Honors & Awards


  • Elected Member, National Academy of Sciences (2024)
  • Momentum Award, International Society for Stem Cell Research (2022)
  • Elected Member, EMBO (2019)
  • Lorry Lokey Endowed Chair, Stanford University (2019)
  • Elected Member, American Academy of Arts and Science (2018)
  • Valkhof Chair Award, Radboud University Nijmegen, the Netherlands (2017)
  • Investigator Award, Howard Hughes Medical Institute (2015)
  • Harland Winfield Mossman Award in Developmental Biology, American Association of Anatomists (2013)
  • Vilcek Prize for Creative Promise, Vilcek Foundation (2013)
  • 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)
  • Postdoctoral Fellowship, Damon Runyon Cancer Research Foundation (2004-2006)

Professional Education


  • 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


We are employing a broad combination of genomic, genetic, biochemical, biophysical, single-cell and embryological approaches in number of cellular and organismal models to investigate functions of the non-coding parts of the genome, understand regulatory mechanisms underlying cellular plasticity and differentiation, investigate how quantitative changes in gene expression dictate differences in human traits, and study craniofacial development, evolution and disease.

MECHANISMS OF LONG RANGE GENE REGULATION
Central to the cell type-specific transcriptional regulation are distal cis-regulatory elements called enhancers. A major area of investigation in our laboratory is focused on general mechanisms of long-range gene regulation by enhancers, which can activate their target genes over tens of even hundreds of kilobases of genomic distances. We are striving to understand how enhancers are activated in response to developmental stimuli, how they communicate with target promoters, what is the dynamics of this process in living cells, and what is the role of chromatin context in priming or restricting enhancer activity. 

HUMAN NEURAL CREST DEVELOPMENT, DISEASE AND EVOLUTION
Our laboratory uses Cranial Neural Crest Cells (CNCCs) as a paradigm to study how genetic information harbored by regulatory elements is decoded into a diversity of functions, behaviors and morphologies. CNCCs are a transient embryonic cell group which delaminates from the neural tube, migrates long distances and acquires an extraordinarily broad differentiation potential, ultimately giving rise to most of the craniofacial structures and determining their individual and species-specific variation. Over a third of human congenital malformations is linked to CNCC dysfunction, including over 700 syndromes with craniofacial manifestations.

The goal of our ongoing research effort is to understand how variation in gene dosage and expression translates into differences in CNCC behavior, leading to the emergence of normal-range and disease-associated morphological diversity in the craniofacial form. This gene expression variation can result both from the trans-regulatory differences, such as those associated with mutations of transcriptional and chromatin regulators in craniofacial syndromes, and from the variation in cis-regulatory sequences like enhancers. To understand both mechanisms of variation and their cell type specificity, we are using human pluripotent stem cell differentiation models that recapitulate induction, migration and differentiation of CNCCs in the dish and facilitate modeling of human neurocristopathies. To study impact of regulatory changes on facial morphology, we are combining these in vitro models with the in vivo work in mice and, in collaboration with human geneticists and anthropologists, with the genetic analysis and morphometric measurements of craniofacial features in human populations.

EXPLORING GENOMIC DARK MATTER: TRANSPOSABLE ELEMENTS
Transposable element (TE) derived sequences comprise nearly half of the human genome. It is not always appreciated, however, that most TEs that are present in modern humans invaded the ancestral genome at various points of primate evolution, but are typically not shared with more distal mammals such as rodents. Thus, TE derived sequences form a vast reservoir of largely primate-specific sequences from which novel regulatory functions can evolve. We are interested in understanding how TEs may serve as a substrate for evolution of species- and tissue-specific cis-regulatory elements for the host genes, and we are investigating a developmental aspect of transposon regulation.

2023-24 Courses


Stanford Advisees


All Publications


  • DNA-guided transcription factor cooperativity shapes face and limb mesenchyme. Cell Kim, S., Morgunova, E., Naqvi, S., Goovaerts, S., Bader, M., Koska, M., Popov, A., Luong, C., Pogson, A., Swigut, T., Claes, P., Taipale, J., Wysocka, J. 2024

    Abstract

    Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.

    View details for DOI 10.1016/j.cell.2023.12.032

    View details for PubMedID 38262408

  • Precise modulation of transcription factor levels identifies features underlying dosage sensitivity. Nature genetics Naqvi, S., Kim, S., Hoskens, H., Matthews, H. S., Spritz, R. A., Klein, O. D., Hallgrimsson, B., Swigut, T., Claes, P., Pritchard, J. K., Wysocka, J. 2023

    Abstract

    Transcriptional regulation exhibits extensive robustness, but human genetics indicates sensitivity to transcription factor (TF) dosage. Reconciling such observations requires quantitative studies of TF dosage effects at trait-relevant ranges, largely lacking so far. TFs play central roles in both normal-range and disease-associated variation in craniofacial morphology; we therefore developed an approach to precisely modulate TF levels in human facial progenitor cells and applied it to SOX9, a TF associated with craniofacial variation and disease (Pierre Robin sequence (PRS)). Most SOX9-dependent regulatory elements (REs) are buffered against small decreases in SOX9 dosage, but REs directly and primarily regulated by SOX9 show heightened sensitivity to SOX9 dosage; these RE responses partially predict gene expression responses. Sensitive REs and genes preferentially affect functional chondrogenesis and PRS-like craniofacial shape variation. We propose that such REs and genes underlie the sensitivity of specific phenotypes to TF dosage, while buffering of other genes leads to robust, nonlinear dosage-to-phenotype relationships.

    View details for DOI 10.1038/s41588-023-01366-2

    View details for PubMedID 37024583

  • Structural elements promote architectural stripe formation and facilitate ultra-long-range gene regulation at a human disease locus. Molecular cell Chen, L. F., Long, H. K., Park, M., Swigut, T., Boettiger, A. N., Wysocka, J. 2023

    Abstract

    Enhancer clusters overlapping disease-associated mutations in Pierre Robin sequence (PRS) patients regulate SOX9 expression at genomic distances over 1.25 Mb. We applied optical reconstruction of chromatin architecture (ORCA) imaging to trace 3D locus topology during PRS-enhancer activation. We observed pronounced changes in locus topology between cell types. Subsequent analysis of single-chromatin fiber traces revealed that these ensemble-average differences arise through changes in the frequency of commonly sampled topologies. We further identified two CTCF-bound elements, internal to the SOX9 topologically associating domain, which promote stripe formation, are positioned near the domain's 3D geometric center, and bridge enhancer-promoter contacts in a series of chromatin loops. Ablation of these elements results in diminished SOX9 expression and altered domain-wide contacts. Polymer models with uniform loading across the domain and frequent cohesin collisions recapitulate this multi-loop, centrally clustered geometry. Together, we provide mechanistic insights into architectural stripe formation and gene regulation over ultra-long genomic ranges.

    View details for DOI 10.1016/j.molcel.2023.03.009

    View details for PubMedID 36996812

  • Deciphering the multi-scale, quantitative cis-regulatory code. Molecular cell Kim, S., Wysocka, J. 2023

    Abstract

    Uncovering the cis-regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state remains a central goal of biology. Here, we discuss major layers of regulation that influence how transcriptional outputs are encoded by DNA sequence and cellular context. We first discuss how transcription factors bind specific DNA sequences in a dosage-dependent and cooperative manner and then proceed to the cofactors that facilitate transcription factor function and mediate the activity of modular cis-regulatory elements such as enhancers, silencers, and promoters. We then consider the complex and poorly understood interplay of these diverse elements within regulatory landscapes and its relationships with chromatin states and nuclear organization. We propose that a mechanistically informed, quantitative model of transcriptional regulation that integrates these multiple regulatory layers will be the key to ultimately cracking the cis-regulatory code.

    View details for DOI 10.1016/j.molcel.2022.12.032

    View details for PubMedID 36693380

  • Reactivation of the pluripotency program precedes formation of the cranial neural crest. Science (New York, N.Y.) Zalc, A., Sinha, R., Gulati, G. S., Wesche, D. J., Daszczuk, P., Swigut, T., Weissman, I. L., Wysocka, J. 2021; 371 (6529)

    Abstract

    During development, cells progress from a pluripotent state to a more restricted fate within a particular germ layer. However, cranial neural crest cells (CNCCs), a transient cell population that generates most of the craniofacial skeleton, have much broader differentiation potential than their ectodermal lineage of origin. Here, we identify a neuroepithelial precursor population characterized by expression of canonical pluripotency transcription factors that gives rise to CNCCs and is essential for craniofacial development. Pluripotency factor Oct4 is transiently reactivated in CNCCs and is required for the subsequent formation of ectomesenchyme. Furthermore, open chromatin landscapes of Oct4+ CNCC precursors resemble those of epiblast stem cells, with additional features suggestive of priming for mesenchymal programs. We propose that CNCCs expand their developmental potential through a transient reacquisition of molecular signatures of pluripotency.

    View details for DOI 10.1126/science.abb4776

    View details for PubMedID 33542111

  • Single Amino Acid Change Underlies Distinct Roles of H2A.Z Subtypes in Human Syndrome. Cell Greenberg, R. S., Long, H. K., Swigut, T. n., Wysocka, J. n. 2019; 178 (6): 1421–36.e24

    Abstract

    The developmental disorder Floating-Harbor syndrome (FHS) is caused by heterozygous truncating mutations in SRCAP, a gene encoding a chromatin remodeler mediating incorporation of histone variant H2A.Z. Here, we demonstrate that FHS-associated mutations result in loss of SRCAP nuclear localization, alter neural crest gene programs in human in vitro models and Xenopus embryos, and cause craniofacial defects. These defects are mediated by one of two H2A.Z subtypes, H2A.Z.2, whose knockdown mimics and whose overexpression rescues the FHS phenotype. Selective rescue by H2A.Z.2 is conferred by one of the three amino acid differences between the H2A.Z subtypes, S38/T38. We further show that H2A.Z.1 and H2A.Z.2 genomic occupancy patterns are qualitatively similar, but quantitatively distinct, and H2A.Z.2 incorporation at AT-rich enhancers and expression of their associated genes are both sensitized to SRCAP truncations. Altogether, our results illuminate the mechanism underlying a human syndrome and uncover selective functions of H2A.Z subtypes during development.

    View details for DOI 10.1016/j.cell.2019.08.002

    View details for PubMedID 31491386

  • Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements SCIENCE Gu, B., Swigut, T., Spencley, A., Bauer, M. R., Chung, M., Meyer, T., Wysocka, J. 2018; 359 (6379): 1050–55

    Abstract

    To achieve guide RNA (gRNA) multiplexing and an efficient delivery of tens of distinct gRNAs into single cells, we developed a molecular assembly strategy termed chimeric array of gRNA oligonucleotides (CARGO). We coupled CARGO with dCas9 (catalytically dead Cas9) imaging to quantitatively measure the movement of enhancers and promoters that undergo differentiation-associated activity changes in live embryonic stem cells. Whereas all examined functional elements exhibited subdiffusive behavior, their relative mobility increased concurrently with transcriptional activation. Furthermore, acute perturbation of RNA polymerase II activity can reverse these activity-linked increases in loci mobility. Through quantitative CARGO-dCas9 imaging, we provide direct measurements of cis-regulatory element dynamics in living cells and distinct cellular and activity states and uncover an intrinsic connection between cis-regulatory element mobility and transcription.

    View details for PubMedID 29371426

  • Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators. Nature Liu, N. n., Lee, C. H., Swigut, T. n., Grow, E. n., Gu, B. n., Bassik, M. n., Wysocka, J. n. 2017

    Abstract

    Transposable elements (TEs) are now recognized not only as parasitic DNA, whose spread in the genome must be controlled by the host, but also as major players in genome evolution and regulation1-6. Long INterspersed Element-1 (LINE-1 or L1), the only currently autonomous mobile transposon in humans, occupies 17% of the genome and continues to generate inter- and intra-individual genetic variation, in some cases resulting in disease1-7. Nonetheless, how L1 activity is controlled and what function L1s play in host gene regulation remain incompletely understood. Here, we use CRISPR/Cas9 screening strategies in two distinct human cell lines to provide the first genome-wide survey of genes involved in L1 retrotransposition control. We identified functionally diverse genes that either promote or restrict L1 retrotransposition. These genes, often associated with human diseases, control the L1 lifecycle at transcriptional or post-transcriptional levels and in a manner that can depend on the endogenous L1 sequence, underscoring the complexity of L1 regulation. We further investigated L1 restriction by MORC2 and human silencing hub (HUSH) complex subunits MPP8 and TASOR8. HUSH/MORC2 selectively bind evolutionarily young, full-length L1s located within transcriptionally permissive euchromatic environment, and promote H3K9me3 deposition for transcriptional silencing. Interestingly, these silencing events often occur within introns of transcriptionally active genes and lead to down-regulation of host gene expression in a HUSH/MORC2-dependent manner. Together, we provide a rich resource for studies of L1 retrotransposition, elucidate a novel L1 restriction pathway, and illustrate how epigenetic silencing of TEs rewires host gene expression programs.

    View details for PubMedID 29211708

  • Enhancer Divergence and cis-Regulatory Evolution in the Human and Chimp Neural Crest. Cell Prescott, S. L., Srinivasan, R., Marchetto, M. C., Grishina, I., Narvaiza, I., Selleri, L., Gage, F. H., Swigut, T., Wysocka, J. 2015; 163 (1): 68-83

    View details for DOI 10.1016/j.cell.2015.08.036

    View details for PubMedID 26365491

  • Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells. Nature Grow, E. J., Flynn, R. A., Chavez, S. L., Bayless, N. L., Wossidlo, M., Wesche, D. J., Martin, L., Ware, C. B., Blish, C. A., Chang, H. Y., Pera, R. A., Wysocka, J. 2015; 522 (7555): 221-225

    Abstract

    Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development.

    View details for DOI 10.1038/nature14308

    View details for PubMedID 25896322

  • A commoncis-regulatory variant impacts normal-range and disease-associated human facial shape through regulation ofPKDCCduring chondrogenesis. eLife Mohammed, J., Arora, N., Matthews, H. S., Hansen, K., Bader, M., Walsh, S., Shaffer, J. R., Weinberg, S. M., Swigut, T., Claes, P., Selleri, L., Wysocka, J. 2024; 13

    Abstract

    Genome-wide association studies (GWAS) identified thousands of genetic variants linked to phenotypic traits and disease risk. However, mechanistic understanding of how GWAS variants influence complex morphological traits and can, in certain cases, simultaneously confer normal-range phenotypic variation and disease predisposition, is still largely lacking. Here, we focus on rs6740960, a single nucleotide polymorphism (SNP) at the 2p21 locus, which in GWAS studies has been associated both with normal-range variation in jaw shape and with an increased risk of non-syndromic orofacial clefting. Using in vitro derived embryonic cell types relevant for human facial morphogenesis, we show that this SNP resides in an enhancer that regulates chondrocytic expression of PKDCC - a gene encoding a tyrosine kinase involved in chondrogenesis and skeletal development. In agreement, we demonstrate that the rs6740960 SNP is sufficient to confer chondrocyte-specific differences in PKDCC expression. By deploying dense landmark morphometric analysis of skull elements in mice, we show that changes in Pkdcc dosage are associated with quantitative changes in the maxilla, mandible, and palatine bone shape that are concordant with the facial phenotypes and disease predisposition seen in humans. We further demonstrate that the frequency of the rs6740960 variant strongly deviated among different human populations, and that the activity of its cognate enhancer diverged in hominids. Our study provides a mechanistic explanation of how a common SNP can mediate normal-range and disease-associated morphological variation, with implications for the evolution of human facial features.

    View details for DOI 10.7554/eLife.82564

    View details for PubMedID 38483448

  • Structural elements facilitate 3D locus topology and regulation of a human disease gene Long, H., Chen, L., Boettiger, A., Wysocka, J. SPRINGERNATURE. 2024: 56-57
  • Joint multi-ancestry and admixed GWAS on 3D facial shape derived from MRI Goovaerts, S., Hoskens, H., Matthews, H., Herrick, N., Vandermeulen, D., Yuan, M., Shaffer, J., Shriver, M., Wysocka, J., Walsh, S., Weinberg, S., Claes, P. SPRINGERNATURE. 2024: 784
  • Joint multi-ancestry and admixed GWAS reveals the complex genetics behind human cranial vault shape. Nature communications Goovaerts, S., Hoskens, H., Eller, R. J., Herrick, N., Musolf, A. M., Justice, C. M., Yuan, M., Naqvi, S., Lee, M. K., Vandermeulen, D., Szabo-Rogers, H. L., Romitti, P. A., Boyadjiev, S. A., Marazita, M. L., Shaffer, J. R., Shriver, M. D., Wysocka, J., Walsh, S., Weinberg, S. M., Claes, P. 2023; 14 (1): 7436

    Abstract

    The cranial vault in humans is highly variable, clinically relevant, and heritable, yet its genetic architecture remains poorly understood. Here, we conduct a joint multi-ancestry and admixed multivariate genome-wide association study on 3D cranial vault shape extracted from magnetic resonance images of 6772 children from the ABCD study cohort yielding 30 genome-wide significant loci. Follow-up analyses indicate that these loci overlap with genomic risk loci for sagittal craniosynostosis, show elevated activity cranial neural crest cells, are enriched for processes related to skeletal development, and are shared with the face and brain. We present supporting evidence of regional localization for several of the identified genes based on expression patterns in the cranial vault bones of E15.5 mice. Overall, our study provides a comprehensive overview of the genetics underlying normal-range cranial vault shape and its relevance for understanding modern human craniofacial diversity and the etiology of congenital malformations.

    View details for DOI 10.1038/s41467-023-43237-8

    View details for PubMedID 37973980

    View details for PubMedCentralID 137471

  • A genome-wide genetic screen uncovers determinants of human pigmentation. Science (New York, N.Y.) Bajpai, V. K., Swigut, T., Mohammed, J., Naqvi, S., Arreola, M., Tycko, J., Kim, T. C., Pritchard, J. K., Bassik, M. C., Wysocka, J. 2023; 381 (6658): eade6289

    Abstract

    Skin color, one of the most diverse human traits, is determined by the quantity, type, and distribution of melanin. In this study, we leveraged the light-scattering properties of melanin to conduct a genome-wide screen for regulators of melanogenesis. We identified 169 functionally diverse genes that converge on melanosome biogenesis, endosomal transport, and gene regulation, of which 135 represented previously unknown associations with pigmentation. In agreement with their melanin-promoting function, the majority of screen hits were up-regulated in melanocytes from darkly pigmented individuals. We further unraveled functions of KLF6 as a transcription factor that regulates melanosome maturation and pigmentation in vivo, and of the endosomal trafficking protein COMMD3 in modulating melanosomal pH. Our study reveals a plethora of melanin-promoting genes, with broad implications for human variation, cell biology, and medicine.

    View details for DOI 10.1126/science.ade6289

    View details for PubMedID 37561850

  • DNA-guided transcription factor cooperativity shapes face and limb mesenchyme. bioRxiv : the preprint server for biology Kim, S., Morgunova, E., Naqvi, S., Bader, M., Koska, M., Popov, A., Luong, C., Pogson, A., Claes, P., Taipale, J., Wysocka, J. 2023

    Abstract

    Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how 'Coordinator', a long DNA motif comprised of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, while HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in shared regulation of genes involved in cell-type and positional identities, and ultimately shapes facial morphology and evolution.

    View details for DOI 10.1101/2023.05.29.541540

    View details for PubMedID 37398193

    View details for PubMedCentralID PMC10312427

  • Extreme long-range gene regulatory perturbation drives a human craniofacial disorder Long, H., Chen, L., Swigut, T., Boettiger, A., Wysocka, J. SPRINGERNATURE. 2023: 14
  • Co-transcriptional genome surveillance by HUSH is coupled to termination machinery. Molecular cell Spencley, A. L., Bar, S., Swigut, T., Flynn, R. A., Lee, C. H., Chen, L., Bassik, M. C., Wysocka, J. 2023

    Abstract

    The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and-via its component MPP8-with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.

    View details for DOI 10.1016/j.molcel.2023.04.014

    View details for PubMedID 37164018

  • C. David Allis (1951-2023). Cell Bernstein, E., Dent, S., Dou, Y., Duncan, E. M., Hake, S. B., Strahl, B. D., Wysocka, J. 2023; 186 (4): 663-667

    View details for DOI 10.1016/j.cell.2023.01.031

    View details for PubMedID 36803594

  • C. David Allis (1951-2023). Molecular cell Bernstein, E., Dent, S., Dou, Y., Duncan, E. M., Hake, S. B., Strahl, B. D., Wysocka, J. 2023; 83 (4): 497-499

    View details for DOI 10.1016/j.molcel.2023.02.002

    View details for PubMedID 36805122

  • Making the Human Face: Elucidating the Role of Enhancers in Hominid Craniofacial Evolution. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Mohammed, J., Hansen, K., Claes, P., Weinberg, S., Selleri, L., Swigut, T., Wysocka, J. 2022; 36 Suppl 1

    Abstract

    There is increasing evidence that morphological differences among individuals of the same species and between species are driven by changes in gene expression mediated by non-coding mutations that are enriched at enhancer elements. The identification and comparative evaluation of these cis-regulatory variants specific to human facial morphology have been facilitated by several recent advances. Among these are access to high-quality genome sequences, especially from archaic hominins such as the Neanderthals, by expanded catalogs of SNPs implicated in normal range human facial variation from Genome-wide Association Studies (GWAS), and by the curation of active human and chimp Cranial Neural Crest Cell (CNCC) enhancers that are important for facial development. To identify functional variants that mediate change in enhancer activity in recent human evolution and normal range variation and to nominate their target genes we employed two methods: (1) STARR-seq, an enhancer reporter assay that permits accurate, comparative analysis of regulatory activity across enhancer homologs and their constituent cis-mutations, and (2) H3K27ac HiChIP, a method to characterize the three-dimensional interactome between enhancers and their target genes. We performed STARR-seq and HiChIP experiments in the in vitro derived CNCCs, and early proliferative cranial chondrocytes. Comparisons of enhancer activity among the orthologs present in homo- or hominin species uncovered several classes of enhancers with species- or clade- restricted activity. Furthermore, for enhancers associated with facial shape GWAS signals, we identified genetic variants associated with changes in regulatory activity. By incorporating HiChIP labeled target genes, we found that biased intra-species and GWAS enhancer homologs target important developmental transcription factors and signaling molecules. Our experiments have begun to provide insights into how evolutionarily important and functionally relevant enhancer cis-variants can modulate gene regulatory programs at the individual- or species-centric level with an outcome on facial phenotypes.

    View details for DOI 10.1096/fasebj.2022.36.S1.0I629

    View details for PubMedID 35553087

  • Decoding the Human Face: Challenges and Progress in Understanding the Genetics of Craniofacial Morphology. Annual review of genomics and human genetics Naqvi, S., Hoskens, H., Wilke, F., Weinberg, S. M., Shaffer, J. R., Walsh, S., Shriver, M. D., Wysocka, J., Claes, P. 2022

    Abstract

    Variations in the form of the human face, which plays a role in our individual identities and societal interactions and exhibits extreme forms in a broad range of craniofacial syndromes and birth defects, have fascinated both geneticists and developmental biologists. Here, we review our current understanding of the genetics underlying variation in craniofacial morphology and dysmorphology, synthesizing decades of progress on Mendelian syndromes in addition to more recent results from genome-wide association studies of human facial shape and disease risk. We also discuss the various approaches used to phenotype and quantify facial shape, which are of particular importance due to the complex, multipartite nature of the craniofacial form. We close by discussing how experimental studies have contributed and will further contribute to our understanding of human genetic variation and then proposing future directions and applications for the field.Expected final online publication date for the Annual Review of Genomics and Human Genetics, Volume 23 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

    View details for DOI 10.1146/annurev-genom-120121-102607

    View details for PubMedID 35483406

  • Roles of transposable elements in the regulation of mammalian transcription. Nature reviews. Molecular cell biology Fueyo, R., Judd, J., Feschotte, C., Wysocka, J. 2022

    Abstract

    Transposable elements (TEs) comprise about half of the mammalian genome. TEs often contain sequences capable of recruiting the host transcription machinery, which they use to express their own products and promote transposition. However, the regulatory sequences carried by TEs may affect host transcription long after the TEs have lost the ability to transpose. Recent advances in genome analysis and engineering have facilitated systematic interrogation of the regulatory activities of TEs. In this Review, we discuss diverse mechanisms by which TEs contribute to transcription regulation. Notably, TEs can donate enhancer and promoter sequences that influence the expression of host genes, modify 3D chromatin architecture and give rise to novel regulatory genes, including non-coding RNAs and transcription factors. We discuss how TEs spur regulatory evolution and facilitate the emergence of genetic novelties in mammalian physiology and development. By virtue of their repetitive and interspersed nature, TEs offer unique opportunities to dissect the effects of mutation and genomic context on the function and evolution of cis-regulatory elements. We argue that TE-centric studies hold the key to unlocking general principles of transcription regulation and evolution.

    View details for DOI 10.1038/s41580-022-00457-y

    View details for PubMedID 35228718

  • Enhancer-associated H3K4 methylation safeguards in vitro germline competence. Nature communications Bleckwehl, T., Crispatzu, G., Schaaf, K., Respuela, P., Bartusel, M., Benson, L., Clark, S. J., Dorighi, K. M., Barral, A., Laugsch, M., van IJcken, W. F., Manzanares, M., Wysocka, J., Reik, W., Rada-Iglesias, A. 2021; 12 (1): 5771

    Abstract

    Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naive pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.

    View details for DOI 10.1038/s41467-021-26065-6

    View details for PubMedID 34599190

  • Genome scans of facial features in East Africans and cross-population comparisons reveal novel associations. PLoS genetics Liu, C., Lee, M. K., Naqvi, S., Hoskens, H., Liu, D., White, J. D., Indencleef, K., Matthews, H., Eller, R. J., Li, J., Mohammed, J., Swigut, T., Richmond, S., Manyama, M., Hallgrimsson, B., Spritz, R. A., Feingold, E., Marazita, M. L., Wysocka, J., Walsh, S., Shriver, M. D., Claes, P., Weinberg, S. M., Shaffer, J. R. 2021; 17 (8): e1009695

    Abstract

    Facial morphology is highly variable, both within and among human populations, and a sizable portion of this variation is attributable to genetics. Previous genome scans have revealed more than 100 genetic loci associated with different aspects of normal-range facial variation. Most of these loci have been detected in Europeans, with few studies focusing on other ancestral groups. Consequently, the degree to which facial traits share a common genetic basis across diverse sets of humans remains largely unknown. We therefore investigated the genetic basis of facial morphology in an East African cohort. We applied an open-ended data-driven phenotyping approach to a sample of 2,595 3D facial images collected on Tanzanian children. This approach segments the face into hierarchically arranged, multivariate features that capture the shape variation after adjusting for age, sex, height, weight, facial size and population stratification. Genome scans of these multivariate shape phenotypes revealed significant (p < 2.5 * 10-8) signals at 20 loci, which were enriched for active chromatin elements in human cranial neural crest cells and embryonic craniofacial tissue, consistent with an early developmental origin of the facial variation. Two of these associations were in highly conserved regions showing craniofacial-specific enhancer activity during embryological development (5q31.1 and 12q21.31). Six of the 20 loci surpassed a stricter threshold accounting for multiple phenotypes with study-wide significance (p < 6.25 * 10-10). Cross-population comparisons indicated 10 association signals were shared with Europeans (seven sharing the same associated SNP), and facilitated fine-mapping of causal variants at previously reported loci. Taken together, these results may point to both shared and population-specific components to the genetic architecture of facial variation.

    View details for DOI 10.1371/journal.pgen.1009695

    View details for PubMedID 34411106

  • 3D facial phenotyping by biometric sibling matching used in contemporary genomic methodologies. PLoS genetics Hoskens, H., Liu, D., Naqvi, S., Lee, M. K., Eller, R. J., Indencleef, K., White, J. D., Li, J., Larmuseau, M. H., Hens, G., Wysocka, J., Walsh, S., Richmond, S., Shriver, M. D., Shaffer, J. R., Peeters, H., Weinberg, S. M., Claes, P. 2021; 17 (5): e1009528

    Abstract

    The analysis of contemporary genomic data typically operates on one-dimensional phenotypic measurements (e.g. standing height). Here we report on a data-driven, family-informed strategy to facial phenotyping that searches for biologically relevant traits and reduces multivariate 3D facial shape variability into amendable univariate measurements, while preserving its structurally complex nature. We performed a biometric identification of siblings in a sample of 424 children, defining 1,048 sib-shared facial traits. Subsequent quantification and analyses in an independent European cohort (n = 8,246) demonstrated significant heritability for a subset of traits (0.17-0.53) and highlighted 218 genome-wide significant loci (38 also study-wide) associated with facial variation shared by siblings. These loci showed preferential enrichment for active chromatin marks in cranial neural crest cells and embryonic craniofacial tissues and several regions harbor putative craniofacial genes, thereby enhancing our knowledge on the genetic architecture of normal-range facial variation.

    View details for DOI 10.1371/journal.pgen.1009528

    View details for PubMedID 33983923

  • Shared heritability of human face and brain shape. Nature genetics Naqvi, S., Sleyp, Y., Hoskens, H., Indencleef, K., Spence, J. P., Bruffaerts, R., Radwan, A., Eller, R. J., Richmond, S., Shriver, M. D., Shaffer, J. R., Weinberg, S. M., Walsh, S., Thompson, J., Pritchard, J. K., Sunaert, S., Peeters, H., Wysocka, J., Claes, P. 2021

    Abstract

    Evidence from model organisms and clinical genetics suggests coordination between the developing brain and face, but the role of this link in common genetic variation remains unknown. We performed a multivariate genome-wide association study of cortical surface morphology in 19,644 individuals of European ancestry, identifying 472 genomic loci influencing brain shape, of which 76 are also linked to face shape. Shared loci include transcription factors involved in craniofacial development, as well as members of signaling pathways implicated in brain-face cross-talk. Brain shape heritability is equivalently enriched near regulatory regions active in either forebrain organoids or facial progenitors. However, we do not detect significant overlap between shared brain-face genome-wide association study signals and variants affecting behavioral-cognitive traits. These results suggest that early in embryogenesis, the face and brain mutually shape each other through both structural effects and paracrine signaling, but this interplay may not impact later brain development associated with cognitive function.

    View details for DOI 10.1038/s41588-021-00827-w

    View details for PubMedID 33821002

  • Publisher Correction: Human-chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution. Nature genetics Gokhman, D. n., Agoglia, R. M., Kinnebrew, M. n., Gordon, W. n., Sun, D. n., Bajpai, V. K., Naqvi, S. n., Chen, C. n., Chan, A. n., Chen, C. n., Petrov, D. A., Ahituv, N. n., Zhang, H. n., Mishina, Y. n., Wysocka, J. n., Rohatgi, R. n., Fraser, H. B. 2021

    View details for DOI 10.1038/s41588-021-00849-4

    View details for PubMedID 33762754

  • The Intersection of the Genetic Architectures of Orofacial Clefts and Normal Facial Variation. Frontiers in genetics Indencleef, K., Hoskens, H., Lee, M. K., White, J. D., Liu, C., Eller, R. J., Naqvi, S., Wehby, G. L., Moreno Uribe, L. M., Hecht, J. T., Long, R. E., Christensen, K., Deleyiannis, F. W., Walsh, S., Shriver, M. D., Richmond, S., Wysocka, J., Peeters, H., Shaffer, J. R., Marazita, M. L., Hens, G., Weinberg, S. M., Claes, P. 2021; 12: 626403

    Abstract

    Unaffected relatives of individuals with non-syndromic cleft lip with or without cleft palate (NSCL/P) show distinctive facial features. The presence of this facial endophenotype is potentially an expression of underlying genetic susceptibility to NSCL/P in the larger unselected population. To explore this hypothesis, we first partitioned the face into 63 partially overlapping regions representing global-to-local facial morphology and then defined endophenotypic traits by contrasting the 3D facial images from 264 unaffected parents of individuals with NSCL/P versus 3,171 controls. We observed distinct facial features between parents and controls across 59 global-to-local facial segments at nominal significance (p ≤ 0.05) and 52 segments at Bonferroni corrected significance (p < 1.2 * 10-3), respectively. Next, we quantified these distinct facial features as univariate traits in another dataset of 8,246 unaffected European individuals and performed a genome-wide association study. We identified 29 independent genetic loci that were associated (p < 5 * 10-8) with at least one of the tested endophenotypic traits, and nine genetic loci also passed the study-wide threshold (p < 8.47 * 10-10). Of the 29 loci, 22 were in proximity of loci previously associated with normal facial variation, 18 were near genes that show strong evidence in orofacial clefting (OFC), and another 10 showed some evidence in OFC. Additionally, polygenic risk scores for NSCL/P showed associations with the endophenotypic traits. This study thus supports the hypothesis of a shared genetic architecture of normal facial development and OFC.

    View details for DOI 10.3389/fgene.2021.626403

    View details for PubMedID 33692830

  • Temporal dissection of an enhancer cluster reveals distinct temporal and functional contributions of individual elements. Molecular cell Thomas, H. F., Kotova, E. n., Jayaram, S. n., Pilz, A. n., Romeike, M. n., Lackner, A. n., Penz, T. n., Bock, C. n., Leeb, M. n., Halbritter, F. n., Wysocka, J. n., Buecker, C. n. 2021

    Abstract

    Many genes are regulated by multiple enhancers that often simultaneously activate their target gene. However, how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the functions and interdependencies of five enhancer elements that together activate Fgf5 expression during exit from naive murine pluripotency. Four intergenic elements form a super-enhancer, and most of the elements contribute to Fgf5 induction at distinct time points. A fifth, poised enhancer located in the first intron contributes to Fgf5 expression at every time point by amplifying overall Fgf5 expression levels. Despite low individual enhancer activity, together these elements strongly induce Fgf5 expression in a super-additive fashion that involves strong accumulation of RNA polymerase II at the intronic enhancer. Finally, we observe a strong anti-correlation between RNA polymerase II levels at enhancers and their distance to the closest promoter, and we identify candidate elements with properties similar to the intronic enhancer.

    View details for DOI 10.1016/j.molcel.2020.12.047

    View details for PubMedID 33482114

  • Human-chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution. Nature genetics Gokhman, D. n., Agoglia, R. M., Kinnebrew, M. n., Gordon, W. n., Sun, D. n., Bajpai, V. K., Naqvi, S. n., Chen, C. n., Chan, A. n., Chen, C. n., Petrov, D. A., Ahituv, N. n., Zhang, H. n., Mishina, Y. n., Wysocka, J. n., Rohatgi, R. n., Fraser, H. B. 2021

    Abstract

    Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.

    View details for DOI 10.1038/s41588-021-00804-3

    View details for PubMedID 33731941

  • Insights into the genetic architecture of the human face. Nature genetics White, J. D., Indencleef, K., Naqvi, S., Eller, R. J., Hoskens, H., Roosenboom, J., Lee, M. K., Li, J., Mohammed, J., Richmond, S., Quillen, E. E., Norton, H. L., Feingold, E., Swigut, T., Marazita, M. L., Peeters, H., Hens, G., Shaffer, J. R., Wysocka, J., Walsh, S., Weinberg, S. M., Shriver, M. D., Claes, P. 2020

    Abstract

    The human face is complex and multipartite, and characterization of its genetic architecture remains challenging. Using a multivariate genome-wide association study meta-analysis of 8,246 European individuals, we identified 203 genome-wide-significant signals (120 also study-wide significant) associated with normal-range facial variation. Follow-up analyses indicate that the regions surrounding these signals are enriched for enhancer activity in cranial neural crest cells and craniofacial tissues, several regions harbor multiple signals with associations to different facial phenotypes, and there is evidence for potential coordinated actions of variants. In summary, our analyses provide insights into the understanding of how complex morphological traits are shaped by both individual and coordinated genetic actions.

    View details for DOI 10.1038/s41588-020-00741-7

    View details for PubMedID 33288918

  • FaceBase 3: analytical tools and FAIR resources for craniofacial and dental research. Development (Cambridge, England) Samuels, B. D., Aho, R., Brinkley, J. F., Bugacov, A., Feingold, E., Fisher, S., Gonzalez-Reiche, A. S., Hacia, J. G., Hallgrimsson, B., Hansen, K., Harris, M. P., Ho, T., Holmes, G., Hooper, J. E., Jabs, E. W., Jones, K. L., Kesselman, C., Klein, O. D., Leslie, E. J., Li, H., Liao, E. C., Long, H., Lu, N., Maas, R. L., Marazita, M. L., Mohammed, J., Prescott, S., Schuler, R., Selleri, L., Spritz, R. A., Swigut, T., van Bakel, H., Visel, A., Welsh, I., Williams, C., Williams, T. J., Wysocka, J., Yuan, Y., Chai, Y. 2020; 147 (18)

    Abstract

    The FaceBase Consortium was established by the National Institute of Dental and Craniofacial Research in 2009 as a 'big data' resource for the craniofacial research community. Over the past decade, researchers have deposited hundreds of annotated and curated datasets on both normal and disordered craniofacial development in FaceBase, all freely available to the research community on the FaceBase Hub website. The Hub has developed numerous visualization and analysis tools designed to promote integration of multidisciplinary data while remaining dedicated to the FAIR principles of data management (findability, accessibility, interoperability and reusability) and providing a faceted search infrastructure for locating desired data efficiently. Summaries of the datasets generated by the FaceBase projects from 2014 to 2019 are provided here. FaceBase 3 now welcomes contributions of data on craniofacial and dental development in humans, model organisms and cell lines. Collectively, the FaceBase Consortium, along with other NIH-supported data resources, provide a continuously growing, dynamic and current resource for the scientific community while improving data reproducibility and fulfilling data sharing requirements.

    View details for DOI 10.1242/dev.191213

    View details for PubMedID 32958507

  • Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder. Cell stem cell Long, H. K., Osterwalder, M. n., Welsh, I. C., Hansen, K. n., Davies, J. O., Liu, Y. E., Koska, M. n., Adams, A. T., Aho, R. n., Arora, N. n., Ikeda, K. n., Williams, R. M., Sauka-Spengler, T. n., Porteus, M. H., Mohun, T. n., Dickel, D. E., Swigut, T. n., Hughes, J. R., Higgs, D. R., Visel, A. n., Selleri, L. n., Wysocka, J. n. 2020

    Abstract

    Non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation.

    View details for DOI 10.1016/j.stem.2020.09.001

    View details for PubMedID 32991838

  • Epigenomic and Transcriptomic Changes During Human RPE EMT in a Stem Cell Model of Epiretinal Membrane Pathogenesis and Prevention by Nicotinamide. Stem cell reports Boles, N. C., Fernandes, M. n., Swigut, T. n., Srinivasan, R. n., Schiff, L. n., Rada-Iglesias, A. n., Wang, Q. n., Saini, J. S., Kiehl, T. n., Stern, J. H., Wysocka, J. n., Blenkinsop, T. A., Temple, S. n. 2020

    Abstract

    Epithelial to mesenchymal transition (EMT) is a biological process involved in tissue morphogenesis and disease that causes dramatic changes in cell morphology, migration, proliferation, and gene expression. The retinal pigment epithelium (RPE), which supports the neural retina, can undergo EMT, producing fibrous epiretinal membranes (ERMs) associated with vision-impairing clinical conditions, such as macular pucker and proliferative vitreoretinopathy (PVR). We found that co-treatment with TGF-β and TNF-α (TNT) accelerates EMT in adult human RPE stem cell-derived RPE cell cultures. We captured the global epigenomic and transcriptional changes elicited by TNT treatment of RPE and identified putative active enhancers associated with actively transcribed genes, including a set of upregulated transcription factors that are candidate regulators. We found that the vitamin B derivative nicotinamide downregulates these key transcriptional changes, and inhibits and partially reverses RPE EMT, revealing potential therapeutic routes to benefit patients with ERM, macular pucker and PVR.

    View details for DOI 10.1016/j.stemcr.2020.03.009

    View details for PubMedID 32243845

  • Transposable elements as a potent source of diverse cis-regulatory sequences in mammalian genomes. Philosophical transactions of the Royal Society of London. Series B, Biological sciences Sundaram, V. n., Wysocka, J. n. 2020; 375 (1795): 20190347

    Abstract

    Eukaryotic gene regulation is mediated by cis-regulatory elements, which are embedded within the vast non-coding genomic space and recognized by the transcription factors in a sequence- and context-dependent manner. A large proportion of eukaryotic genomes, including at least half of the human genome, are composed of transposable elements (TEs), which in their ancestral form carried their own cis-regulatory sequences able to exploit the host trans environment to promote TE transcription and facilitate transposition. Although not all present-day TE copies have retained this regulatory function, the preexisting regulatory potential of TEs can provide a rich source of cis-regulatory innovation for the host. Here, we review recent evidence documenting diverse contributions of TE sequences to gene regulation by functioning as enhancers, promoters, silencers and boundary elements. We discuss how TE-derived enhancer sequences can rapidly facilitate changes in existing gene regulatory networks and mediate species- and cell-type-specific regulatory innovations, and we postulate a unique contribution of TEs to species-specific gene expression divergence in pluripotency and early embryogenesis. With advances in genome-wide technologies and analyses, systematic investigation of TEs' cis-regulatory potential is now possible and our understanding of the biological impact of genomic TEs is increasing. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

    View details for DOI 10.1098/rstb.2019.0347

    View details for PubMedID 32075564

  • Opposing Effects of Cohesin and Transcription on CTCF Organization Revealed by Super-resolution Imaging. Molecular cell Gu, B. n., Comerci, C. J., McCarthy, D. G., Saurabh, S. n., Moerner, W. E., Wysocka, J. n. 2020

    Abstract

    CCCTC-binding factor (CTCF) and cohesin play critical roles in organizing mammalian genomes into topologically associating domains (TADs). Here, by combining genetic engineering with quantitative super-resolution stimulated emission depletion (STED) microscopy, we demonstrate that in living cells, CTCF forms clusters typically containing 2-8 molecules. A fraction of CTCF clusters, enriched for those with ≥3 molecules, are coupled with cohesin complexes with a characteristic physical distance suggestive of a defined molecular interaction. Acute degradation of the cohesin unloader WAPL or transcriptional inhibition (TI) result in increased CTCF clustering. Furthermore, the effect of TI on CTCF clusters is alleviated by the acute loss of the cohesin subunit SMC3. Our study provides quantitative characterization of CTCF clusters in living cells, uncovers the opposing effects of cohesin and transcription on CTCF clustering, and highlights the power of quantitative super-resolution microscopy as a tool to bridge the gap between biochemical and genomic methodologies in chromatin research.

    View details for DOI 10.1016/j.molcel.2020.10.001

    View details for PubMedID 33091336

  • Zscan4 binds nucleosomal microsatellite DNA and protects mouse two-cell embryos from DNA damage. Science advances Srinivasan, R. n., Nady, N. n., Arora, N. n., Hsieh, L. J., Swigut, T. n., Narlikar, G. J., Wossidlo, M. n., Wysocka, J. n. 2020; 6 (12): eaaz9115

    Abstract

    Zinc finger protein Zscan4 is selectively expressed in mouse two-cell (2C) embryos undergoing zygotic genome activation (ZGA) and in a rare subpopulation of embryonic stem cells with 2C-like features. Here, we show that Zscan4 specifically recognizes a subset of (CA)n microsatellites, repeat sequences prone to genomic instability. Zscan4-associated microsatellite regions are characterized by low nuclease sensitivity and high histone occupancy. In vitro, Zscan4 binds nucleosomes and protects them from disassembly upon torsional strain. Furthermore, Zscan4 depletion leads to elevated DNA damage in 2C mouse embryos in a transcription-dependent manner. Together, our results identify Zscan4 as a DNA sequence-dependent microsatellite binding factor and suggest a developmentally regulated mechanism, which protects fragile genomic regions from DNA damage at a time of embryogenesis associated with high transcriptional burden and genomic stress.

    View details for DOI 10.1126/sciadv.aaz9115

    View details for PubMedID 32219172

    View details for PubMedCentralID PMC7083622

  • The Spatiotemporal Pattern and Intensity of p53 Activation Dictates Phenotypic Diversity in p53-Driven Developmental Syndromes. Developmental cell Bowen, M. E., McClendon, J., Long, H. K., Sorayya, A., Van Nostrand, J. L., Wysocka, J., Attardi, L. D. 2019

    Abstract

    Inappropriate activation of the p53 transcription factor contributes to numerous developmental syndromes characterized by distinct constellations of phenotypes. How p53 drives exquisitely specific sets of symptoms in diverse syndromes, however, remains enigmatic. Here, we deconvolute the basis of p53-driven developmental syndromes by leveraging an array of mouse strains to modulate the spatial expression pattern, temporal profile, and magnitude of p53 activation during embryogenesis. We demonstrate that inappropriate p53 activation in the neural crest, facial ectoderm, anterior heart field, and endothelium induces distinct spectra of phenotypes. Moreover, altering the timing and degree of p53 hyperactivation substantially affects the phenotypic outcomes. Phenotypes are associated with p53-driven cell-cycle arrest or apoptosis, depending on the cell type, with gene expression programs, rather than extent of mitochondrial priming, largely governing the specific response. Together, our findings provide a critical framework for decoding the role of p53 as a mediator of diverse developmental syndromes.

    View details for DOI 10.1016/j.devcel.2019.05.015

    View details for PubMedID 31178404

  • Hunting for genes that shape human faces: Initial successes and challenges for the future Weinberg, S. M., Roosenboom, J., Shaffer, J. R., Shriver, M. D., Wysocka, J., Claes, P. WILEY. 2019: 207–12

    View details for DOI 10.1111/ocr.12268

    View details for Web of Science ID 000467585100032

  • SNPs discovered in European GWAS shape worldwide facial diversity Shriver, M. D., White, J. D., Lasisi, T., Eller, R. J., Gonzalez-Zarzar, T., Indencleef, K., Li, J., Hoskens, H., Ortega-Castrillon, A., Zaidi, A., Weinberg, S. M., Wysocka, J., Walsh, S., Akey, J. M., Claes, P. WILEY. 2019: 227
  • Meta-analysis identifies 48 SNPs with multiple independent effects on human facial features White, J. D., Roosenboom, J., Indencleef, K., Mohammed, J., Li, J., Ortega-Castrillon, A., Swigut, T., Lee, M., Gonzalez-Zarzar, T., Zaidi, A. A., Shaffer, J. R., Feingold, E., Richmond, S., Eller, R. J., Walsh, S., Marazita, M. L., Wysocka, J., Weinberg, S. M., Claes, P., Shriver, M. D. WILEY. 2019: 267
  • Hunting for genes that shape human faces: Initial successes and challenges for the future. Orthodontics & craniofacial research Weinberg, S. M., Roosenboom, J. n., Shaffer, J. R., Shriver, M. D., Wysocka, J. n., Claes, P. n. 2019; 22 Suppl 1: 207–12

    Abstract

    There is ample evidence from heritability studies, genetic syndromes and experimental animal models that facial morphology is strongly influenced by genes. In this brief review, we present an up-to-date overview of the efforts to identify genes associated with the size and shape of human facial features. We discuss recent methodological advances that have led to breakthroughs, but also the multitude of challenges facing the field. We offer perspective on possible applications of this line of research, particularly in the context of the precision genomics movement.

    View details for PubMedID 31074157

  • 2018 ISSCR Strategic Planning: Looking to the Future. Stem cell reports Wysocka, J. n., Rossant, J. n. 2019; 12 (6): 1183–85

    Abstract

    In 2018, the ISSCR underwent a strategic planning process to identify current priorities and plan for future initiatives. Key recommendations will help the society better serve its members and fulfill its mission of promoting excellence in stem cell science and applications to human health.

    View details for DOI 10.1016/j.stemcr.2019.05.015

    View details for PubMedID 31189091

  • Heterogeneity in old fibroblasts is linked to variability in reprogramming and wound healing. Nature Mahmoudi, S. n., Mancini, E. n., Xu, L. n., Moore, A. n., Jahanbani, F. n., Hebestreit, K. n., Srinivasan, R. n., Li, X. n., Devarajan, K. n., Prélot, L. n., Ang, C. E., Shibuya, Y. n., Benayoun, B. A., Chang, A. L., Wernig, M. n., Wysocka, J. n., Longaker, M. T., Snyder, M. P., Brunet, A. n. 2019; 574 (7779): 553–58

    Abstract

    Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing1, but its influence on specific cells remains largely unknown. Fibroblasts are present in most tissues and contribute to wound healing2,3. They are also the most widely used cell type for reprogramming to induced pluripotent stem (iPS) cells, a process that has implications for regenerative medicine and rejuvenation strategies4. Here we show that fibroblast cultures from old mice secrete inflammatory cytokines and exhibit increased variability in the efficiency of iPS cell reprogramming between mice. Variability between individuals is emerging as a feature of old age5-8, but the underlying mechanisms remain unknown. To identify drivers of this variability, we performed multi-omics profiling of fibroblast cultures from young and old mice that have different reprogramming efficiencies. This approach revealed that fibroblast cultures from old mice contain 'activated fibroblasts' that secrete inflammatory cytokines, and that the proportion of activated fibroblasts in a culture correlates with the reprogramming efficiency of that culture. Experiments in which conditioned medium was swapped between cultures showed that extrinsic factors secreted by activated fibroblasts underlie part of the variability between mice in reprogramming efficiency, and we have identified inflammatory cytokines, including TNF, as key contributors. Notably, old mice also exhibited variability in wound healing rate in vivo. Single-cell RNA-sequencing analysis identified distinct subpopulations of fibroblasts with different cytokine expression and signalling in the wounds of old mice with slow versus fast healing rates. Hence, a shift in fibroblast composition, and the ratio of inflammatory cytokines that they secrete, may drive the variability between mice in reprogramming in vitro and influence wound healing rate in vivo. This variability may reflect distinct stochastic ageing trajectories between individuals, and could help in developing personalized strategies to improve iPS cell generation and wound healing in elderly individuals.

    View details for DOI 10.1038/s41586-019-1658-5

    View details for PubMedID 31645721

  • Systematic perturbation of retroviral LTRs reveals widespread long-range effects on human gene regulation. eLife Fuentes, D. R., Swigut, T., Wysocka, J. 2018; 7

    Abstract

    Recent work suggests extensive adaptation of transposable elements (TEs) for host gene regulation. However, high numbers of integrations typical of TEs, coupled with sequence divergence within families, have made systematic interrogation of the regulatory contributions of TEs challenging. Here, we employ CARGO, our recent method for CRISPR gRNA multiplexing, to facilitate targeting of LTR5HS, an ape-specific class of HERVK (HML-2) LTRs that is active during early development and present in ~700 copies throughout the human genome. We combine CARGO with CRISPR activation or interference to, respectively, induce or silence LTR5HS en masse, and demonstrate that this system robustly targets the vast majority of LTR5HS insertions. Remarkably, activation/silencing of LTR5HS is associated with reciprocal up- and down-regulation of hundreds of human genes. These effects require presence of retroviral sequences, but occur over long genomic distances, consistent with a pervasive function of LTR5HS elements as early embryonic enhancers in apes.

    View details for PubMedID 30070637

  • Nicotinamide protects against PVR by preventing RPE cells to undergo EMT Fernandes, M., Schiff, L., Swigut, T., Boles, N., Srinivasan, R., Rada-Iglesias, A., Wang, Q., Saini, J., Kiehl, T., Stern, J., Wysocka, J., Temple, S., Blenkinsop, T. A. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018
  • Single cell expression analysis of primate-specific retroviruses-derived HPAT lincRNAs in viable human blastocysts identifies embryonic cells co-expressing genetic markers of multiple lineages HELIYON Glinsky, G., Durruthy-Durruthy, J., Wossidlo, M., Grow, E. J., Weirather, J. L., Au, K., Wysocka, J., Sebastiano, V. 2018; 4 (6): e00667

    Abstract

    Chromosome instability and aneuploidies occur very frequently in human embryos, impairing proper embryogenesis and leading to cell cycle arrest, loss of cell viability, and developmental failures in 50-80% of cleavage-stage embryos. This high frequency of cellular extinction events represents a significant experimental obstacle challenging analyses of individual cells isolated from human preimplantation embryos. We carried out single cell expression profiling of 241 individual cells recovered from 32 human embryos during the early and late stages of viable human blastocyst (VHB) differentiation. Classification of embryonic cells was performed solely based on expression patterns of human pluripotency-associated transcripts (HPAT), which represent a family of primate-specific transposable element-derived lincRNAs highly expressed in human embryonic stem cells and regulating nuclear reprogramming and pluripotency induction. We then validated our findings by analyzing transcriptomes of 1,708 individual cells recovered from more than 100 human embryos and 259 mouse cells from more than 40 mouse embryos at different stages of preimplantation embryogenesis. HPAT's expression-guided spatiotemporal reconstruction of human embryonic development inferred from single-cell expression analysis of VHB differentiation enabled identification of telomerase-positive embryonic cells co-expressing key pluripotency regulatory genes and genetic markers of three major lineages. Follow-up validation analyses confirmed the emergence in human embryos prior to lineage segregation of telomerase-positive cells co-expressing genetic markers of multiple lineages. Observations reported in this contribution support the hypothesis of a developmental pathway of creation embryonic lineages and extraembryonic tissues from telomerase-positive pre-lineage cells manifesting multi-lineage precursor phenotype.

    View details for PubMedID 30003161

  • Histone H3 lysine 4 monomethylation modulates longrange chromatin interactions at enhancers (vol 28, pg 204, 2018) CELL RESEARCH Yan, J., Chen, S. A., Local, A., Liu, T., Qiu, Y., Dorighi, K. M., Preissl, S., Rivera, C. M., Wang, C., Ye, Z., Ge, K., Hu, M., Wysocka, J., Ren, B. 2018; 28 (3): 387

    Abstract

    This corrects the article DOI: 10.1038/cr.2018.1.

    View details for PubMedID 29497152

    View details for PubMedCentralID PMC5835777

  • Genome-wide mapping of global-to-local genetic effects on human facial shape NATURE GENETICS Claes, P., Roosenboom, J., White, J. D., Swigut, T., Sero, D., Li, J., Lee, M., Zaidi, A., Mattern, B. C., Liebowitz, C., Pearson, L., Gonzalez, T., Leslie, E. J., Carlson, J. C., Orlova, E., Suetens, P., Vandermeulen, D., Feingold, E., Marazita, M. L., Shaffer, J. R., Wysocka, J., Shriver, M. D., Weinberg, S. M. 2018; 50 (3): 414-+

    Abstract

    Genome-wide association scans of complex multipartite traits like the human face typically use preselected phenotypic measures. Here we report a data-driven approach to phenotyping facial shape at multiple levels of organization, allowing for an open-ended description of facial variation while preserving statistical power. In a sample of 2,329 persons of European ancestry, we identified 38 loci, 15 of which replicated in an independent European sample (n = 1,719). Four loci were completely new. For the others, additional support (n = 9) or pleiotropic effects (n = 2) were found in the literature, but the results reported here were further refined. All 15 replicated loci highlighted distinctive patterns of global-to-local genetic effects on facial shape and showed enrichment for active chromatin elements in human cranial neural crest cells, suggesting an early developmental origin of the facial variation captured. These results have implications for studies of facial genetics and other complex morphological traits.

    View details for PubMedID 29459680

    View details for PubMedCentralID PMC5937280

  • Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders NATURE Calo, E., Gu, B., Bowen, M. E., Aryan, F., Zalc, A., Liang, J., Flynn, R. A., Swigut, T., Chang, H. Y., Attardi, L. D., Wysocka, J. 2018; 554 (7690): 112-+

    Abstract

    Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond-Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and dosage-dependent effects on craniofacial development. Taken together, our findings illustrate how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations.

    View details for PubMedID 29364875

  • Histone H3 lysine 4 monomethylation modulates long-range chromatin interactions at enhancers. Cell research Yan, J. n., Chen, S. A., Local, A. n., Liu, T. n., Qiu, Y. n., Dorighi, K. M., Preissl, S. n., Rivera, C. M., Wang, C. n., Ye, Z. n., Ge, K. n., Hu, M. n., Wysocka, J. n., Ren, B. n. 2018

    Abstract

    Long-range chromatin interactions between enhancers and promoters are essential for transcription of many developmentally controlled genes in mammals and other metazoans. Currently, the exact mechanisms that connect distal enhancers to their specific target promoters remain to be fully elucidated. Here, we show that the enhancer-specific histone H3 lysine 4 monomethylation (H3K4me1) and the histone methyltransferases MLL3 and MLL4 (MLL3/4) play an active role in this process. We demonstrate that in differentiating mouse embryonic stem cells, MLL3/4-dependent deposition of H3K4me1 at enhancers correlates with increased levels of chromatin interactions, whereas loss of this histone modification leads to reduced levels of chromatin interactions and defects in gene activation during differentiation. H3K4me1 facilitates recruitment of the Cohesin complex, a known regulator of chromatin organization, to chromatin in vitro and in vivo, providing a potential mechanism for MLL3/4 to promote chromatin interactions between enhancers and promoters. Taken together, our results support a role for MLL3/4-dependent H3K4me1 in orchestrating long-range chromatin interactions at enhancers in mammalian cells.Cell Research advance online publication 9 January 2018; doi:10.1038/cr.2018.1.

    View details for PubMedID 29313530

  • Mll3 and Mll4 Facilitate Enhancer RNA Synthesis and Transcription from Promoters Independently of H3K4 Monomethylation MOLECULAR CELL Dorighi, K. M., Swigut, T., Henriques, T., Bhanu, N. V., Scruggs, B. S., Nady, N., Still, C. D., Garcia, B. A., Adelman, K., Wysocka, J. 2017; 66 (4): 568-?

    Abstract

    Monomethylation of histone H3 at lysine 4 (H3K4me1) and acetylation of histone H3 at lysine 27 (H3K27ac) are correlated with transcriptionally engaged enhancer elements, but the functional impact of these modifications on enhancer activity is not well understood. Here we used CRISPR/Cas9 genome editing to separate catalytic activity-dependent and independent functions of Mll3 (Kmt2c) and Mll4 (Kmt2d, Mll2), the major enhancer H3K4 monomethyltransferases. Loss of H3K4me1 from enhancers in Mll3/4 catalytically deficient cells causes partial reduction of H3K27ac, but has surprisingly minor effects on transcription from either enhancers or promoters. In contrast, loss of Mll3/4 proteins leads to strong depletion of enhancer Pol II occupancy and eRNA synthesis, concomitant with downregulation of target genes. Interestingly, downregulated genes exhibit reduced polymerase levels in gene bodies, but not at promoters, suggestive of pause-release defects. Altogether, our results suggest that enhancer H3K4me1 provides only a minor contribution to the long-range coactivator function of Mll3/4.

    View details for DOI 10.1016/j.molcel.2017.04.018

    View details for Web of Science ID 000401512200014

    View details for PubMedID 28483418

  • Transcriptional Dependencies in Diffuse Intrinsic Pontine Glioma CANCER CELL Nagaraja, S., Vitanza, N. A., Woo, P. J., Taylor, K. R., Liu, F., Zhang, L., Li, M., Meng, W., Ponnuswami, A., Sun, W., Ma, J., Hulleman, E., Swigut, T., Wysocka, J., Tang, Y., Monje, M. 2017; 31 (5): 635-?

    Abstract

    Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric cancer with limited therapeutic options. The majority of cases of DIPG exhibit a mutation in histone-3 (H3K27M) that results in oncogenic transcriptional aberrancies. We show here that DIPG is vulnerable to transcriptional disruption using bromodomain inhibition or CDK7 blockade. Targeting oncogenic transcription through either of these methods synergizes with HDAC inhibition, and DIPG cells resistant to HDAC inhibitor therapy retain sensitivity to CDK7 blockade. Identification of super-enhancers in DIPG provides insights toward the cell of origin, highlighting oligodendroglial lineage genes, and reveals unexpected mechanisms mediating tumor viability and invasion, including potassium channel function and EPH receptor signaling. The findings presented demonstrate transcriptional vulnerabilities and elucidate previously unknown mechanisms of DIPG pathobiology.

    View details for DOI 10.1016/j.ccell.2017.03.011

    View details for Web of Science ID 000400738600008

    View details for PubMedID 28434841

  • Corrigendum: HIPSTR and thousands of lncRNAs are heterogeneously expressed in human embryos, primordial germ cells and stable cell lines. Scientific reports Yunusov, D., Anderson, L., DaSilva, L. F., Wysocka, J., Ezashi, T., Roberts, R. M., Verjovski-Almeida, S. 2017; 7: 44632-?

    View details for DOI 10.1038/srep44632

    View details for PubMedID 28300214

  • CSNK1a1 Regulates PRMT1 to Maintain the Progenitor State in Self-Renewing Somatic Tissue. Developmental cell Bao, X. n., Siprashvili, Z. n., Zarnegar, B. J., Shenoy, R. M., Rios, E. J., Nady, N. n., Qu, K. n., Mah, A. n., Webster, D. E., Rubin, A. J., Wozniak, G. G., Tao, S. n., Wysocka, J. n., Khavari, P. A. 2017; 43 (2): 227–39.e5

    Abstract

    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 PubMedID 28943242

  • CHARGE syndrome modeling using patient-iPSCs reveals defective migration of neural crest cells harboring CHD7 mutations. eLife Okuno, H. n., Renault Mihara, F. n., Ohta, S. n., Fukuda, K. n., Kurosawa, K. n., Akamatsu, W. n., Sanosaka, T. n., Kohyama, J. n., Hayashi, K. n., Nakajima, K. n., Takahashi, T. n., Wysocka, J. n., Kosaki, K. n., Okano, H. n. 2017; 6

    Abstract

    CHARGE syndrome is caused by heterozygous mutations in the chromatin remodeler, CHD7, and is characterized by a set of malformations that, on clinical grounds, were historically postulated to arise from defects in neural crest formation during embryogenesis. To better delineate neural crest defects in CHARGE syndrome, we generated induced pluripotent stem cells (iPSCs) from two patients with typical syndrome manifestations, and characterized neural crest cells differentiated in vitro from these iPSCs (iPSC-NCCs). We found that expression of genes associated with cell migration was altered in CHARGE iPSC-NCCs compared to control iPSC-NCCs. Consistently, CHARGE iPSC-NCCs showed defective delamination, migration and motility in vitro, and their transplantation in ovo revealed overall defective migratory activity in the chick embryo. These results support the historical inference that CHARGE syndrome patients exhibit defects in neural crest migration, and provide the first successful application of patient-derived iPSCs in modeling craniofacial disorders.

    View details for PubMedID 29179815

    View details for PubMedCentralID PMC5705211

  • Ever-Changing Landscapes: Transcriptional Enhancers in Development and Evolution CELL Long, H. K., Prescott, S. L., Wysocka, J. 2016; 167 (5): 1170-1187

    Abstract

    A class of cis-regulatory elements, called enhancers, play a central role in orchestrating spatiotemporally precise gene-expression programs during development. Consequently, divergence in enhancer sequence and activity is thought to be an important mediator of inter- and intra-species phenotypic variation. Here, we give an overview of emerging principles of enhancer function, current models of enhancer architecture, genomic substrates from which enhancers emerge during evolution, and the influence of three-dimensional genome organization on long-range gene regulation. We discuss intricate relationships between distinct elements within complex regulatory landscapes and consider their potential impact on specificity and robustness of transcriptional regulation.

    View details for DOI 10.1016/j.cell.2016.09.018

    View details for Web of Science ID 000389470100011

    View details for PubMedID 27863239

    View details for PubMedCentralID PMC5123704

  • Zika Virus Infection Induces Cranial Neural Crest Cells to Produce Cytokines at Levels Detrimental for Neurogenesis. Cell host & microbe Bayless, N. L., Greenberg, R. S., Swigut, T., Wysocka, J., Blish, C. A. 2016; 20 (4): 423-428

    Abstract

    Zika virus (ZIKV) infection during pregnancy is linked to microcephaly, which is attributed to infection of developing brain structures. ZIKV infects neural progenitor cells in vitro, though its effects on other developmentally relevant stem cell populations, including cranial neural crest cells (CNCCs), have not been assessed. CNCCs give rise to most cranial bones and exert paracrine effects on the developing brain. Here, we report that CNCCs are productively infected by ZIKV, but not by the related dengue virus. ZIKV-infected CNCCs undergo limited apoptosis but secrete cytokines that promote death and drive aberrant differentiation of neural progenitor cultures. Addition of two such cytokines, LIF or VEGF, at levels comparable to those secreted by ZIKV-infected CNCCs is sufficient to recapitulate premature neuronal differentiation and apoptotic death of neural progenitors. Thus, our results suggest that CNCC infection by ZIKV may contribute to associated embryopathies through signaling crosstalk between developing face and brain structures.

    View details for DOI 10.1016/j.chom.2016.09.006

    View details for PubMedID 27693308

    View details for PubMedCentralID PMC5113290

  • The FaceBase Consortium: a comprehensive resource for craniofacial researchers DEVELOPMENT Brinkley, J. F., Fisher, S., Harris, M. P., Holmes, G., Hooper, J. E., Jabs, E. W., Jones, K. L., Kesselman, C., Klein, O. D., Maas, R. L., Marazita, M. L., Selleri, L., Spritz, R. A., van Bakel, H., Visel, A., Williams, T. J., Wysocka, J., Consortium, F., Chai, Y. 2016; 143 (14): 2677-2688

    View details for DOI 10.1242/dev.135434

    View details for Web of Science ID 000392715600019

  • The FaceBase Consortium: a comprehensive resource for craniofacial researchers. Development Brinkley, J. F., Fisher, S., Harris, M. P., Holmes, G., Hooper, J. E., Jabs, E. W., Jones, K. L., Kesselman, C., Klein, O. D., Maas, R. L., Marazita, M. L., Selleri, L., Spritz, R. A., van Bakel, H., Visel, A., Williams, T. J., Wysocka, J., Chai, Y. 2016; 143 (14): 2677-2688

    Abstract

    The FaceBase Consortium, funded by the National Institute of Dental and Craniofacial Research, National Institutes of Health, is designed to accelerate understanding of craniofacial developmental biology by generating comprehensive data resources to empower the research community, exploring high-throughput technology, fostering new scientific collaborations among researchers and human/computer interactions, facilitating hypothesis-driven research and translating science into improved health care to benefit patients. The resources generated by the FaceBase projects include a number of dynamic imaging modalities, genome-wide association studies, software tools for analyzing human facial abnormalities, detailed phenotyping, anatomical and molecular atlases, global and specific gene expression patterns, and transcriptional profiling over the course of embryonic and postnatal development in animal models and humans. The integrated data visualization tools, faceted search infrastructure, and curation provided by the FaceBase Hub offer flexible and intuitive ways to interact with these multidisciplinary data. In parallel, the datasets also offer unique opportunities for new collaborations and training for researchers coming into the field of craniofacial studies. Here, we highlight the focus of each spoke project and the integration of datasets contributed by the spokes to facilitate craniofacial research.

    View details for DOI 10.1242/dev.135434

    View details for PubMedID 27287806

    View details for PubMedCentralID PMC4958338

  • 7SK-BAF axis controls pervasive transcription at enhancers. Nature structural & molecular biology Flynn, R. A., Do, B. T., Rubin, A. J., Calo, E., Lee, B., Kuchelmeister, H., Rale, M., Chu, C., Kool, E. T., Wysocka, J., Khavari, P. A., Chang, H. Y. 2016; 23 (3): 231-238

    Abstract

    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

  • Foxd3 Promotes Exit from Naive Pluripotency through Enhancer Decommissioning and Inhibits Germline Specification CELL STEM CELL Respuela, P., Nikolic, M., Tan, M., Frommolt, P., Zhao, Y., Wysocka, J., Rada-Iglesias, A. 2016; 18 (1): 118-133
  • Foxd3 Promotes Exit from Naive Pluripotency through Enhancer Decommissioning and Inhibits Germline Specification. Cell stem cell Respuela, P., Nikolic, M., Tan, M., Frommolt, P., Zhao, Y., Wysocka, J., Rada-Iglesias, A. 2016; 18 (1): 118-133

    Abstract

    Following implantation, mouse epiblast cells transit from a naive to a primed state in which they are competent for both somatic and primordial germ cell (PGC) specification. Using mouse embryonic stem cells as an in vitro model to study the transcriptional regulatory principles orchestrating peri-implantation development, here we show that the transcription factor Foxd3 is necessary for exit from naive pluripotency and progression to a primed pluripotent state. During this transition, Foxd3 acts as a repressor that dismantles a significant fraction of the naive pluripotency expression program through decommissioning of active enhancers associated with key naive pluripotency and early germline genes. Subsequently, Foxd3 needs to be silenced in primed pluripotent cells to allow re-activation of relevant genes required for proper PGC specification. Our findings therefore uncover a cycle of activation and deactivation of Foxd3 required for exit from naive pluripotency and subsequent PGC specification.

    View details for DOI 10.1016/j.stem.2015.09.010

    View details for PubMedID 26748758

  • HIPSTR and thousands of lncRNAs are heterogeneously expressed in human embryos, primordial germ cells and stable cell lines. Scientific reports Yunusov, D., Anderson, L., DaSilva, L. F., Wysocka, J., Ezashi, T., Roberts, R. M., Verjovski-Almeida, S. 2016; 6: 32753-?

    Abstract

    Eukaryotic genomes are transcribed into numerous regulatory long non-coding RNAs (lncRNAs). Compared to mRNAs, lncRNAs display higher developmental stage-, tissue-, and cell-subtype-specificity of expression, and are generally less abundant in a population of cells. Despite the progress in single-cell-focused research, the origins of low population-level expression of lncRNAs in homogeneous populations of cells are poorly understood. Here, we identify HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel lncRNA gene in the developmentally regulated TFAP2A locus. HIPSTR has evolutionarily conserved expression patterns, its promoter is most active in undifferentiated cells, and depletion of HIPSTR in HEK293 and in pluripotent H1BP cells predominantly affects the genes involved in early organismal development and cell differentiation. Most importantly, we find that HIPSTR is specifically induced and heterogeneously expressed in the 8-cell-stage human embryos during the major wave of embryonic genome activation. We systematically explore the phenomenon of cell-to-cell variation of gene expression and link it to low population-level expression of lncRNAs, showing that, similar to HIPSTR, the expression of thousands of lncRNAs is more highly heterogeneous than the expression of mRNAs in the individual, otherwise indistinguishable cells of totipotent human embryos, primordial germ cells, and stable cell lines.

    View details for DOI 10.1038/srep32753

    View details for PubMedID 27605307

    View details for PubMedCentralID PMC5015059

  • The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming. Nature genetics Durruthy-Durruthy, J., Sebastiano, V., Wossidlo, M., Cepeda, D., Cui, J., Grow, E. J., Davila, J., Mall, M., Wong, W. H., Wysocka, J., Au, K. F., Reijo Pera, R. A. 2016; 48 (1): 44-52

    Abstract

    Long intergenic noncoding RNAs (lincRNAs) are derived from thousands of loci in mammalian genomes and are frequently enriched in transposable elements (TEs). Although families of TE-derived lincRNAs have recently been implicated in the regulation of pluripotency, little is known of the specific functions of individual family members. Here we characterize three new individual TE-derived human lincRNAs, human pluripotency-associated transcripts 2, 3 and 5 (HPAT2, HPAT3 and HPAT5). Loss-of-function experiments indicate that HPAT2, HPAT3 and HPAT5 function in preimplantation embryo development to modulate the acquisition of pluripotency and the formation of the inner cell mass. CRISPR-mediated disruption of the genes for these lincRNAs in pluripotent stem cells, followed by whole-transcriptome analysis, identifies HPAT5 as a key component of the pluripotency network. Protein binding and reporter-based assays further demonstrate that HPAT5 interacts with the let-7 microRNA family. Our results indicate that unique individual members of large primate-specific lincRNA families modulate gene expression during development and differentiation to reinforce cell fate.

    View details for DOI 10.1038/ng.3449

    View details for PubMedID 26595768

  • The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming NATURE GENETICS Durruthy-Durruthy, J., Sebastiano, V., Wossidlo, M., Cepeda, D., Cui, J., Grow, E. J., Davila, J., Mall, M., Wong, W. H., Wysocka, J., Au, K. F., Pera, R. A. 2016; 48 (1): 44-?

    View details for DOI 10.1038/ng.3449

    View details for Web of Science ID 000367255300012

  • RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature Calo, E., Flynn, R. A., Martin, L., Spitale, R. C., Chang, H. Y., Wysocka, J. 2015; 518 (7538): 249-253

    Abstract

    DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle, but the molecular underpinnings of their involvement, particularly in mammalian cells, remain poorly understood. Here we show that the DEAD-box RNA helicase DDX21 can sense the transcriptional status of both RNA polymerase (Pol) I and II to control multiple steps of ribosome biogenesis in human cells. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of RNA, most prominently with non-coding RNAs involved in the formation of ribonucleoprotein complexes, including ribosomal RNA, small nucleolar RNAs (snoRNAs) and 7SK RNA. Although broad, these molecular interactions, both at the chromatin and RNA level, exhibit remarkable specificity for the regulation of ribosomal genes. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and snoRNAs, and promotes rRNA transcription, processing and modification. In the nucleoplasm, DDX21 binds 7SK RNA and, as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, is recruited to the promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. Promoter-bound DDX21 facilitates the release of the positive transcription elongation factor b (P-TEFb) from the 7SK snRNP in a manner that is dependent on its helicase activity, thereby promoting transcription of its target genes. Our results uncover the multifaceted role of DDX21 in multiple steps of ribosome biogenesis, and provide evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control.

    View details for DOI 10.1038/nature13923

    View details for PubMedID 25470060

  • RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature Calo, E., Flynn, R. A., Martin, L., Spitale, R. C., Chang, H. Y., Wysocka, J. 2015; 518 (7538): 249-253

    View details for DOI 10.1038/nature13923

    View details for PubMedID 25470060

  • ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation. eLife Nady, N., Gupta, A., Ma, Z., Swigut, T., Koide, A., Koide, S., Wysocka, J. 2015; 4

    Abstract

    Prdm14 is a sequence-specific transcriptional regulator of embryonic stem cell (ESC) pluripotency and primordial germ cell (PGC) formation. It exerts its function, at least in part, through repressing genes associated with epigenetic modification and cell differentiation. Here, we show that this repressive function is mediated through an ETO-family co-repressor Mtgr1, which tightly binds to the pre-SET/SET domains of Prdm14 and co-occupies its genomic targets in mouse ESCs. We generated two monobodies, synthetic binding proteins, targeting the Prdm14 SET domain and demonstrate their utility, respectively, in facilitating crystallization and structure determination of the Prdm14-Mtgr1 complex, or as genetically encoded inhibitor of the Prdm14-Mtgr1 interaction. Structure-guided point mutants and the monobody abrogated the Prdm14-Mtgr1 association and disrupted Prdm14's function in mESC gene expression and PGC formation in vitro. Altogether, our work uncovers the molecular mechanism underlying Prdm14-mediated repression and provides renewable reagents for studying and controlling Prdm14 functions.

    View details for DOI 10.7554/eLife.10150

    View details for PubMedID 26523391

    View details for PubMedCentralID PMC4749557

  • Inappropriate p53 activation during development induces features of CHARGE syndrome NATURE Van Nostrand, J. L., Brady, C. A., Jung, H., Fuentes, D. R., Kozak, M. M., Johnson, T. M., Lin, C., Lin, C., Swiderski, D. L., Vogel, H., Bernstein, J. A., Attie-Bitach, T., Chang, C., Wysocka, J., Martin, D. M., Attardi, L. D. 2014; 514 (7521): 228-?

    Abstract

    CHARGE syndrome is a multiple anomaly disorder in which patients present with a variety of phenotypes, including ocular coloboma, heart defects, choanal atresia, retarded growth and development, genitourinary hypoplasia and ear abnormalities. Despite 70-90% of CHARGE syndrome cases resulting from mutations in the gene CHD7, which encodes an ATP-dependent chromatin remodeller, the pathways underlying the diverse phenotypes remain poorly understood. Surprisingly, our studies of a knock-in mutant mouse strain that expresses a stabilized and transcriptionally dead variant of the tumour-suppressor protein p53 (p53(25,26,53,54)), along with a wild-type allele of p53 (also known as Trp53), revealed late-gestational embryonic lethality associated with a host of phenotypes that are characteristic of CHARGE syndrome, including coloboma, inner and outer ear malformations, heart outflow tract defects and craniofacial defects. We found that the p53(25,26,53,54) mutant protein stabilized and hyperactivated wild-type p53, which then inappropriately induced its target genes and triggered cell-cycle arrest or apoptosis during development. Importantly, these phenotypes were only observed with a wild-type p53 allele, as p53(25,26,53,54)(/-) embryos were fully viable. Furthermore, we found that CHD7 can bind to the p53 promoter, thereby negatively regulating p53 expression, and that CHD7 loss in mouse neural crest cells or samples from patients with CHARGE syndrome results in p53 activation. Strikingly, we found that p53 heterozygosity partially rescued the phenotypes in Chd7-null mouse embryos, demonstrating that p53 contributes to the phenotypes that result from CHD7 loss. Thus, inappropriate p53 activation during development can promote CHARGE phenotypes, supporting the idea that p53 has a critical role in developmental syndromes and providing important insight into the mechanisms underlying CHARGE syndrome.

    View details for DOI 10.1038/nature13585

    View details for Web of Science ID 000342663100042

  • Inappropriate p53 activation during development induces features of CHARGE syndrome. Nature Van Nostrand, J. L., Brady, C. A., Jung, H., Fuentes, D. R., Kozak, M. M., Johnson, T. M., Lin, C., Lin, C., Swiderski, D. L., Vogel, H., Bernstein, J. A., Attié-Bitach, T., Chang, C., Wysocka, J., Martin, D. M., Attardi, L. D. 2014; 514 (7521): 228-232

    Abstract

    CHARGE syndrome is a multiple anomaly disorder in which patients present with a variety of phenotypes, including ocular coloboma, heart defects, choanal atresia, retarded growth and development, genitourinary hypoplasia and ear abnormalities. Despite 70-90% of CHARGE syndrome cases resulting from mutations in the gene CHD7, which encodes an ATP-dependent chromatin remodeller, the pathways underlying the diverse phenotypes remain poorly understood. Surprisingly, our studies of a knock-in mutant mouse strain that expresses a stabilized and transcriptionally dead variant of the tumour-suppressor protein p53 (p53(25,26,53,54)), along with a wild-type allele of p53 (also known as Trp53), revealed late-gestational embryonic lethality associated with a host of phenotypes that are characteristic of CHARGE syndrome, including coloboma, inner and outer ear malformations, heart outflow tract defects and craniofacial defects. We found that the p53(25,26,53,54) mutant protein stabilized and hyperactivated wild-type p53, which then inappropriately induced its target genes and triggered cell-cycle arrest or apoptosis during development. Importantly, these phenotypes were only observed with a wild-type p53 allele, as p53(25,26,53,54)(/-) embryos were fully viable. Furthermore, we found that CHD7 can bind to the p53 promoter, thereby negatively regulating p53 expression, and that CHD7 loss in mouse neural crest cells or samples from patients with CHARGE syndrome results in p53 activation. Strikingly, we found that p53 heterozygosity partially rescued the phenotypes in Chd7-null mouse embryos, demonstrating that p53 contributes to the phenotypes that result from CHD7 loss. Thus, inappropriate p53 activation during development can promote CHARGE phenotypes, supporting the idea that p53 has a critical role in developmental syndromes and providing important insight into the mechanisms underlying CHARGE syndrome.

    View details for DOI 10.1038/nature13585

    View details for PubMedID 25119037

  • Reorganization of enhancer patterns in transition from naive to primed pluripotency. Cell stem cell Buecker, C., Srinivasan, R., Wu, Z., Calo, E., Acampora, D., Faial, T., Simeone, A., Tan, M., Swigut, T., Wysocka, J. 2014; 14 (6): 838-853

    Abstract

    Naive and primed pluripotency is characterized by distinct signaling requirements, transcriptomes, and developmental properties, but both cellular states share key transcriptional regulators: Oct4, Sox2, and Nanog. Here, we demonstrate that transition between these two pluripotent states is associated with widespread Oct4 relocalization, mirrored by global rearrangement of enhancer chromatin landscapes. Our genomic and biochemical analyses identified candidate mediators of primed state-specific Oct4 binding, including Otx2 and Zic2/3. Even when differentiation cues are blocked, premature Otx2 overexpression is sufficient to exit the naive state, induce transcription of a substantial subset of primed pluripotency-associated genes, and redirect Oct4 to previously inaccessible enhancer sites. However, the ability of Otx2 to engage new enhancer regions is determined by its levels, cis-encoded properties of the sites, and the signaling environment. Our results illuminate regulatory mechanisms underlying pluripotency and suggest that the capacity of transcription factors such as Otx2 and Oct4 to pioneer new enhancer sites is highly context dependent.

    View details for DOI 10.1016/j.stem.2014.04.003

    View details for PubMedID 24905168

  • Modification of Enhancer Chromatin: What, How, and Why? MOLECULAR CELL Calo, E., Wysocka, J. 2013; 49 (5): 825-837

    Abstract

    Emergence of form and function during embryogenesis arises in large part through cell-type- and cell-state-specific variation in gene expression patterns, mediated by specialized cis-regulatory elements called enhancers. Recent large-scale epigenomic mapping revealed unexpected complexity and dynamics of enhancer utilization patterns, with 400,000 putative human enhancers annotated by the ENCODE project alone. These large-scale efforts were largely enabled through the understanding that enhancers share certain stereotypical chromatin features. However, an important question still lingers: what is the functional significance of enhancer chromatin modification? Here we give an overview of enhancer-associated modifications of histones and DNA and discuss enzymatic activities involved in their dynamic deposition and removal. We describe potential downstream effectors of these marks and propose models for exploring functions of chromatin modification in regulating enhancer activity during development.

    View details for DOI 10.1016/j.molcel.2013.01.038

    View details for Web of Science ID 000316168000005

    View details for PubMedID 23473601

  • The chromatin remodeling factor Chd1l is required in the preimplantation embryo. Biology open Snider, A. C., Leong, D., Wang, Q. T., Wysocka, J., Yao, M. W., Scott, M. P. 2013; 2 (2): 121-131

    Abstract

    During preimplantation development, the embryo must establish totipotency and enact the earliest differentiation choices, processes that involve extensive chromatin modification. To identify novel developmental regulators, we screened for genes that are preferentially transcribed in the pluripotent inner cell mass (ICM) of the mouse blastocyst. Genes that encode chromatin remodeling factors were prominently represented in the ICM, including Chd1l, a member of the Snf2 gene family. Chd1l is developmentally regulated and expressed in embryonic stem (ES) cells, but its role in development has not been investigated. Here we show that inhibiting Chd1l protein production by microinjection of antisense morpholinos causes arrest prior to the blastocyst stage. Despite this important function in vivo, Chd1l is non-essential for cultured ES cell survival, pluripotency, or differentiation, suggesting that Chd1l is vital for events in embryos that are distinct from events in ES cells. Our data reveal a novel role for the chromatin remodeling factor Chd1l in the earliest cell divisions of mammalian development.

    View details for DOI 10.1242/bio.20122949

    View details for PubMedID 23429299

    View details for PubMedCentralID PMC3575647

  • The chromatin remodeling factor Chd1l is required in the preimplantation embryo BIOLOGY OPEN Snider, A. C., Leong, D., Wang, Q. T., Wysocka, J., Yao, M. W., Scott, M. P. 2013; 2 (2): 121-131

    Abstract

    During preimplantation development, the embryo must establish totipotency and enact the earliest differentiation choices, processes that involve extensive chromatin modification. To identify novel developmental regulators, we screened for genes that are preferentially transcribed in the pluripotent inner cell mass (ICM) of the mouse blastocyst. Genes that encode chromatin remodeling factors were prominently represented in the ICM, including Chd1l, a member of the Snf2 gene family. Chd1l is developmentally regulated and expressed in embryonic stem (ES) cells, but its role in development has not been investigated. Here we show that inhibiting Chd1l protein production by microinjection of antisense morpholinos causes arrest prior to the blastocyst stage. Despite this important function in vivo, Chd1l is non-essential for cultured ES cell survival, pluripotency, or differentiation, suggesting that Chd1l is vital for events in embryos that are distinct from events in ES cells. Our data reveal a novel role for the chromatin remodeling factor Chd1l in the earliest cell divisions of mammalian development.

    View details for DOI 10.1242/bio.20122949

    View details for Web of Science ID 000209205900003

    View details for PubMedCentralID PMC3575647

  • Human genetic variation within neural crest enhancers: molecular and phenotypic implications. Philosophical transactions of the Royal Society of London. Series B, Biological sciences Rada-Iglesias, A., Prescott, S. L., Wysocka, J. 2013; 368 (1620): 20120360-?

    Abstract

    Developmental gene expression programmes are coordinated by the specialized distal cis-regulatory elements called enhancers, which integrate lineage- and signalling-dependent inputs to guide morphogenesis. In previous work, we characterized the genome-wide repertoire of active enhancers in human neural crest cells (hNCC), an embryonic cell population with critical roles in craniofacial development. We showed that in hNCC, co-occupancy of a master regulator TFAP2A with nuclear receptors NR2F1 and NR2F2 correlates with the presence of permissive enhancer chromatin states. Here, we take advantage of pre-existing human genetic variation to further explore potential cooperation between TFAP2A and NR2F1/F2. We demonstrate that isolated single nucleotide polymorphisms affecting NR2F1/F2-binding sites within hNCC enhancers can alter TFAP2A occupancy and overall chromatin features at the same enhancer allele. We propose that a similar strategy can be used to elucidate other cooperative relationships between transcription factors involved in developmental transitions. Using the neural crest and its major contribution to human craniofacial phenotypes as a paradigm, we discuss how genetic variation might modulate the molecular properties and activity of enhancers, and ultimately impact human phenotypic diversity.

    View details for DOI 10.1098/rstb.2012.0360

    View details for PubMedID 23650634

  • Human genetic variation within neural crest enhancers: molecular and phenotypic implications. Philosophical transactions of the Royal Society of London. Series B, Biological sciences Rada-Iglesias, A., Prescott, S. L., Wysocka, J. 2013; 368 (1620): 20120360-?

    View details for DOI 10.1098/rstb.2012.0360

    View details for PubMedID 23650634

  • Epigenomic Annotation of Enhancers Predicts Transcriptional Regulators of Human Neural Crest CELL STEM CELL Rada-Iglesias, A., Bajpai, R., Prescott, S., Brugmann, S. A., Swigut, T., Wysocka, J. 2012; 11 (5): 633-648

    Abstract

    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 PubMedID 22981823

  • Enhancers as information integration hubs in development: lessons from genomics TRENDS IN GENETICS Buecker, C., Wysocka, J. 2012; 28 (6): 276-284

    Abstract

    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

  • Identification of 67 Histone Marks and Histone Lysine Crotonylation as a New Type of Histone Modification CELL Tan, M., Luo, H., Lee, S., Jin, F., Yang, J. S., Montellier, E., Buchou, T., Cheng, Z., Rousseaux, S., Rajagopal, N., Lu, Z., Ye, Z., Zhu, Q., Wysocka, J., Ye, Y., Khochbin, S., Ren, B., Zhao, Y. 2011; 146 (6): 1015-1027

    Abstract

    We report the identification of 67 previously undescribed histone modifications, increasing the current number of known histone marks by about 70%. We further investigated one of the marks, lysine crotonylation (Kcr), confirming that it represents an evolutionarily-conserved histone posttranslational modification. The unique structure and genomic localization of histone Kcr suggest that it is mechanistically and functionally different from histone lysine acetylation (Kac). Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in postmeiotic male germ cells.

    View details for DOI 10.1016/j.cell.2011.08.008

    View details for Web of Science ID 000295258100022

    View details for PubMedCentralID PMC3176443

  • Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell Tan, M., Luo, H., Lee, S., Jin, F., Yang, J. S., Montellier, E., Buchou, T., Cheng, Z., Rousseaux, S., Rajagopal, N., Lu, Z., Ye, Z., Zhu, Q., Wysocka, J., Ye, Y., Khochbin, S., Ren, B., Zhao, Y. 2011; 146 (6): 1016-1028

    Abstract

    We report the identification of 67 previously undescribed histone modifications, increasing the current number of known histone marks by about 70%. We further investigated one of the marks, lysine crotonylation (Kcr), confirming that it represents an evolutionarily-conserved histone posttranslational modification. The unique structure and genomic localization of histone Kcr suggest that it is mechanistically and functionally different from histone lysine acetylation (Kac). Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in postmeiotic male germ cells.

    View details for DOI 10.1016/j.cell.2011.08.008

    View details for PubMedID 21925322

  • A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression NATURE Wang, K. C., Yang, Y. W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B. R., Protacio, A., Flynn, R. A., Gupta, R. A., Wysocka, J., Lei, M., Dekker, J., Helms, J. A., Chang, H. Y. 2011; 472 (7341): 120-U158

    Abstract

    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 PubMedID 21423168

  • Long noncoding RNA programs active chromatin domain to coordinate homeotic gene expression 71st Annual Meeting of the Society-for-Investigative-Dermatology Wang, K. C., Yang, Y. W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B. R., Protacio, A., Flynn, R. A., Gupta, R. A., Wysocka, J., Lei, M., Dekker, J., Helms, J. A., Chang, H. Y. NATURE PUBLISHING GROUP. 2011: S63–S63
  • A unique chromatin signature uncovers early developmental enhancers in humans NATURE Rada-Iglesias, A., Bajpai, R., Swigut, T., Brugmann, S. A., Flynn, R. A., Wysocka, J. 2011; 470 (7333): 279-?

    Abstract

    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 Ma, Z., Swigut, T., Valouev, A., Rada-Iglesias, A., Wysocka, J. 2011; 18 (2): 120-U175

    Abstract

    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

  • Epigenomics of human embryonic stem cells and induced pluripotent stem cells: insights into pluripotency and implications for disease. Genome medicine Rada-Iglesias, A., Wysocka, J. 2011; 3 (6): 36-?

    Abstract

    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

    View details for PubMedCentralID PMC3218810

  • Epigenomics of human embryonic stem cells and induced pluripotent stem cells: insights into pluripotency and implications for disease GENOME MEDICINE Rada-Iglesias, A., Wysocka, J. 2011; 3

    View details for DOI 10.1186/gm252

    View details for Web of Science ID 000208627400036

  • Flipping MLL1's Switch One Proline at a Time CELL Grow, E. J., Wysocka, J. 2010; 141 (7): 1108-1110

    Abstract

    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

  • CHD7 cooperates with PBAF to control multipotent neural crest formation NATURE Bajpai, R., Chen, D. A., Rada-Iglesias, A., Zhang, J., Xiong, Y., Helms, J., Chang, C., Zhao, Y., Swigut, T., Wysocka, J. 2010; 463 (7283): 958-U135

    Abstract

    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

    View details for PubMedCentralID PMC2890258

  • MS/MS/MS Reveals False Positive Identification of Histone Serine Methylation JOURNAL OF PROTEOME RESEARCH Zhang, J., Chen, Y., Zhang, Z., Xing, G., Wysocka, J., Zhao, Y. 2010; 9 (1): 585-594

    Abstract

    Methylation of lysine and arginine residues is known to play a key role in regulating histone structure and function. However, methylation of other amino acid residues in histones has not been previously described. Using exhaustive nano-HPLC/MS/MS and blind protein sequence database searches, we tentatively assigned methylation to serine 28 of histone H3 from calf thymus. The assignment was in agreement with our stringent manual verification rules, coelution in HPLC/MS/MS with its corresponding synthetic peptide, the dynamic nature of such methylation in distinct cell lines, and isotopic labeling. However, careful inspection of the MS/MS and MS/MS/MS spectra of a series of synthetic peptides confirmed that methylation actually occurs on K27 rather than on S28. The misassignment was caused by the fact that the (y(9) + 14) of the putative S28-methylated peptide and (b(9) + 18) ions of the K27 methylated peptide share the same m/z value (m/z 801). This MS/MS peak was used as the major evidence to assign methylation to S28 (consecutive y(8) and (y(9) + 14) ions). MS/MS/MS analysis revealed the false positive nature of serine methylation: the ambiguous ion at m/z 801 is indeed (b(9) + 18), an ion resulting from an in vitro reaction in the gas phase during collisionally activated dissociation (CAD). When lysine (K27) was acetylated, the degree of such in vitro reactions was greatly reduced, and such reactions were completely eliminated when the C-terminus was blocked by carboxylic group derivatization. Moreover, such side-chain assisted C-terminal rearrangement was found to be charge dependent. In aggregate, these results suggest that extra caution should be taken in interpretation of post-translational modification (PTM) data and that MS/MS as well as MS/MS/MS of synthetic peptides are needed for verifying the identity of peptides bearing a novel PTM.

    View details for DOI 10.1021/pr900864s

    View details for Web of Science ID 000273267900055

    View details for PubMedID 19877717

    View details for PubMedCentralID PMC2801770

  • Jarid2/Jumonji Coordinates Control of PRC2 Enzymatic Activity and Target Gene Occupancy in Pluripotent Cells CELL Peng, J. C., Valouev, A., Swigut, T., Zhang, J., Zhao, Y., Sidow, A., Wysocka, J. 2009; 139 (7): 1290-1302

    Abstract

    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

    View details for PubMedCentralID PMC2911953

  • Fallen Immortals CELL Swigut, T., Wysocka, J. 2009; 137 (2): 203-205

    Abstract

    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

  • Chromatin remodelling factor Mll1 is essential for neurogenesis from postnatal neural stem cells NATURE Lim, D. A., Huang, Y., Swigut, T., Mirick, A. L., Manuel Garcia-Verdugo, J., Wysocka, J., Ernst, P., Alvarez-Buylla, A. 2009; 458 (7237): 529-U9

    Abstract

    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

  • It takes a PHD to SUMO TRENDS IN BIOCHEMICAL SCIENCES Peng, J., Wysocka, J. 2008; 33 (5): 191-194

    Abstract

    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 PLOS ONE Lee, S., Horn, V., Julien, E., Liu, Y., Wysocka, J., Bowerman, B., Hengartner, M. O., Herr, W. 2007; 2 (11)

    Abstract

    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

    View details for PubMedCentralID PMC2082077

  • H3K27 demethylases, at long last CELL Swigut, T., Wysocka, J. 2007; 131 (1): 29-32

    Abstract

    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 MOLECULAR CELL Tyagi, S., Chabes, A. L., Wysocka, J., Herr, W. 2007; 27 (1): 107-119

    Abstract

    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 MOLECULAR CELL Ruthenburg, A. J., Allis, C. D., Wysocka, J. 2007; 25 (1): 15-30

    Abstract

    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

  • Identifying novel proteins recognizing histone modifications using peptide pull-down assay METHODS Wysocka, J. 2006; 40 (4): 339-343

    Abstract

    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 NATURE Li, H., Ilin, S., Wang, W., Duncan, E. M., Wysocka, J., Allis, C. D., Patel, D. J. 2006; 442 (7098): 91-95

    Abstract

    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

  • A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling NATURE Wysocka, J., Swigut, T., Xiao, H., Milne, T. A., Kwon, S. Y., Landry, J., Kauer, M., Tackett, A. J., Chait, B. T., Badenhorst, P., Wu, C., Allis, C. D. 2006; 442 (7098): 86-90

    Abstract

    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

  • Histone arginine methylation and its dynamic regulation FRONTIERS IN BIOSCIENCE-LANDMARK Wysocka, J., Allis, C. D., Coonrod, S. 2006; 11: 344-355

    Abstract

    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 CELL Wysocka, J., Milne, T. A., Allis, C. D. 2005; 122 (5): 654-658

    Abstract

    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 CELL Dou, Y. L., Milne, T. A., Tackett, A. J., Smith, E. R., Fukuda, A., Wysocka, J., Allis, C. D., Chait, B. T., Hess, J. L., Roeder, R. G. 2005; 121 (6): 873-885

    Abstract

    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

  • WDR5 associates with histone H3 methylated at K4 and is essential for H3K4 methylation and vertebrate development CELL Wysocka, J., Swigut, T., Milne, T. A., Dou, Y. L., Zhang, X., Burlingame, A. L., Roeder, R. G., Brivanlou, A. H., Allis, C. D. 2005; 121 (6): 859-872

    Abstract

    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

  • Human PAD4 regulates histone arginine methylation levels via demethylimination SCIENCE Wang, Y., Wysocka, J., Sayegh, J., Lee, Y. H., Perlin, J. R., Leonelli, L., Sonbuchner, L. S., McDonald, C. H., COOK, R. G., Dou, Y., Roeder, R. G., Clarke, S., Stallcup, M. R., Allis, C. D., Coonrod, S. A. 2004; 306 (5694): 279-283

    Abstract

    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 Yokoyama, A., Wang, Z., Wysocka, J., Sanyal, M., Aufiero, D. J., Kitabayashi, I., Herr, W., Cleary, M. L. 2004; 24 (13): 5639-5649

    Abstract

    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

    View details for PubMedCentralID PMC480881

  • Linking covalent histone modifications to epigenetics: The rigidity and plasticity of the marks 69th Cold Spring Harbor Symposium on Quantitative Biology Wang, Y., Wysocka, J., Perlin, J. R., Leonelli, L., Allis, C. D., Coonrod, S. A. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 2004: 161–169

    View details for Web of Science ID 000232227200021

    View details for PubMedID 16117646

  • The herpes simplex virus VP16-induced complex: the makings of a regulatory switch TRENDS IN BIOCHEMICAL SCIENCES Wysocka, J., Herr, W. 2003; 28 (6): 294-304

    Abstract

    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 Wysocka, J., Myers, M. P., Laherty, C. D., Eisenman, R. N., Herr, W. 2003; 17 (7): 896-911

    Abstract

    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 Reilly, P. T., Wysocka, J., Herr, W. 2002; 22 (19): 6767-6778

    Abstract

    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 Wysocka, J., Reilly, P. T., Herr, W. 2001; 21 (11): 3820-3829

    Abstract

    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 BIOCHEMISTRY Wysocka, J., Liu, Y., Kobayashi, R., Herr, W. 2001; 40 (19): 5786-5794

    Abstract

    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