molecular explanation for the closely correlated distribution of H3 K4 methylation and H4 K16 acetylation on active genes.
View details for DOI 10.1016/j.cell.2005.04.031
View details for Web of Science ID 000230011200010
View details for PubMedID 15960975
Human PAD4 regulates histone arginine methylation levels via demethylimination
2004; 306 (5694): 279-283
Methylation of arginine (Arg) and lysine residues in histones has been correlated with epigenetic forms of gene regulation. Although histone methyltransferases are known, enzymes that demethylate histones have not been identified. Here, we demonstrate that human peptidylarginine deiminase 4 (PAD4) regulates histone Arg methylation by converting methyl-Arg to citrulline and releasing methylamine. PAD4 targets multiple sites in histones H3 and H4, including those sites methylated by coactivators CARM1 (H3 Arg17) and PRMT1 (H4 Arg3). A decrease of histone Arg methylation, with a concomitant increase of citrullination, requires PAD4 activity in human HL-60 granulocytes. Moreover, PAD4 activity is linked with the transcriptional regulation of estrogen-responsive genes in MCF-7 cells. These data suggest that PAD4 mediates gene expression by regulating Arg methylation and citrullination in histones.
View details for DOI 10.1126/science.1101400
View details for Web of Science ID 000224419700041
View details for PubMedID 15345777
Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression
MOLECULAR AND CELLULAR BIOLOGY
2004; 24 (13): 5639-5649
MLL (for mixed-lineage leukemia) is a proto-oncogene that is mutated in a variety of human leukemias. Its product, a homolog of Drosophila melanogaster trithorax, displays intrinsic histone methyltransferase activity and functions genetically to maintain embryonic Hox gene expression. Here we report the biochemical purification of MLL and demonstrate that it associates with a cohort of proteins shared with the yeast and human SET1 histone methyltransferase complexes, including a homolog of Ash2, another Trx-G group protein. Two other members of the novel MLL complex identified here are host cell factor 1 (HCF-1), a transcriptional coregulator, and the related HCF-2, both of which specifically interact with a conserved binding motif in the MLL(N) (p300) subunit of MLL and provide a potential mechanism for regulating its antagonistic transcriptional properties. Menin, a product of the MEN1 tumor suppressor gene, is also a component of the 1-MDa MLL complex. Abrogation of menin expression phenocopies loss of MLL and reveals a critical role for menin in the maintenance of Hox gene expression. Oncogenic mutant forms of MLL retain an ability to interact with menin but not other identified complex components. These studies link the menin tumor suppressor protein with the MLL histone methyltransferase machinery, with implications for Hox gene expression in development and leukemia pathogenesis.
View details for DOI 10.1128/MCB.24.13.5639-5649.2004
View details for Web of Science ID 000222149200001
View details for PubMedID 15199122
- Linking covalent histone modifications to epigenetics: The rigidity and plasticity of the marks COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY 2004; 69: 161-169
The herpes simplex virus VP16-induced complex: the makings of a regulatory switch
TRENDS IN BIOCHEMICAL SCIENCES
2003; 28 (6): 294-304
When herpes simplex virus (HSV) infects human cells, it is able to enter two modes of infection: lytic and latent. A key activator of lytic infection is a virion protein called VP16, which, upon infection of a permissive cell, forms a transcriptional regulatory complex with two cellular proteins - the POU-domain transcription factor Oct-1 and the cell-proliferation factor HCF-1 - to activate transcription of the first set of expressed viral genes. This regulatory complex, called the VP16-induced complex, reveals mechanisms of combinatorial control of transcription. The activities of Oct-1 and HCF-1 - two important regulators of cellular gene expression and proliferation - illuminate strategies by which HSV might coexist with its host.
View details for DOI 10.1016/S0968-0004(03)00088-4
View details for Web of Science ID 000183948400004
View details for PubMedID 12826401
Human Sin3 deacetylase and trithorax-related Set1/Ash2 histone H3-K4 methyltransferase are tethered together selectively by the cell-proliferation factor HCF-1
GENES & DEVELOPMENT
2003; 17 (7): 896-911
The abundant and chromatin-associated protein HCF-1 is a critical player in mammalian cell proliferation as well as herpes simplex virus (HSV) transcription. We show here that separate regions of HCF-1 critical for its role in cell proliferation associate with the Sin3 histone deacetylase (HDAC) and a previously uncharacterized human trithorax-related Set1/Ash2 histone methyltransferase (HMT). The Set1/Ash2 HMT methylates histone H3 at Lys 4 (K4), but not if the neighboring K9 residue is already methylated. HCF-1 tethers the Sin3 and Set1/Ash2 transcriptional regulatory complexes together even though they are generally associated with opposite transcriptional outcomes: repression and activation of transcription, respectively. Nevertheless, this tethering is context-dependent because the transcriptional activator VP16 selectively binds HCF-1 associated with the Set1/Ash2 HMT complex in the absence of the Sin3 HDAC complex. These results suggest that HCF-1 can broadly regulate transcription, both positively and negatively, through selective modulation of chromatin structure.
View details for DOI 10.1101/gad.252103
View details for Web of Science ID 000182044700010
View details for PubMedID 12670868
Inactivation of the retinoblastoma protein family can bypass the HCF-1 defect in tsBN67 cell proliferation and cytokinesis
MOLECULAR AND CELLULAR BIOLOGY
2002; 22 (19): 6767-6778
Owing to a single missense mutation in the cell proliferation factor HCF-1, the temperature-sensitive tsBN67 hamster cell line arrests proliferation at nonpermissive temperatures, primarily in a G(0)/G(1) state, and displays temperature-sensitive cytokinesis defects. The HCF-1 mutation in tsBN67 cells also causes a temperature-sensitive dissociation of HCF-1 from chromatin prior to cell proliferation arrest, suggesting that HCF-1-chromatin association is important for mammalian-cell proliferation. Here, we report that the simian virus 40 (SV40) early region, in particular, large T antigen (Tag), and the adenovirus oncoprotein E1A can rescue the tsBN67 cell proliferation defect at nonpermissive temperatures. The SV40 early region rescues the tsBN67 cell proliferation defect without restoring the HCF-1-chromatin association, indicating that these oncoproteins bypass a requirement for HCF-1 function. The SV40 early region also rescues the tsBN67 cytokinesis defect, suggesting that the roles of HCF-1 in cell proliferation and proper cytokinesis are intimately linked. The ability of SV40 Tag and adenovirus E1A to inactivate members of the pRb protein family-pRb, p107, and p130-is important for the bypass of HCF-1 function. These results suggest that HCF-1 regulates mammalian-cell proliferation and cytokinesis, at least in part, by either directly or indirectly opposing pRb family member function.
View details for DOI 10.1128/MCB.22.19.6767-6778.2002
View details for Web of Science ID 000177961900012
View details for PubMedID 12215534
Loss of HCF-1-chromatin association precedes temperature-induced growth arrest of tsBN67 cells
MOLECULAR AND CELLULAR BIOLOGY
2001; 21 (11): 3820-3829
Human HCF-1 is a large, highly conserved, and abundant nuclear protein that plays an important but unknown role in cell proliferation. It also plays a role in activation of herpes simplex virus immediate-early gene transcription by the viral regulatory protein VP16. A single proline-to-serine substitution in the HCF-1 VP16 interaction domain causes a temperature-induced arrest of cell proliferation in hamster tsBN67 cells and prevents transcriptional activation by VP16. We show here that HCF-1 is naturally bound to chromatin in uninfected cells through its VP16 interaction domain. HCF-1 is chromatin bound in tsBN67 cells at permissive temperature but dissociates from chromatin before tsBN67 cells stop proliferating at the nonpermissive temperature, suggesting that loss of HCF-1 chromatin association is the primary cause of the temperature-induced tsBN67 cell proliferation arrest. We propose that the role of HCF-1 in cell proliferation is to regulate gene transcription by associating with a multiplicity of DNA-bound transcription factors through its VP16 interaction domain.
View details for Web of Science ID 000168706600019
View details for PubMedID 11340173
Developmental and cell-cycle regulation of Caenorhabditis elegans HCF phosphorylation
2001; 40 (19): 5786-5794
HCF-1 is a mammalian protein required for cell proliferation. It is also involved in transcriptional activation of herpes-simplex-virus immediate-early gene transcription in association with the viral transactivator VP16. HCF-1 and a related protein called HCF-2 possess a homologue in Caenorhabditis elegans that can associate with and activate VP16. Here, we demonstrate developmental regulation of C. elegans HCF (CeHCF) phosphorylation: a hyperphosphorylated form of CeHCF is present in embryos, whereas a hypophosphorylated form is present in L1 larvae. The phosphorylation patterns of endogenous CeHCF in worms and ectopically synthesized CeHCF in mammalian cells are remarkably similar, suggesting that the way CeHCF can be recognized by kinases is conserved in animals. Phosphorylation-site mapping of endogenous CeHCF, however, revealed that phosphorylation occurs at four clustered sites in the region of the protein that is not highly conserved among HCF proteins and is not required for VP16-induced complex formation. Indeed, phosphorylation of either CeHCF or human HCF-1 appears to be dispensable for association with VP16. All four CeHCF phosphorylation sites match the consensus recognition site for the cell-cycle kinases CDC2 and CDK2. Consistent with this similarity and with the developmental phosphorylation of CeHCF in C. elegans embryos, CeHCF phosphorylation is cell-cycle-regulated in mammalian cells.
View details for Web of Science ID 000168635900023
View details for PubMedID 11341844