Dr. Khavari only sees U.S. veteran patients at the VA Palo Alto Healthcare System
- Cancer > Cutaneous (Dermatologic) Oncology
- General Dermatology
Chair Department of Dermatology, Stanford University School of Medicine (2010 - Present)
Co-Director, Stanford Program in Epithelial Biology (1999 - Present)
Fellowship:Stanford University School of Medicine Registrar (1994) CA
Residency:Stanford University School of Medicine Registrar (1991) CA
Medical Education:Yale School Of Medicine Office of Student Affairs (1988) CT
Internship:Yale-New Haven Hospital (1989) CT
Board Certification: Dermatology, American Board of Dermatology (1992)
Residency:Yale - New Haven Hospital (1990) CT
Current Research and Scholarly Interests
Our experimental focus is on the mammalian setting, including mouse genetics, human genetics, single cell studies, and new human tissue platforms. The latter encompass human skin regenerated on immune deficient mice as well as organotypic constructs with epithelial and stromal cells embedded within architecturally faithful mesenchyma in vitro. These new models, which we term Multi-Functional Human Tissue Genetics, allow up to 10 alleles or more to be altered simultaneously, permitting genetic experiments with an unprecedented degree of rapidity and complexity.
Stem cell biology and differentiation
In stratified epithelia proliferative basal cells adherent to the underlying basement membrane undergo cell cycle arrest then outward migration and terminal differentiation. This process is mediated by 2 mutually exclusive programs of gene expression: 1) an undifferentiated program supporting proliferation by stem cells within the basal layer and 2) a differentiation program instructing growth arrest and differentiation-associated programmed cell death in suprabasal layers. The control of this transition from epithelial stem cell to differentiated corneocyte, which is abnormal in epidermal cancers, is not well understood. We are currently pursuing studies of the dominant signaling and gene regulatory networks that control this process, including the Ras/MAPK cascade, which is required for stem cell-mediated self-renewal and the p53 transcription factor family member, p63, which is required for epidermal differentiation.
Epigenetic regulation by histone modifying proteins and noncoding RNA
In addition to classical gene regulatory networks noted above, we have recently identified a central role for additional biologic mechanisms, namely gene regulation by chromatin regulators and by noncoding RNAs. Epigenetic control of gene expression lasts through multiple cell divisions without alterations in primary DNA sequence and can occur via mechanisms that include histone modification and DNA methylation. Noncoding RNA sequences can regulate gene expression via interactions with epigenetic and other control mechanisms. The function of histone modifying epigenetic regulators and noncoding RNA as central mediators of epithelial stem cell renewal and differentiation represent major emerging areas of study in the lab.
Skin malignancies, including epidermal squamous cell carcinoma (SCC), alone account for nearly as many cancers as all other tissues combined. Progress in understanding epithelial carcinogenesis has been hindered in the past by a lack of models that faithfully recapitulate the 3-dimensional architecture of tumor-stroma co-evolution. To address this and to also study the oncogenic potential of unregulated function of dominant regulators of epithelial homeostasis noted above, we developed Multi-Functional Human Tissue Genetics noted above which, when combined with skin tissue regeneration on immune deficient mice, has permitted the molecular reconstruction of events sufficient to trigger human cancer. These models are being used to systematically elucidate proteins required for cutaneous carcinogenesis and to test their potential role as therapeutic targets.
Epithelial tissues in general and skin in particular offer an attractive site for development of new approaches in molecular therapeutics. A family of human genetic skin diseases is characterized by defective epithelial gene expression. Among the most severe of these are subtypes of epidermolysis bullosa (EB) and lamellar ichthyosis (LI). We have developed approaches for high efficiency gene transfer to EB and LI patient skin tissue that are corrective at biochemical, histologic, clinical and functional levels. In addition to EB subtypes and LI, similar corrective efforts have also been undertaken with a number of other genetic skin disorders.
Characteristics of Patients With Recessive Dystrophic Epidermolysis Bullosa
Recessive dystrophic epidermolysis bullosa (RDEB) is a disease caused by genetic mutations in the gene for type VII collagen. Patients with RDEB develop large, severely painful blisters and open wounds from minor trauma to their skin. We are screening subjects with RDEB to evaluate characteristics of the subjects and their cells in order to develop new strategies of therapy and determine whether subjects could be candidates for treatment studies.
Analysis of Cutaneous and Hematologic Disorders by High-Throughput Nucleic Acid Sequencing
The goal of this study is to identify genetic changes associated with the initiation, progression, and treatment response of response of cutaneous and hematologic disorders using recently developed high-throughput sequencing technologies. The improved understanding of the genetic changes associated with cutaneous and hematologic disorders may lead to improved diagnostic, prognostic and therapeutic options for these disorders.
Stanford is currently not accepting patients for this trial. For more information, please contact Alexander Ungewickell, 650-723-6661.
Characteristics of Adult Patients With Recessive Dystrophic Epidermolysis Bullosa
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited blistering disease caused by the absence of type VII collagen. Patients with RDEB develop large, severely painful blisters and open wounds from minor trauma to their skin. We are screening RDEB subjects to determine additional characteristics of patients who survive to adulthood.
Stanford is currently not accepting patients for this trial. For more information, please contact Emily Gorell, MS, 650-721-7166.
Gene Transfer for Recessive Dystrophic Epidermolysis Bullosa
This trial will create a skin graft, which the investigators call "LEAES," using the patient's own skin cells that have been genetically engineered in the lab to express a missing protein called type VII collagen. The corrected cells will be transplanted back to the patient.
Stanford is currently not accepting patients for this trial. For more information, please contact Yana Dutt-Singkh, 650-721-7166.
Pilot Trial to Evaluate the Effect of Vitamin D on Melanocyte Biomarkers
The purpose of this study is to determine the signaling pathways and changes in gene expression in melanocytes of subjects with a history of non-melanoma skin cancer who are exposed to oral vitamin D. If vitamin D is found to inhibit a signaling pathway involved in the development of melanoma such as BRAF, a protein involved in cell proliferation, then oral vitamin D could be explored further as a chemoprevention for melanoma skin cancer.
Stanford is currently not accepting patients for this trial. For more information, please contact Irene Bailey, 650-498-7061.
- Cellular and Clinical Aspects of Cancer
CBIO 242 (Spr)
Independent Studies (14)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Dermatology
DERM 299 (Aut, Win, Spr, Sum)
- Directed Reading in Stem Cell Biology and Regenerative Medicine
STEMREM 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Dermatology
DERM 280 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
DERM 399 (Aut, Win, Spr, Sum)
- Graduate Research
STEMREM 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
DERM 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
STEMREM 370 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Sum)
- Out-of-Department Graduate Research
BIO 300X (Sum)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Undergraduate Research
DERM 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
STEMREM 199 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
- Prior Year Courses
Postdoctoral Faculty Sponsor
Ibilola Adekunle, Meghan Halley, Andrew Ji, Namyoung Jung, Jordan Meyers, Smarajit Mondal, Douglas Porter, David Reynolds, Yuning Wei
Doctoral Dissertation Advisor (AC)
Robin Meyers, Xue Yang
Postdoctoral Research Mentor
Ibilola Adekunle, Meghan Halley, Andrew Ji, Namyoung Jung, Muthukumar Ramanathan, Rose Tran
Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks.
Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in 4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human Blymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFsuncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.
View details for DOI 10.1016/j.cell.2018.11.022
View details for PubMedID 30580963
RNA-protein interaction detection in living cells.
RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min.
View details for DOI 10.1038/nmeth.4601
View details for PubMedID 29400715
Lineage-specific dynamic and pre-established enhancer-promoter contacts cooperate in terminal differentiation.
2017; 49 (10): 1522–28
Chromosome conformation is an important feature of metazoan gene regulation; however, enhancer-promoter contact remodeling during cellular differentiation remains poorly understood. To address this, genome-wide promoter capture Hi-C (CHi-C) was performed during epidermal differentiation. Two classes of enhancer-promoter contacts associated with differentiation-induced genes were identified. The first class ('gained') increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class ('stable') were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation. Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation.
View details for DOI 10.1038/ng.3935
View details for PubMedID 28805829
CSNK1a1 Regulates PRMT1 to Maintain the Progenitor State in Self-Renewing Somatic Tissue.
2017; 43 (2): 227–39.e5
Somatic progenitors sustain tissue self-renewal while suppressing premature differentiation. Protein arginine methyltransferases (PRMTs) affect many processes; however, their role in progenitor function is incompletely understood. PRMT1 was found to be the most highly expressed PRMT in epidermal progenitors and the most downregulated PRMT during differentiation. In targeted mouse knockouts and in long-term regenerated human mosaic epidermis in vivo, epidermal PRMT1 loss abolished progenitor self-renewal and led to premature differentiation. Mass spectrometry of the PRMT1 protein interactome identified the CSNK1a1 kinase, which also proved essential for progenitor maintenance. CSNK1a1 directly bound and phosphorylated PRMT1 to control its genomic targeting to PRMT1-sustained proliferation genes as well as PRMT1-suppressed differentiation genes. Among the latter were GRHL3, whose derepression was required for the premature differentiation seen with PRMT1 and CSNK1a1 loss. Maintenance of the progenitors thus requires cooperation by PRMT1 and CSNK1a1 to sustain proliferation gene expression and suppress premature differentiation driven by GRHL3.
View details for DOI 10.1016/j.devcel.2017.08.021
View details for PubMedID 28943242
View details for PubMedCentralID PMC5659279
irCLIP platform for efficient characterization of protein-RNA interactions
2016; 13 (6): 489-?
The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein-RNA interactions using far less material than standard protocols.
View details for DOI 10.1038/NMETH.3840
View details for Web of Science ID 000377480100014
View details for PubMedID 27111506
The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer.
2016; 48 (1): 53-58
Small nucleolar RNAs (snoRNAs) are conserved noncoding RNAs best studied as ribonucleoprotein (RNP) guides in RNA modification. To explore their role in cancer, we compared 5,473 tumor-normal genome pairs to identify snoRNAs with frequent copy number loss. The SNORD50A-SNORD50B snoRNA locus was deleted in 10-40% of 12 common cancers, where its loss was associated with reduced survival. A human protein microarray screen identified direct SNORD50A and SNORD50B RNA binding to K-Ras. Loss of SNORD50A and SNORD50B increased the amount of GTP-bound, active K-Ras and hyperactivated Ras-ERK1/ERK2 signaling. Loss of these snoRNAs also increased binding by farnesyltransferase to K-Ras and increased K-Ras prenylation, suggesting that KRAS mutation might synergize with SNORD50A and SNORD50B loss in cancer. In agreement with this hypothesis, CRISPR-mediated deletion of SNORD50A and SNORD50B in KRAS-mutant tumor cells enhanced tumorigenesis, and SNORD50A and SNORD50B deletion and oncogenic KRAS mutation co-occurred significantly in multiple human tumor types. SNORD50A and SNORD50B snoRNAs thus directly bind and inhibit K-Ras and are recurrently deleted in human cancer.
View details for DOI 10.1038/ng.3452
View details for PubMedID 26595770
View details for PubMedCentralID PMC5324971
Network Analysis Identifies Mitochondria! Regulation of Epidermal Differentiation by MPZL3 and FDXR
2015; 35 (4): 444-457
Current gene expression network approaches commonly focus on transcription factors (TFs), biasing network-based discovery efforts away from potentially important non-TF proteins. We developed proximity analysis, a network reconstruction method that uses topological constraints of scale-free, small-world biological networks to reconstruct relationships in eukaryotic systems, independent of subcellular localization. Proximity analysis identified MPZL3 as a highly connected hub that is strongly induced during epidermal differentiation. MPZL3 was essential for normal differentiation, acting downstream of p63, ZNF750, KLF4, and RCOR1, each of which bound near the MPZL3 gene and controlled its expression. MPZL3 protein localized to mitochondria, where it interacted with FDXR, which was itself also found to be essential for differentiation. Together, MPZL3 and FDXR increased reactive oxygen species (ROS) to drive epidermal differentiation. ROS-induced differentiation is dependent upon promotion of FDXR enzymatic activity by MPZL3. ROS induction by the MPZL3 and FDXR mitochondrial proteins is therefore essential for epidermal differentiation.
View details for DOI 10.1016/j.devcel.2015.10.023
View details for Web of Science ID 000365099300009
View details for PubMedID 26609959
Genomic analysis of mycosis fungoides and Sézary syndrome identifies recurrent alterations in TNFR2.
2015; 47 (9): 1056-1060
Mycosis fungoides and Sézary syndrome comprise the majority of cutaneous T cell lymphomas (CTCLs), disorders notable for their clinical heterogeneity that can present in skin or peripheral blood. Effective treatment options for CTCL are limited, and the genetic basis of these T cell lymphomas remains incompletely characterized. Here we report recurrent point mutations and genomic gains of TNFRSF1B, encoding the tumor necrosis factor receptor TNFR2, in 18% of patients with mycosis fungoides and Sézary syndrome. Expression of the recurrent TNFR2 Thr377Ile mutant in T cells leads to enhanced non-canonical NF-κB signaling that is sensitive to the proteasome inhibitor bortezomib. Using an integrative genomic approach, we additionally discovered a recurrent CTLA4-CD28 fusion, as well as mutations in downstream signaling mediators of these receptors.
View details for DOI 10.1038/ng.3370
View details for PubMedID 26258847
A LncRNA-MAF:MAFB Transcription Factor Network Regulates Epidermal Differentiation
2015; 32 (6): 693-706
Progenitor differentiation requires remodeling of genomic expression; however, in many tissues, such as epidermis, the spectrum of remodeled genes and the transcription factors (TFs) that control them are not fully defined. We performed kinetic transcriptome analysis during regeneration of differentiated epidermis and identified gene sets enriched in progenitors (594 genes), in early (159 genes), and in late differentiation (387 genes). Module mapping of 1,046 TFs identified MAF and MAFB as necessary and sufficient for progenitor differentiation. MAF:MAFB regulated 393 genes altered in this setting. Integrative analysis identified ANCR and TINCR lncRNAs as essential upstream MAF:MAFB regulators. ChIP-seq analysis demonstrated MAF:MAFB binding to known epidermal differentiation TF genes whose expression they controlled, including GRHL3, ZNF750, KLF4, and PRDM1. Each of these TFs rescued expression of specific MAF:MAFB target gene subsets in the setting of MAF:MAFB loss, indicating they act downstream of MAF:MAFB. A lncRNA-TF network is thus essential for epidermal differentiation.
View details for DOI 10.1016/j.devcel.2015.01.028
View details for Web of Science ID 000351841900008
View details for PubMedID 25805135
- Advances in skin grafting and treatment of cutaneous wounds SCIENCE 2014; 346 (6212): 941-945
Recurrent point mutations in the kinetochore gene KNSTRN in cutaneous squamous cell carcinoma
2014; 46 (10): 1060-1062
Here we report the discovery of recurrent mutations concentrated at an ultraviolet signature hotspot in KNSTRN, which encodes a kinetochore protein, in 19% of cutaneous squamous cell carcinomas (SCCs). Cancer-associated KNSTRN mutations, most notably those encoding p.Ser24Phe, disrupt chromatid cohesion in normal cells, occur in SCC precursors, correlate with increased aneuploidy in primary tumors and enhance tumorigenesis in vivo. These findings suggest a role for KNSTRN mutagenesis in SCC development.
View details for DOI 10.1038/ng.3091
View details for Web of Science ID 000342554100007
View details for PubMedCentralID PMC4324615
IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors.
2013; 19 (5): 626-630
Upregulation of the ERK1 and ERK2 (ERK1/2) MAP kinase (MAPK) cascade occurs in >30% of cancers, often through mutational activation of receptor tyrosine kinases or other upstream genes, including KRAS and BRAF. Efforts to target endogenous MAPKs are challenged by the fact that these kinases are required for viability in mammals. Additionally, the effectiveness of new inhibitors of mutant BRAF has been diminished by acquired tumor resistance through selection for BRAF-independent mechanisms of ERK1/2 induction. Furthermore, recently identified ERK1/2-inducing mutations in MEK1 and MEK2 (MEK1/2) MAPK genes in melanoma confer resistance to emerging therapeutic MEK inhibitors, underscoring the challenges facing direct kinase inhibition in cancer. MAPK scaffolds, such as IQ motif-containing GTPase activating protein 1 (IQGAP1), assemble pathway kinases to affect signal transmission, and disrupting scaffold function therefore offers an orthogonal approach to MAPK cascade inhibition. Consistent with this, we found a requirement for IQGAP1 in RAS-driven tumorigenesis in mouse and human tissue. In addition, the ERK1/2-binding IQGAP1 WW domain peptide disrupted IQGAP1-ERK1/2 interactions, inhibited RAS- and RAF-driven tumorigenesis, bypassed acquired resistance to the BRAF inhibitor vemurafenib (PLX-4032) and acted as a systemically deliverable therapeutic to significantly increase the lifespan of tumor-bearing mice. Scaffold-kinase interaction blockade acts by a mechanism distinct from direct kinase inhibition and may be a strategy to target overactive oncogenic kinase cascades in cancer.
View details for DOI 10.1038/nm.3165
View details for PubMedID 23603816
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
Control of somatic tissue differentiation by the long non-coding RNA TINCR.
2013; 493 (7431): 231-235
Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high messenger RNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a range of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25-nucleotide 'TINCR box' motif that is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyse TINCR binding capacity to approximately 9,400 human recombinant proteins revealed direct binding of TINCR RNA to the staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay, however, did not have differentiation effects. Instead, the TINCR-STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression.
View details for DOI 10.1038/nature11661
View details for PubMedID 23201690
View details for PubMedCentralID PMC3674581
ZNF750 Is a p63 Target Gene that Induces KLF4 to Drive Terminal Epidermal Differentiation
2012; 22 (3): 669-677
Disrupted epidermal differentiation characterizes numerous diseases that impact >25% of the population. In a search for dominant mediators of differentiation, we defined a requirement for ZNF750 in terminal epidermal differentiation. ZNF750 controlled genes mutated in numerous human skin diseases, including FLG, LOR, LCE3B, ALOXE3, and SPINK5. ZNF750 induced progenitor differentiation via an evolutionarily conserved C2H2 zinc finger motif. The epidermal master regulator, p63, bound the ZNF750 promoter and was necessary for its induction. ZNF750 restored differentiation to p63-deficient tissue, suggesting that it acts downstream of p63. A search for functionally important ZNF750 targets via analysis of ZNF750-regulated genes identified KLF4, a transcription factor that activates late epidermal differentiation. ZNF750 binds to KLF4 at multiple sites flanking the transcriptional start site and controls its expression. ZNF750 thus directly links a tissue-specifying factor, p63, to an effector of terminal differentiation, KLF4, and represents a potential future target for disorders of this process.
View details for DOI 10.1016/j.devcel.2011.12.001
View details for Web of Science ID 000301701600020
View details for PubMedID 22364861
View details for PubMedCentralID PMC3306457
DNMT1 maintains progenitor function in self-renewing somatic tissue
2010; 463 (7280): 563-U189
Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, UHRF1 (refs 9, 10), a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A and B, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.
View details for DOI 10.1038/nature08683
View details for Web of Science ID 000273981100056
View details for PubMedID 20081831
Methods to study RNA-protein interactions.
2019; 16 (3): 225–34
Noncoding RNA sequences, including long noncoding RNAs, small nucleolar RNAs, and untranslated mRNA regions, accomplish many of their diverse functions through direct interactions with RNA-binding proteins (RBPs). Recent efforts have identified hundreds of new RBPs that lack known RNA-binding domains, thus underscoring the complexity and diversity of RNA-protein complexes. Recent progress has expanded the number of methods for studying RNA-protein interactions in two general categories: approaches that characterize proteins bound to an RNA of interest (RNA-centric), and those that examine RNAs bound to a protein of interest (protein-centric). Each method has unique strengths and limitations, which makes it important to select optimal approaches for the biological question being addressed. Here we review methods for the study of RNA-protein interactions, with a focus on their suitability for specific applications.
View details for DOI 10.1038/s41592-019-0330-1
View details for PubMedID 30804549
The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2.
Proximity-dependent biotin labeling (BioID) may identify new targets for cancers driven by difficult-to-drug oncogenes such as Ras. Therefore, BioID was used with wild-type (WT) and oncogenic mutant (MT) H-,K-, and N-Ras, identifying known interactors, including Raf and PI3K, as well as a common set of 130 novel proteins proximal to all Ras isoforms. A CRISPR screen of these proteins for Ras dependence identified mTOR, which was also found proximal to MT Ras in human tumors. Oncogenic Ras directly bound two mTOR complex 2 (mTORC2) components, mTOR and MAPKAP1, to promote mTORC2 kinase activity at the plasma membrane. mTORC2 enabled the Ras pro-proliferative cell cycletranscriptional program, and perturbing the Ras-mTORC2 interaction impaired Ras-dependent neoplasia invivo. Combining proximity-dependent proteomics with CRISPR screening identified a new set of functional Ras-associated proteins, defined mTORC2 as a new direct Ras effector, and offers a strategy for finding new proteins that cooperate with dominant oncogenes.
View details for DOI 10.1016/j.molcel.2018.12.001
View details for PubMedID 30639242
- Cancer-Associated Long Noncoding RNA SMRT-2 Controls Epidermal Differentiation JOURNAL OF INVESTIGATIVE DERMATOLOGY 2018; 138 (6): 1445–49
Single-cell RNA-sequencing reveals SCC intratumoral heterogeneity
ELSEVIER SCIENCE INC. 2018: S31
View details for Web of Science ID 000431188500182
Dynamic morphogen-p63 chromatin interactions that guide epigenetic changes and p63 activity in surface ectoderm commitment
ELSEVIER SCIENCE INC. 2018: S243
View details for Web of Science ID 000431498600698
KRAS regulation by small non-coding RNAs and SNARE proteins
ELSEVIER SCIENCE INC. 2018: S27
View details for Web of Science ID 000431188500162
Metabolomic analysis reveals an essential role for glucose in epidermal differentiation
ELSEVIER SCIENCE INC. 2018: S123
View details for Web of Science ID 000431188500728
Small non-coding RNAs control the MAPK/ERK pathway
ELSEVIER SCIENCE INC. 2018: S27
View details for Web of Science ID 000431188500159
Decoding regulatory sequence across skin differentiation with deep learning
ELSEVIER SCIENCE INC. 2018: S135
View details for Web of Science ID 000431498600057
The functions and unique features of long intergenic non-coding RNA.
Nature reviews. Molecular cell biology
2018; 19 (3): 143–57
Long intergenic non-coding RNA (lincRNA) genes have diverse features that distinguish them from mRNA-encoding genes and exercise functions such as remodelling chromatin and genome architecture, RNA stabilization and transcription regulation, including enhancer-associated activity. Some genes currently annotated as encoding lincRNAs include small open reading frames (smORFs) and encode functional peptides and thus may be more properly classified as coding RNAs. lincRNAs may broadly serve to fine-tune the expression of neighbouring genes with remarkable tissue specificity through a diversity of mechanisms, highlighting our rapidly evolving understanding of the non-coding genome.
View details for DOI 10.1038/nrm.2017.104
View details for PubMedID 29138516
View details for PubMedCentralID PMC5889127
Research Techniques Made Simple: Emerging Methods to Elucidate Protein Interactions through Spatial Proximity
JOURNAL OF INVESTIGATIVE DERMATOLOGY
2017; 137 (12): E197–E203
Interactions between proteins are essential for fundamental cellular processes, and the diversity of such interactions enables the vast variety of functions essential for life. A persistent goal in biological research is to develop assays that can faithfully capture different types of protein interactions to allow their study. A major step forward in this direction came with a family of methods that delineates spatial proximity of proteins as an indirect measure of protein-protein interaction. A variety of enzyme- and DNA ligation-based methods measure protein co-localization in space, capturing novel interactions that were previously too transient or low affinity to be identified. Here we review some of the methods that have been successfully used to measure spatially proximal protein-protein interactions.
View details for DOI 10.1016/j.jid.2017.09.028
View details for Web of Science ID 000415797600001
View details for PubMedID 29169465
Safety and Wound Outcomes Following Genetically Corrected Autologous Epidermal Grafts in Patients With Recessive Dystrophic Epidermolysis Bullosa.
2016; 316 (17): 1808-1817
Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating, often fatal, inherited blistering disorder caused by mutations in the COL7A1 gene encoding type VII collagen. Support and palliation are the only current therapies.To evaluate the safety and wound outcomes following genetically corrected autologous epidermal grafts in patients with RDEB.Single-center phase 1 clinical trial conducted in the United States of 4 patients with severe RDEB with a measured area of wounds suitable for grafting of at least 100 cm2. Patients with undetectable type VII collagen keratinocyte expression were excluded.Autologous keratinocytes isolated from biopsy samples collected from 4 patients with RDEB were transduced with good manufacturing practice-grade retrovirus carrying full-length human COL7A1 and assembled into epidermal sheet grafts. Type VII collagen gene-corrected grafts (approximately 35 cm2) were transplanted onto 6 wounds in each of the patients (n = 24 grafts).The primary safety outcomes were recombination competent retrovirus, cancer, and autoimmune reaction. Molecular correction was assessed as type VII collagen expression measured by immunofluorescence and immunoelectron microscopy. Wound healing was assessed using serial photographs taken at 3, 6, and 12 months after grafting.The 4 patients (mean age, 23 years [range, 18-32 years]) were all male with an estimated body surface area affected with RDEB of 4% to 30%. All 24 grafts were well tolerated without serious adverse events. Type VII collagen expression at the dermal-epidermal junction was demonstrated on the graft sites by immunofluorescence microscopy in 9 of 10 biopsy samples (90%) at 3 months, in 8 of 12 samples (66%) at 6 months, and in 5 of 12 samples (42%) at 12 months, including correct type VII collagen localization to anchoring fibrils. Wounds with recombinant type VII collagen graft sites displayed 75% or greater healing at 3 months (21 intact graft sites of 24 wound sites; 87%), 6 months (16/24; 67%), and 12 months (12/24; 50%) compared with baseline wound sites.In this preliminary study of 4 patients with RDEB, there was wound healing in some type VII collagen gene-corrected grafts, but the response was variable among patients and among grafted sites and generally declined over 1 year. Long-term follow-up is necessary for these patients, and controlled trials are needed with a broader range of patients to better understand the potential long-term efficacy of genetically corrected autologous epidermal grafts.clinicaltrials.gov Identifier: NCT01263379.
View details for DOI 10.1001/jama.2016.15588
View details for PubMedID 27802546
HiChIP: efficient and sensitive analysis of protein-directed genome architecture.
2016; 13 (11): 919-922
Genome conformation is central to gene control but challenging to interrogate. Here we present HiChIP, a protein-centric chromatin conformation method. HiChIP improves the yield of conformation-informative reads by over 10-fold and lowers the input requirement over 100-fold relative to that of ChIA-PET. HiChIP of cohesin reveals multiscale genome architecture with greater signal-to-background ratios than those of in situ Hi-C.
View details for DOI 10.1038/nmeth.3999
View details for PubMedID 27643841
RAC1 activation drives pathologic interactions between the epidermis and immune cells
JOURNAL OF CLINICAL INVESTIGATION
2016; 126 (7): 2661-2677
Interactions between the epidermis and the immune system govern epidermal tissue homeostasis. These epidermis-immune interactions are altered in the inflammatory disease psoriasis; however, the pathways that underlie this aberrant immune response are not well understood. Here, we determined that Ras-related C3 botulinum toxin substrate 1 (RAC1) is a key mediator of epidermal dysfunction. RAC1 activation was consistently elevated in psoriatic epidermis and primary psoriatic human keratinocytes (PHKCs) exposed to psoriasis-related stimuli, but not in skin from patients with basal or squamous cell carcinoma. Expression of a constitutively active form of RAC1 (RACV12) in mice resulted in the development of lesions similar to those of human psoriasis that required the presence of an intact immune system. RAC1V12-expressing mice and human psoriatic skin showed similar RAC1-dependent signaling as well as transcriptional overlap of differentially expressed epidermal and immune pathways. Coculture of PHKCs with immunocytes resulted in the upregulation of RAC1-dependent proinflammatory cytokines, an effect that was reproduced by overexpressing RAC1 in normal human keratinocytes. In keratinocytes, modulating RAC1 activity altered differentiation, proliferation, and inflammatory pathways, including STAT3, NFκB, and zinc finger protein 750 (ZNF750). Finally, RAC1 inhibition in xenografts composed of human PHKCs and immunocytes abolished psoriasiform hyperplasia and inflammation in vivo. These studies implicate RAC1 as a potential therapeutic target for psoriasis and as a key orchestrator of pathologic epidermis-immune interactions.
View details for DOI 10.1172/JCI85738
View details for Web of Science ID 000379094800028
View details for PubMedID 27294528
View details for PubMedCentralID PMC4922704
7SK-BAF axis controls pervasive transcription at enhancers.
Nature structural & molecular biology
2016; 23 (3): 231-238
RNA functions at enhancers remain mysterious. Here we show that the 7SK small nuclear RNA (snRNA) inhibits enhancer transcription by modulating nucleosome position. 7SK occupies enhancers and super enhancers genome wide in mouse and human cells, and it is required to limit enhancer-RNA initiation and synthesis in a manner distinct from promoter pausing. Clustered elements at super enhancers uniquely require 7SK to prevent convergent transcription and DNA-damage signaling. 7SK physically interacts with the BAF chromatin-remodeling complex, recruits BAF to enhancers and inhibits enhancer transcription by modulating chromatin structure. In turn, 7SK occupancy at enhancers coincides with that of Brd4 and is exquisitely sensitive to the bromodomain inhibitor JQ1. Thus, 7SK uses distinct mechanisms to counteract the diverse consequences of pervasive transcription that distinguish super enhancers, enhancers and promoters.
View details for DOI 10.1038/nsmb.3176
View details for PubMedID 26878240
View details for PubMedCentralID PMC4982704
CALML5 is a ZNF750-and TINCR-induced protein that binds stratifin to regulate epidermal differentiation
GENES & DEVELOPMENT
2015; 29 (21): 2225-2230
Outward migration of epidermal progenitors occurs with induction of hundreds of differentiation genes, but the identities of all regulators required for this process are unknown. We used laser capture microdissection followed by RNA sequencing to identify calmodulin-like 5 (CALML5) as the most enriched gene in differentiating outer epidermis. CALML5 mRNA was up-regulated by the ZNF750 transcription factor and then stabilized by the long noncoding RNA TINCR. CALML5 knockout impaired differentiation, abolished keratohyalin granules, and disrupted epidermal barrier function. Mass spectrometry identified SFN (stratifin/14-3-3σ) as a CALML5-binding protein. CALML5 interacts with SFN in suprabasal epidermis, cocontrols 13% of late differentiation genes, and modulates interaction of SFN to some of its binding partners. A ZNF750-TINCR-CALML5-SFN network is thus essential for epidermal differentiation.
View details for DOI 10.1101/gad.267708.115
View details for Web of Science ID 000364853500002
View details for PubMedID 26545810
View details for PubMedCentralID PMC4647556
Dissecting noncoding and pathogen RNA-protein interactomes
RNA-A PUBLICATION OF THE RNA SOCIETY
2015; 21 (1): 135-143
RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.
View details for DOI 10.1261/rna.047803.114
View details for Web of Science ID 000346896200011
View details for PubMedID 25411354
View details for PubMedCentralID PMC4274633
A novel ATAC-seq approach reveals lineage-specific reinforcement of the open chromatin landscape via cooperation between BAF and p63.
2015; 16 (1): 284-?
Open chromatin regions are correlated with active regulatory elements in development and are dysregulated in diseases. The BAF (SWI/SNF) complex is essential for development, and has been demonstrated to remodel reconstituted chromatin in vitro and to control the accessibility of a few individual regions in vivo. However, it remains unclear where and how BAF controls the open chromatin landscape to regulate developmental processes, such as human epidermal differentiation.Using a novel "on-plate" ATAC-sequencing approach for profiling open chromatin landscapes with a low number of adherent cells, we demonstrate that the BAF complex is essential for maintaining 11.6 % of open chromatin regions in epidermal differentiation. These BAF-dependent open chromatin regions are highly cell-type-specific and are strongly enriched for binding sites for p63, a master epidermal transcription factor. The DNA sequences of p63 binding sites intrinsically favor nucleosome formation and are inaccessible in other cell types without p63 to prevent ectopic activation. In epidermal cells, BAF and p63 mutually recruit each other to maintain 14,853 open chromatin regions. We further demonstrate that BAF and p63 cooperatively position nucleosomes away from p63 binding sites and recruit transcriptional machinery to control tissue differentiation.BAF displays high specificity in controlling the open chromatin landscape during epidermal differentiation by cooperating with the master transcription factor p63 to maintain lineage-specific open chromatin regions.
View details for DOI 10.1186/s13059-015-0840-9
View details for PubMedID 26683334
View details for PubMedCentralID PMC4699366
Recurrent point mutations in the kinetochore gene KNSTRN in cutaneous squamous cell carcinoma.
2014; 46 (10): 1060-1062
Here we report the discovery of recurrent mutations concentrated at an ultraviolet signature hotspot in KNSTRN, which encodes a kinetochore protein, in 19% of cutaneous squamous cell carcinomas (SCCs). Cancer-associated KNSTRN mutations, most notably those encoding p.Ser24Phe, disrupt chromatid cohesion in normal cells, occur in SCC precursors, correlate with increased aneuploidy in primary tumors and enhance tumorigenesis in vivo. These findings suggest a role for KNSTRN mutagenesis in SCC development.
View details for DOI 10.1038/ng.3091
View details for PubMedID 25194279
- ZNF750 interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 chromatin regulators to repress epidermal progenitor genes and induce differentiation genes GENES & DEVELOPMENT 2014; 28 (18): 2013-2026
ZNF750 interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 chromatin regulators to repress epidermal progenitor genes and induce differentiation genes.
Genes & development
2014; 28 (18): 2013-2026
ZNF750 controls epithelial homeostasis by inhibiting progenitor genes while inducing differentiation genes, a role underscored by pathogenic ZNF750 mutations in cancer and psoriasis. How ZNF750 accomplishes these dual gene regulatory impacts is unknown. Here, we characterized ZNF750 as a transcription factor that binds both the progenitor and differentiation genes that it controls at a CCNNAGGC DNA motif. ZNF750 interacts with the pluripotency transcription factor KLF4 and chromatin regulators RCOR1, KDM1A, and CTBP1/2 through conserved PLNLS sequences. ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) and gene depletion revealed that KLF4 colocalizes ∼10 base pairs from ZNF750 at differentiation target genes to facilitate their activation but is unnecessary for ZNF750-mediated progenitor gene repression. In contrast, KDM1A colocalizes with ZNF750 at progenitor genes and facilitates their repression but is unnecessary for ZNF750-driven differentiation. ZNF750 thus controls differentiation in concert with RCOR1 and CTBP1/2 by acting with either KDM1A to repress progenitor genes or KLF4 to induce differentiation genes.
View details for DOI 10.1101/gad.246579.114
View details for PubMedID 25228645
- Activating HRAS Mutation in Nevus Spilus. journal of investigative dermatology 2014; 134 (6): 1766-1768
Enhancer-targeted genome editing selectively blocks innate resistance to oncokinase inhibition
2014; 24 (5): 751-760
Thousands of putative enhancers are characterized in the human genome, yet few have been shown to have a functional role in cancer progression. Inhibiting oncokinases, such as EGFR, ALK, ERBB2, and BRAF, is a mainstay of current cancer therapy but is hindered by innate drug resistance mediated by up-regulation of the HGF receptor, MET. The mechanisms mediating such genomic responses to targeted therapy are unknown. Here, we identify lineage-specific enhancers at the MET locus for multiple common tumor types, including a melanoma lineage-specific enhancer 63 kb downstream from the MET TSS. This enhancer displays inducible chromatin looping with the MET promoter to up-regulate MET expression upon BRAF inhibition. Epigenomic analysis demonstrated that the melanocyte-specific transcription factor, MITF, mediates this enhancer function. Targeted genomic deletion (<7 bp) of the MITF motif within the MET enhancer suppressed inducible chromatin looping and innate drug resistance, while maintaining MITF-dependent, inhibitor-induced melanoma cell differentiation. Epigenomic analysis can thus guide functional disruption of regulatory DNA to decouple pro- and anti-oncogenic functions of a dominant transcription factor and block innate resistance to oncokinase therapy.
View details for DOI 10.1101/gr.166231.113
View details for Web of Science ID 000335365600005
View details for PubMedID 24443471
View details for PubMedCentralID PMC4009605
Quantitative analysis of mammalian translation initiation sites by FACS-seq.
Molecular systems biology
2014; 10: 748-?
An approach combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS-seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the -6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed by a single motif, were also important for modeling TIS efficiency. Our dataset combined with modeling allowed us to predict genome-wide translation initiation efficiency for all mRNA transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to identify candidate driver mutations consistent with known tumor expression patterns. Finally, we implemented a quantitative leaky scanning model to predict alternative initiation sites that produce truncated protein isoforms and compared predictions with ribosome footprint profiling data. The comprehensive analysis of the TIS sequence space enables quantitative predictions of translation initiation based on genome sequence.
View details for DOI 10.15252/msb.20145136
View details for PubMedID 25170020
Activating HRAS Mutation in Agminated Spitz Nevi Arising in a Nevus Spilus.
2013; 149 (9): 1077-1081
IMPORTANCE Spitz nevi are benign melanocytic proliferations that can sometimes be clinically and histopathologically difficult to distinguish from melanoma. Agminated Spitz nevi have been reported to arise spontaneously, in association with an underlying nevus spilus, or after radiation or chemotherapy. However, to our knowledge, the genetic mechanism for this eruption has not been described. OBSERVATIONS We report a case of agminated Spitz nevi arising in a nevus spilus and use exome sequencing to identify a clonal activating point mutation in HRAS (GenBank 3265) (c.37G→C) in the Spitz nevi and underlying nevus spilus. We also identify a secondary copy number increase involving HRAS on chromosome 11p, which occurs during the development of the Spitz nevi. CONCLUSIONS AND RELEVANCE Our results reveal an activating HRAS mutation in a nevus spilus that predisposes to the formation of Spitz nevi. In addition, we demonstrate a copy number increase in HRAS as a "second hit" during the formation of agminated Spitz nevi, which suggests that both multiple Spitz nevi and solitary Spitz nevi may arise through similar molecular pathways. In addition, we describe a unique investigative approach for the discovery of genetic alterations in Spitz nevi.
View details for DOI 10.1001/jamadermatol.2013.4745
View details for PubMedID 23884457
Genomic Profiling of a Human Organotypic Model of AEC Syndrome Reveals ZNF750 as an Essential Downstream Target of Mutant TP63
AMERICAN JOURNAL OF HUMAN GENETICS
2012; 91 (3): 435-443
The basis for impaired differentiation in TP63 mutant ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome is unknown. Human epidermis harboring AEC TP63 mutants recapitulated this impairment, along with downregulation of differentiation activators, including HOPX, GRHL3, KLF4, PRDM1, and ZNF750. Gene-set enrichment analysis indicated that disrupted expression of epidermal differentiation programs under the control of ZNF750 and KLF4 accounted for the majority of disrupted epidermal differentiation resulting from AEC mutant TP63. Chromatin immunoprecipitation (ChIP) analysis and ChIP-sequencing of TP63 binding in differentiated keratinocytes revealed ZNF750 as a direct target of wild-type and AEC mutant TP63. Restoring ZNF750 to AEC model tissue rescued activator expression and differentiation, indicating that AEC TP63-mediated ZNF750 inhibition contributes to differentiation defects in AEC. Incorporating disease-causing mutants into regenerated human tissue can thus dissect pathomechanisms and identify targets that reverse disease features.
View details for DOI 10.1016/j.ajhg.2012.07.007
View details for Web of Science ID 000308683100004
View details for PubMedCentralID PMC3511987
- Invasive three-dimensional organotypic neoplasia from multiple normal human epithelia Nature Medicine 2010; 16: 1450-1455
Modeling Inducible Human Tissue Neoplasia Identifies an Extracellular Matrix Interaction Network Involved in Cancer Progression
2009; 15 (6): 477-488
To elucidate mechanisms of cancer progression, we generated inducible human neoplasia in three-dimensionally intact epithelial tissue. Gene expression profiling of both epithelia and stroma at specific time points during tumor progression revealed sequential enrichment of genes mediating discrete biologic functions in each tissue compartment. A core cancer progression signature was distilled using the increased signaling specificity of downstream oncogene effectors and subjected to network modeling. Network topology predicted that tumor development depends on specific extracellular matrix-interacting network hubs. Blockade of one such hub, the beta1 integrin subunit, disrupted network gene expression and attenuated tumorigenesis in vivo. Thus, integrating network modeling and temporal gene expression analysis of inducible human neoplasia provides an approach to prioritize and characterize genes functioning in cancer progression.
View details for DOI 10.1016/j.ccr.2009.04.002
View details for Web of Science ID 000266686500006
View details for PubMedID 19477427
View details for PubMedCentralID PMC3050547
Use of human tissue to assess the oncogenic activity of melanoma-associated mutations
2005; 37 (7): 745-749
Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Induction of Ras and Raf can be caused by active N-Ras and B-Raf mutants as well as by gene amplification. Activation of PI3K pathway components occurs by PTEN loss and by AKT3 amplification. Melanomas also commonly show impairment of the p16(INK4A)-CDK4-Rb and ARF-HDM2-p53 tumor suppressor pathways. CDKN2A mutations can produce p16(INK4A) and ARF protein loss. Rb bypass can also occur through activating CDK4 mutations as well as by CDK4 amplification. In addition to ARF deletion, p53 pathway disruption can result from dominant negative TP53 mutations. TERT amplification also occurs in melanoma. The extent to which these mutations can induce human melanocytic neoplasia is unknown. Here we characterize pathways sufficient to generate human melanocytic neoplasia and show that genetically altered human tissue facilitates functional analysis of mutations observed in human tumors.
View details for DOI 10.1038/ng1586
View details for Web of Science ID 000230196400022
View details for PubMedID 15951821
Type VII collagen is required for Ras-driven human epidermal tumorigenesis
2005; 307 (5716): 1773-1776
Type VII collagen defects cause recessive dystrophic epidermolysis bullosa (RDEB), a blistering skin disorder often accompanied by epidermal cancers. To study the role of collagen VII in these cancers, we examined Ras-driven tumorigenesis in RDEB keratinocytes. Cells devoid of collagen VII did not form tumors in mice, whereas those retaining a specific collagen VII fragment (the amino-terminal noncollagenous domain NC1) were tumorigenic. Forced NC1 expression restored tumorigenicity to collagen VII-null epidermis in a non-cell-autonomous fashion. Fibronectin-like sequences within NC1 (FNC1) promoted tumor cell invasion in a laminin 5-dependent manner and were required for tumorigenesis. Tumor-stroma interactions mediated by collagen VII thus promote neoplasia, and retention of NC1 sequences in a subset of RDEB patients may contribute to their increased susceptibility to squamous cell carcinoma.
View details for DOI 10.1126/science.1106209
View details for Web of Science ID 000227883900044
View details for PubMedID 15774758
NF-kappa B blockade and oncogenic Ras trigger invasive human epidermal neoplasia
2003; 421 (6923): 639-643
The nuclear factor NF-kappaB and oncogenic Ras can alter proliferation in epidermis, the most common site of human cancer. These proteins are implicated in epidermal squamous cell carcinoma in mice, however, the potential effects of altering their function are uncertain. Whereas inhibition of NF-kappaB enhances apoptosis in certain tumours, blockade of NF-kappaB predisposes murine skin to squamous cell carcinoma. Because therapeutics inhibiting Ras and NF-kappaB pathways are being developed to treat human cancer, it is essential to assess the effects of altering these regulators. The medical relevance of murine studies is limited, however, by differences between mouse and human skin, and by the greater ease of transforming murine cells. Here we show that in normal human epidermal cells both NF-kappaB and oncogenic Ras trigger cell-cycle arrest. Growth arrest triggered by oncogenic Ras can be bypassed by IkappaBalpha-mediated blockade of NF-kappaB, generating malignant human epidermal tissue resembling squamous cell carcinoma. Human cell tumorigenesis is dependent on laminin 5 and alpha6beta4 integrin. Thus, IkappaBalpha circumvents restraints on growth promotion induced by oncogenic Ras and can act with Ras to induce invasive human tissue neoplasia.
View details for DOI 10.1038/nature01283
View details for Web of Science ID 000180803200044
View details for PubMedID 12571598
CDK4 coexpression with Ras generates malignant human epidermal tumorigenesis
2002; 8 (10): 1105-1114
Ras acts with other proteins to induce neoplasia. By itself, however, strong Ras signaling can suppress proliferation of normal cells. In primary epidermal cells, we found that oncogenic Ras transiently decreases cyclin-dependent kinase (CDK) 4 expression in association with cell cycle arrest in G1 phase. CDK4 co-expression circumvents Ras growth suppression and induces invasive human neoplasia resembling squamous cell carcinoma. Tumorigenesis is dependent on CDK4 kinase function, with cyclin D1 required but not sufficient for this process. In facilitating escape from G1 growth restraints, Ras and CDK4 alter the composition of cyclin D and cyclin E complexes and promote resistance to growth inhibition by INK4 cyclin-dependent kinase inhibitors. These data identify a new role for oncogenic Ras in CDK4 regulation and highlight the functional importance of CDK4 suppression in preventing uncontrolled growth.
View details for DOI 10.1038/nm779
View details for Web of Science ID 000178311000037
View details for PubMedID 12357246
Stable nonviral genetic correction of inherited human skin disease
2002; 8 (10): 1166-1170
Current gene-transfer technologies display limitations in achieving effective gene delivery. Among these limitations are difficulties in stably integrating large corrective sequences into the genomes of long-lived progenitor-cell populations. Current larger-capacity viral vectors suffer from biosafety concerns, whereas plasmid-based approaches have poor efficiency of stable gene transfer. These barriers hinder genetic correction of many severe inherited human diseases, such as the blistering skin disorder recessive dystrophic epidermolysis bullosa (RDEB), caused by mutations in the large COL7A1 gene. To circumvent these barriers, we used the phi C31 bacteriophage integrase, which stably integrates large DNA sequences containing a specific 285-base-pair attB sequence into genomic 'pseudo-attP sites'. phi C31 integrase-based gene transfer stably integrated the COL7A1 cDNA into genomes of primary epidermal progenitor cells from four unrelated RDEB patients. Skin regenerated using these cells displayed stable correction of hallmark RDEB disease features, including Type VII collagen protein expression, anchoring fibril formation and dermal-epidermal cohesion. These findings establish a practical approach to nonviral genetic correction of severe human genetic disorders requiring stable genomic integration of large DNA sequences.
View details for DOI 10.1038/nm766
View details for Web of Science ID 000178311000045
View details for PubMedID 12244305
- Stable nonviral genetic correction of inherited human skin disease Nature Medicine 2002; 8: 1166-1170
Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation
2000; 6 (11): 1253-1257
Many systemically effective drugs such as cyclosporin A are ineffective topically because of their poor penetration into skin. To surmount this problem, we conjugated a heptamer of arginine to cyclosporin A through a pH-sensitive linker to produce R7-CsA. In contrast to unmodified cyclosporin A, which fails to penetrate skin, topically applied R7-CsA was efficiently transported into cells in mouse and human skin. R7-CsA reached dermal T lymphocytes and inhibited cutaneous inflammation. These data establish a general strategy for enhancing delivery of poorly absorbed drugs across tissue barriers and provide a new topical approach to the treatment of inflammatory skin disorders.
View details for Web of Science ID 000165114800033
View details for PubMedID 11062537
Immunization via hair follicles by topical application of naked DNA to normal skin
1999; 17 (9): 870-872
In order to test the immune response generated to small amounts of foreign protein in skin, we applied naked DNA in aqueous solution to untreated normal skin. Topical application of plasmid expression vectors for lacZ and the hepatitis B surface antigen (HBsAg) to intact skin induced antigen-specific immune responses that displayed TH2 features. For HBsAg, specific antibody and cellular responses were induced to the same order of magnitude as those produced by intramuscular injection of the commercially available recombinant HBsAg polypeptide vaccine. Finally, topical gene transfer was dependent on the presence of normal hair follicles.
View details for Web of Science ID 000082365800027
View details for PubMedID 10471927
Sustainable cutaneous gene delivery
1997; 15 (13): 1388-1391
Durable gene delivery to human skin is necessary for lasting correction of human genetic skin disease. Current cutaneous gene-delivery strategies, however, have achieved only transient gene expression, often only within a small percentage of tissue cells. The recent inability to sustain phenotypic correction of human genetic skin disease due to loss of therapeutic gene expression in regenerated epidermal tissue has highlighted this current limitation. In an effort to surmount this problem, we have generated gene delivery vectors that produce more durable gene delivery in human skin tissue in vivo.
View details for Web of Science ID A1997YK36100031
View details for PubMedID 9415892
Induction of basal cell carcinoma features in transgenic human skin expressing Sonic Hedgehog
1997; 3 (7): 788-792
Hedgehog (HH) signaling proteins mediate inductive events during animal development. Mutation of the only known HH receptor gene, Patched (PTC), has recently been implicated in inherited and sporadic forms of the most common human cancer, basal cell carcinoma (BCC). In Drosophila, HH acts by inactivating PTC function, raising the possibility that overexpression of Sonic Hedgehog (SHH) in human epidermis might have a tumorigenic effect equivalent to loss of PTC function. We used retroviral transduction of normal human keratinocytes to constitutively express SHH. SHH-expressing cells demonstrated increased expression of both the known HH target, BMP-2B, as well as bcl-2, a protein prominently expressed by keratinocytes in BCCs. These keratinocytes were then used to regenerate human skin transgenic for long terminal repeat-driven SHH (LTR-SHH) on immune-deficient mice. LTR-SHH human skin consistently displays the abnormal specific histologic features seen in BCCs, including downgrowth of epithelial buds into the dermis, basal cell palisading and separation of epidermis from the underlying dermis. In addition, LTR-SHH skin displays the gene expression abnormalities previously described for human BCCs, including decreased BP180/BPAG2 and laminin 5 adhesion proteins and expression of basal epidermal keratins. These data indicate that expression of SHH in human skin recapitulates features of human BCC in vivo, suggest that activation of this conserved signaling pathway contributes to the development of epithelial neoplasia and describe a new transgenic human tissue model of neoplasia.
View details for Web of Science ID A1997XG76700042
View details for PubMedID 9212109
- Gene therapy: Progress, problems, prospects. NATURE MEDICINE 1997; 3 (6): 612-613
Corrective gene transfer in the human skin disorder lamellar ichthyosis
1996; 2 (11): 1263-1267
Lamellar ichthyosis (LI) is a disfiguring skin disease characterized by abnormal epidermal differentiation and defective cutaneous barrier function. LI has been associated with loss of keratinocyte transglutaminase 1 (TGase1), an enzyme believed necessary for normal formation of the cornified epidermal barrier. Using LI as a prototype for therapeutic cutaneous gene delivery, we have used the human skin/immunodeficient mouse xenograft model to correct the molecular, histologic and functional abnormalities of LI patient skin in vivo. We have used TGase1-deficient primary keratinocytes from LI patients combined with high-efficiency transfer of functional TGase1 to regenerate engineered human LI epidermis on immunodeficient mice. Engineered LI epidermis displayed normal TGase1 expression in vivo, unlike unengineered LI epidermis where TGase1 was absent. Epidermal architecture was also normalized by TGase1 restoration, as was expression of the epidermal differentiation marker filaggrin. Engineered LI skin demonstrated restoration of cutaneous barrier function measures to levels seen in epidermis regenerated by keratinocytes from patients with normal skin, indicating functional correction in vivo of the proposed primary pathophysiologic defect in LI. These results confirm a major role for TGase1 in epidermal differentiation and demonstrate a potential future approach to therapeutic gene delivery in human skin.
View details for Web of Science ID A1996VQ10100046
View details for PubMedID 8898758
THE RETINOBLASTOMA PROTEIN AND BRG1 FORM A COMPLEX AND COOPERATE TO INDUCE CELL-CYCLE ARREST
1994; 79 (1): 119-130
The retinoblastoma tumor suppressor protein (RB) binds several cellular proteins involved in cell cycle progression. Using the yeast two-hybrid system, we found that RB bound specifically to the protein BRG1. BRG1 shares extensive sequence similarity to Drosophila brahma, an activator of homeotic gene expression, and the yeast transcriptional activator SNF2/SW12. BRG1 contains an RB-binding motif found in viral oncoproteins and bound to the A/B pocket and the hypophosphorylated form of RB. BRG1 did not bind RB in viral oncoprotein-transformed cells. Coimmunoprecipitation experiments suggested BRG1 associates with the RB family in vivo. In the human carcinoma cell line SW13, BRG1 exhibited tumor suppressor activity by inducing formation of flat, growth-arrested cells. This activity depended on the ability of BRG1 to cooperate and complex with RB, as both an RB-nonbinding mutant of BRG1 and the sequestration of RB by adenovirus E1A protein abolished flat cell formation.
View details for Web of Science ID A1994PK58500013
View details for PubMedID 7923370
NUCLEOSOME DISRUPTION AND ENHANCEMENT OF ACTIVATOR BINDING BY A HUMAN SW1/SNF COMPLEX
1994; 370 (6489): 477-481
CHROMATIN structure can affect the transcriptional activity of eukaryotic structural genes by blocking access of sequence-specific activator proteins (activators) to their promoter-binding sites. For example, the DNA-binding domain of the yeast GAL4 protein interacts very poorly with nucleosome cores compared with naked DNA2 (and see below), and binding of other activators is even more strongly inhibited. The way in which activators bind to nucleosomal DNA is therefore a critical aspect of transcriptional activation. Genetic studies have suggested that the multi-component SWI/SNF complex of Saccharomyces cerevisiae facilitates transcription by altering the structure of the chromatin. Here we identify and partially purify a human homologue of the yeast SWI/SNF complex (hSWI/SNF complex). We show that a partially purified hSWI/SNF complex mediates the ATP-dependent disruption of a nucleosome, thereby enabling the activators, GAL4-VP16 and GAL4-AH, to bind within a nucleosome core. We conclude that the hSWI/SNF complex acts directly to reorganize chromatin structure so as to facilitate binding of transcription factors.
View details for Web of Science ID A1994PB40700061
View details for PubMedID 8047169
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 Web of Science ID A1993MG21600061
View details for PubMedID 8232556
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 Web of Science ID A1991GW31600034
View details for PubMedID 1763325