Honors & Awards
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)
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, canonically defined as short noncoding DNA sequences that act to drive transcription independent of their relative distance, location or orientation to their cognate promoter. 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 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 frogs and, in collaboration with human geneticists and anthropologists, with the 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.
- Research Seminar
CSB 270 (Aut, Win, Spr)
- The Biology of Chromatin Templated Processes
CSB 250 (Win)
Independent Studies (16)
- Directed Reading in Cancer Biology
CBIO 299 (Win, Spr)
- Directed Reading in Chemical and Systems Biology
CSB 299 (Aut, Win, Spr, Sum)
- Directed Reading in Developmental Biology
DBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Stem Cell Biology and Regenerative Medicine
STEMREM 299 (Win, Spr)
- Graduate Research
CBIO 399 (Win, Spr)
- Graduate Research
CSB 399 (Aut, Win, Spr, Sum)
- Graduate Research
DBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
STEMREM 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
CSB 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
DBIO 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
STEMREM 370 (Win, Spr)
- Out-of-Department Graduate Research
BIO 300X (Aut, Win, Spr, Sum)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Undergraduate Research
CSB 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
DBIO 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
STEMREM 199 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
- Prior Year Courses
Doctoral Dissertation Reader (AC)
Laura Amaya, Nicole DelRosso, TzuChiao Hung, Thomas Lozanoski, Joseph Park, Joydeb Sinha, Ali Wilkening
Postdoctoral Faculty Sponsor
Shiran Bar, Liang-Fu Chen, Tiffany Chern, Raquel Fueyo, Sahin Naqvi
Doctoral Dissertation Advisor (AC)
Alex Adams, Christina Jensen, Naz Koska, Andrew Spencley, Saman Tabatabaee, Jordan Valgardson
Doctoral Dissertation Co-Advisor (AC)
Postdoctoral Research Mentor
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
2022; 36 Suppl 1
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
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
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.
2021; 12 (1): 5771
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.
2021; 17 (8): e1009695
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.
2021; 17 (5): e1009528
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.
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
Reactivation of the pluripotency program precedes formation of the cranial neural crest.
Science (New York, N.Y.)
2021; 371 (6529)
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
The Intersection of the Genetic Architectures of Orofacial Clefts and Normal Facial Variation.
Frontiers in genetics
2021; 12: 626403
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.
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.
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
- Publisher Correction: Human-chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution. Nature genetics 2021
Insights into the genetic architecture of the human face.
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)
2020; 147 (18)
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
Opposing Effects of Cohesin and Transcription on CTCF Organization Revealed by Super-resolution Imaging.
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
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
2020; 375 (1795): 20190347
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
Epigenomic and Transcriptomic Changes During Human RPE EMT in a Stem Cell Model of Epiretinal Membrane Pathogenesis and Prevention by Nicotinamide.
Stem cell reports
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
Zscan4 binds nucleosomal microsatellite DNA and protects mouse two-cell embryos from DNA damage.
2020; 6 (12): eaaz9115
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
Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder.
Cell stem cell
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
The Spatiotemporal Pattern and Intensity of p53 Activation Dictates Phenotypic Diversity in p53-Driven Developmental Syndromes.
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 WILEY. 2019: 207–12
SNPs discovered in European GWAS shape worldwide facial diversity
WILEY. 2019: 227
View details for Web of Science ID 000458409602325
Meta-analysis identifies 48 SNPs with multiple independent effects on human facial features
WILEY. 2019: 267
View details for Web of Science ID 000458409603108
Hunting for genes that shape human faces: Initial successes and challenges for the future.
Orthodontics & craniofacial research
2019; 22 Suppl 1: 207–12
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
2019; 12 (6): 1183–85
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.
2019; 574 (7779): 553–58
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
Single Amino Acid Change Underlies Distinct Roles of H2A.Z Subtypes in Human Syndrome.
2019; 178 (6): 1421–36.e24
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
Systematic perturbation of retroviral LTRs reveals widespread long-range effects on human gene regulation.
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
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018
View details for Web of Science ID 000442912507160
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
2018; 4 (6): e00667
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
Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements
2018; 359 (6379): 1050–55
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
Genome-wide mapping of global-to-local genetic effects on human facial shape
2018; 50 (3): 414-+
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
- Histone H3 lysine 4 monomethylation modulates longrange chromatin interactions at enhancers (vol 28, pg 204, 2018) CELL RESEARCH 2018; 28 (3): 387
Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders
2018; 554 (7690): 112-+
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.
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
CSNK1a1 Regulates PRMT1 to Maintain the Progenitor State in Self-Renewing Somatic Tissue.
2017; 43 (2): 227–39.e5
Somatic progenitors sustain tissue self-renewal while suppressing premature differentiation. Protein arginine methyltransferases (PRMTs) affect many processes; however, their role in progenitor function is incompletely understood. PRMT1 was found to be the most highly expressed PRMT in epidermal progenitors and the most downregulated PRMT during differentiation. In targeted mouse knockouts and in long-term regenerated human mosaic epidermis in vivo, epidermal PRMT1 loss abolished progenitor self-renewal and led to premature differentiation. Mass spectrometry of the PRMT1 protein interactome identified the CSNK1a1 kinase, which also proved essential for progenitor maintenance. CSNK1a1 directly bound and phosphorylated PRMT1 to control its genomic targeting to PRMT1-sustained proliferation genes as well as PRMT1-suppressed differentiation genes. Among the latter were GRHL3, whose derepression was required for the premature differentiation seen with PRMT1 and CSNK1a1 loss. Maintenance of the progenitors thus requires cooperation by PRMT1 and CSNK1a1 to sustain proliferation gene expression and suppress premature differentiation driven by GRHL3.
View details for PubMedID 28943242
CHARGE syndrome modeling using patient-iPSCs reveals defective migration of neural crest cells harboring CHD7 mutations.
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
Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators.
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 2015; 163 (1): 68-83
Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells.
2015; 522 (7555): 221-225
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
Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification.
2011; 146 (6): 1016-1028
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
E2F activation of S phase promoters via association with HCF-1 and the MLL family of histone H3K4 methyltransferases
2007; 27 (1): 107-119
E2F transcriptional regulators control human-cell proliferation by repressing and activating the transcription of genes required for cell-cycle progression, particularly the S phase. E2F proteins repress transcription in association with retinoblastoma pocket proteins, but less is known about how they activate transcription. Here, we show that the human G1 phase regulator HCF-1 associates with both activator (E2F1 and E2F3a) and repressor (E2F4) E2F proteins, properties that are conserved in insect cells. Human HCF-1-E2F interactions are versatile: their associations and binding to E2F-responsive promoters are cell-cycle selective, and HCF-1 displays coactivator properties when bound to the E2F1 activator and corepressor properties when bound to the E2F4 repressor. During the G1-to-S phase transition, HCF-1 recruits the mixed-lineage leukemia (MLL) and Set-1 histone H3 lysine 4 methyltransferases to E2F-responsive promoters and induces histone methylation and transcriptional activation. These results suggest that HCF-1 induces cell-cycle-specific transcriptional activation by E2F proteins to promote cell proliferation.
View details for DOI 10.1016/j.molcel.2007.05.030
View details for Web of Science ID 000248088500010
View details for PubMedID 17612494
Methylation of lysine 4 on histone H3: Intricacy of writing and reading a single epigenetic mark
2007; 25 (1): 15-30
Cells employ elaborate mechanisms to introduce structural and chemical variation into chromatin. Here, we focus on one such element of variation: methylation of lysine 4 in histone H3 (H3K4). We assess a growing body of literature, including treatment of how the mark is established, the patterns of methylation, and the functional consequences of this epigenetic signature. We discuss structural aspects of the H3K4 methyl recognition by the downstream effectors and propose a distinction between sequence-specific recruitment mechanisms and stabilization on chromatin through methyl-lysine recognition. Finally, we hypothesize how the unique properties of the polyvalent chromatin fiber and associated effectors may amplify small differences in methyl-lysine recognition, simultaneously allowing for a dynamic chromatin architecture.
View details for DOI 10.1016/j.molcel.2006.12.014
View details for Web of Science ID 000243566700002
View details for PubMedID 17218268
Identifying novel proteins recognizing histone modifications using peptide pull-down assay
2006; 40 (4): 339-343
Post-translational modifications of histones have been correlated with virtually all chromatin-templated processes, including gene expression regulation, DNA replication, mitosis and meiosis, and DNA repair. In order to better understand the mechanistic basis by which histone modifications participate in the control of cellular processes, it is essential to identify and characterize downstream effector proteins, or "readers", that are responsible for recognizing different marks and translating them into specific biological outcomes. Ideally, identification of potential histone-binding effectors should occur in an unbiased fashion. Although in the recent years much progress has been made in identifying readers of histone modifications, in particular methylation, recognition of the majority of known histone marks is still poorly understood. Here I describe a simple and unbiased biochemical pull-down assay that allows for the identification of novel histone effector proteins and utilizes biotinylated histone peptides modified at various residues. I provide detailed protocols and suggestions for troubleshooting.
View details for DOI 10.1016/j.ymeth.2006.05.028
View details for Web of Science ID 000242902600008
View details for PubMedID 17101446
A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling
2006; 442 (7098): 86-90
Lysine methylation of histones is recognized as an important component of an epigenetic indexing system demarcating transcriptionally active and inactive chromatin domains. Trimethylation of histone H3 lysine 4 (H3K4me3) marks transcription start sites of virtually all active genes. Recently, we reported that the WD40-repeat protein WDR5 is important for global levels of H3K4me3 and control of HOX gene expression. Here we show that a plant homeodomain (PHD) finger of nucleosome remodelling factor (NURF), an ISWI-containing ATP-dependent chromatin-remodelling complex, mediates a direct preferential association with H3K4me3 tails. Depletion of H3K4me3 causes partial release of the NURF subunit, BPTF (bromodomain and PHD finger transcription factor), from chromatin and defective recruitment of the associated ATPase, SNF2L (also known as ISWI and SMARCA1), to the HOXC8 promoter. Loss of BPTF in Xenopus embryos mimics WDR5 loss-of-function phenotypes, and compromises spatial control of Hox gene expression. These results strongly suggest that WDR5 and NURF function in a common biological pathway in vivo, and that NURF-mediated ATP-dependent chromatin remodelling is directly coupled to H3K4 trimethylation to maintain Hox gene expression patterns during development. We also identify a previously unknown function for the PHD finger as a highly specialized methyl-lysine-binding domain.
View details for DOI 10.1038/nature04815
View details for Web of Science ID 000238724500042
View details for PubMedID 16728976
Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF
2006; 442 (7098): 91-95
Mono-, di- and trimethylated states of particular histone lysine residues are selectively found in different regions of chromatin, thereby implying specialized biological functions for these marks ranging from heterochromatin formation to X-chromosome inactivation and transcriptional regulation. A major challenge in chromatin biology has centred on efforts to define the connection between specific methylation states and distinct biological read-outs impacting on function. For example, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with transcription start sites of active genes, but the molecular 'effectors' involved in specific recognition of H3K4me3 tails remain poorly understood. Here we demonstrate the molecular basis for specific recognition of H3(1-15)K4me3 (residues 1-15 of histone H3 trimethylated at K4) by a plant homeodomain (PHD) finger of human BPTF (bromodomain and PHD domain transcription factor), the largest subunit of the ATP-dependent chromatin-remodelling complex, NURF (nucleosome remodelling factor). We report on crystallographic and NMR structures of the bromodomain-proximal PHD finger of BPTF in free and H3(1-15)K4me3-bound states. H3(1-15)K4me3 interacts through anti-parallel beta-sheet formation on the surface of the PHD finger, with the long side chains of arginine 2 (R2) and K4me3 fitting snugly in adjacent pre-formed surface pockets, and bracketing an invariant tryptophan. The observed stapling role by non-adjacent R2 and K4me3 provides a molecular explanation for H3K4me3 site specificity. Binding studies establish that the BPTF PHD finger exhibits a modest preference for K4me3- over K4me2-containing H3 peptides, and discriminates against monomethylated and unmodified counterparts. Furthermore, we identified key specificity-determining residues from binding studies of H3(1-15)K4me3 with PHD finger point mutants. Our findings call attention to the PHD finger as a previously uncharacterized chromatin-binding module found in a large number of chromatin-associated proteins.
View details for DOI 10.1038/nature04802
View details for Web of Science ID 000238724500043
View details for PubMedID 16728978
Histone arginine methylation and its dynamic regulation
FRONTIERS IN BIOSCIENCE-LANDMARK
2006; 11: 344-355
Methylation of histones by protein arginine methyltransferases (PRMTs) is increasingly being found to play an important and dynamic role in gene regulation. In mammals, PRMT1- and CARM1-catalyzed histone asymmetric dimethyl-arginine is involved in gene activation while PRMT5-catalyzed histone symmetric dimethyl-arginine is associated with gene repression. Insight into mechanisms by which histone arginine methylation can be dynamically regulated comes from recent reports demonstrating that conversion of histone methylarginine residues to citrulline by peptidylarginine deiminase 4 (PADI4) leads to transcriptional repression. While the downstream cellular effects of histone arginine methylation remain poorly understood, recent findings indicate that protein arginine methylation, in general, is required for mammalian development and is also likely important for cellular proliferation and differentiation. Given the surge of interest in histone arginine methylation, this review article will focus on recent progress in this area.
View details for Web of Science ID 000232528000028
View details for PubMedID 16146736
Taking LSD1 to a new high
2005; 122 (5): 654-658
Histone modifications mediate changes in gene expression by altering the underlying chromatin structure or by serving as a binding platform to recruit other proteins. One such modification, histone methylation, was thought to be irreversible until last year when Shi and co-workers broke new ground with their discovery of a lysine-specific histone demethylase (LSD 1). They showed that LSD 1, a nuclear amine oxidase homolog, is a bona fide histone H3 lysine 4 demethylase (Shi et al., 2004). Now, a new study from published in a recent issue of Molecular Cell, together with two studies recently published by and in Nature, reveal that LSD 1's specificity and activity is in fact regulated by associated protein cofactors.
View details for DOI 10.1016/j.cell.2005.08.022
View details for Web of Science ID 000231844300006
View details for PubMedID 16143099
Physical association and coordinate function of the H3K4 methyltransferase MLL1 and the H4K16 acetyltransferase MOF
2005; 121 (6): 873-885
A stable complex containing MLL1 and MOF has been immunoaffinity purified from a human cell line that stably expresses an epitope-tagged WDR5 subunit. Stable interactions between MLL1 and MOF were confirmed by reciprocal immunoprecipitation, cosedimentation, and cotransfection analyses, and interaction sites were mapped to MLL1 C-terminal and MOF zinc finger domains. The purified complex has a robust MLL1-mediated histone methyltransferase activity that can effect mono-, di-, and trimethylation of H3 K4 and a MOF-mediated histone acetyltransferase activity that is specific for H4 K16. Importantly, both activities are required for optimal transcription activation on a chromatin template in vitro and on an endogenous MLL1 target gene, Hox a9, in vivo. These results indicate an activator-based mechanism for joint MLL1 and MOF recruitment and targeted methylation and acetylation and provide a molecular explanation for the closely correlated distribution of H3 K4 methylation and H4 K16 acetylation on active genes.
View details for DOI 10.1016/j.cell.2005.04.031
View details for Web of Science ID 000230011200010
View details for PubMedID 15960975
WDR5 associates with histone H3 methylated at K4 and is essential for H3K4 methylation and vertebrate development
2005; 121 (6): 859-872
Histone H3 lysine 4 (K4) methylation has been linked to the transcriptional activation in a variety of eukaryotic species. Here we show that a common component of MLL1, MLL2, and hSet1 H3 K4 methyltransferase complexes, the WD40-repeat protein WDR5, directly associates with histone H3 di- and trimethylated at K4 and with H3-K4-dimethylated nucleosomes. WDR5 is required for binding of the methyltransferase complex to the K4-dimethylated H3 tail as well as for global H3 K4 trimethylation and HOX gene activation in human cells. WDR5 is essential for vertebrate development, in that WDR5-depleted X. laevis tadpoles exhibit a variety of developmental defects and abnormal spatial Hox gene expression. Our results are the first demonstration that a WD40-repeat protein acts as a module for recognition of a specific histone modification and suggest a mechanism for reading and writing an epigenetic mark for gene activation.
View details for DOI 10.1016/j.cell.2005.03.036
View details for Web of Science ID 000230011200009
View details for PubMedID 15960974
Human PAD4 regulates histone arginine methylation levels via demethylimination
2004; 306 (5694): 279-283
Methylation of arginine (Arg) and lysine residues in histones has been correlated with epigenetic forms of gene regulation. Although histone methyltransferases are known, enzymes that demethylate histones have not been identified. Here, we demonstrate that human peptidylarginine deiminase 4 (PAD4) regulates histone Arg methylation by converting methyl-Arg to citrulline and releasing methylamine. PAD4 targets multiple sites in histones H3 and H4, including those sites methylated by coactivators CARM1 (H3 Arg17) and PRMT1 (H4 Arg3). A decrease of histone Arg methylation, with a concomitant increase of citrullination, requires PAD4 activity in human HL-60 granulocytes. Moreover, PAD4 activity is linked with the transcriptional regulation of estrogen-responsive genes in MCF-7 cells. These data suggest that PAD4 mediates gene expression by regulating Arg methylation and citrullination in histones.
View details for DOI 10.1126/science.1101400
View details for Web of Science ID 000224419700041
View details for PubMedID 15345777
Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression
MOLECULAR AND CELLULAR BIOLOGY
2004; 24 (13): 5639-5649
MLL (for mixed-lineage leukemia) is a proto-oncogene that is mutated in a variety of human leukemias. Its product, a homolog of Drosophila melanogaster trithorax, displays intrinsic histone methyltransferase activity and functions genetically to maintain embryonic Hox gene expression. Here we report the biochemical purification of MLL and demonstrate that it associates with a cohort of proteins shared with the yeast and human SET1 histone methyltransferase complexes, including a homolog of Ash2, another Trx-G group protein. Two other members of the novel MLL complex identified here are host cell factor 1 (HCF-1), a transcriptional coregulator, and the related HCF-2, both of which specifically interact with a conserved binding motif in the MLL(N) (p300) subunit of MLL and provide a potential mechanism for regulating its antagonistic transcriptional properties. Menin, a product of the MEN1 tumor suppressor gene, is also a component of the 1-MDa MLL complex. Abrogation of menin expression phenocopies loss of MLL and reveals a critical role for menin in the maintenance of Hox gene expression. Oncogenic mutant forms of MLL retain an ability to interact with menin but not other identified complex components. These studies link the menin tumor suppressor protein with the MLL histone methyltransferase machinery, with implications for Hox gene expression in development and leukemia pathogenesis.
View details for DOI 10.1128/MCB.24.13.5639-5649.2004
View details for Web of Science ID 000222149200001
View details for PubMedID 15199122
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 COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 2004: 161–169
The herpes simplex virus VP16-induced complex: the makings of a regulatory switch
TRENDS IN BIOCHEMICAL SCIENCES
2003; 28 (6): 294-304
When herpes simplex virus (HSV) infects human cells, it is able to enter two modes of infection: lytic and latent. A key activator of lytic infection is a virion protein called VP16, which, upon infection of a permissive cell, forms a transcriptional regulatory complex with two cellular proteins - the POU-domain transcription factor Oct-1 and the cell-proliferation factor HCF-1 - to activate transcription of the first set of expressed viral genes. This regulatory complex, called the VP16-induced complex, reveals mechanisms of combinatorial control of transcription. The activities of Oct-1 and HCF-1 - two important regulators of cellular gene expression and proliferation - illuminate strategies by which HSV might coexist with its host.
View details for DOI 10.1016/S0968-0004(03)00088-4
View details for Web of Science ID 000183948400004
View details for PubMedID 12826401
Human Sin3 deacetylase and trithorax-related Set1/Ash2 histone H3-K4 methyltransferase are tethered together selectively by the cell-proliferation factor HCF-1
GENES & DEVELOPMENT
2003; 17 (7): 896-911
The abundant and chromatin-associated protein HCF-1 is a critical player in mammalian cell proliferation as well as herpes simplex virus (HSV) transcription. We show here that separate regions of HCF-1 critical for its role in cell proliferation associate with the Sin3 histone deacetylase (HDAC) and a previously uncharacterized human trithorax-related Set1/Ash2 histone methyltransferase (HMT). The Set1/Ash2 HMT methylates histone H3 at Lys 4 (K4), but not if the neighboring K9 residue is already methylated. HCF-1 tethers the Sin3 and Set1/Ash2 transcriptional regulatory complexes together even though they are generally associated with opposite transcriptional outcomes: repression and activation of transcription, respectively. Nevertheless, this tethering is context-dependent because the transcriptional activator VP16 selectively binds HCF-1 associated with the Set1/Ash2 HMT complex in the absence of the Sin3 HDAC complex. These results suggest that HCF-1 can broadly regulate transcription, both positively and negatively, through selective modulation of chromatin structure.
View details for DOI 10.1101/gad.252103
View details for Web of Science ID 000182044700010
View details for PubMedID 12670868
Inactivation of the retinoblastoma protein family can bypass the HCF-1 defect in tsBN67 cell proliferation and cytokinesis
MOLECULAR AND CELLULAR BIOLOGY
2002; 22 (19): 6767-6778
Owing to a single missense mutation in the cell proliferation factor HCF-1, the temperature-sensitive tsBN67 hamster cell line arrests proliferation at nonpermissive temperatures, primarily in a G(0)/G(1) state, and displays temperature-sensitive cytokinesis defects. The HCF-1 mutation in tsBN67 cells also causes a temperature-sensitive dissociation of HCF-1 from chromatin prior to cell proliferation arrest, suggesting that HCF-1-chromatin association is important for mammalian-cell proliferation. Here, we report that the simian virus 40 (SV40) early region, in particular, large T antigen (Tag), and the adenovirus oncoprotein E1A can rescue the tsBN67 cell proliferation defect at nonpermissive temperatures. The SV40 early region rescues the tsBN67 cell proliferation defect without restoring the HCF-1-chromatin association, indicating that these oncoproteins bypass a requirement for HCF-1 function. The SV40 early region also rescues the tsBN67 cytokinesis defect, suggesting that the roles of HCF-1 in cell proliferation and proper cytokinesis are intimately linked. The ability of SV40 Tag and adenovirus E1A to inactivate members of the pRb protein family-pRb, p107, and p130-is important for the bypass of HCF-1 function. These results suggest that HCF-1 regulates mammalian-cell proliferation and cytokinesis, at least in part, by either directly or indirectly opposing pRb family member function.
View details for DOI 10.1128/MCB.22.19.6767-6778.2002
View details for Web of Science ID 000177961900012
View details for PubMedID 12215534
Loss of HCF-1-chromatin association precedes temperature-induced growth arrest of tsBN67 cells
MOLECULAR AND CELLULAR BIOLOGY
2001; 21 (11): 3820-3829
Human HCF-1 is a large, highly conserved, and abundant nuclear protein that plays an important but unknown role in cell proliferation. It also plays a role in activation of herpes simplex virus immediate-early gene transcription by the viral regulatory protein VP16. A single proline-to-serine substitution in the HCF-1 VP16 interaction domain causes a temperature-induced arrest of cell proliferation in hamster tsBN67 cells and prevents transcriptional activation by VP16. We show here that HCF-1 is naturally bound to chromatin in uninfected cells through its VP16 interaction domain. HCF-1 is chromatin bound in tsBN67 cells at permissive temperature but dissociates from chromatin before tsBN67 cells stop proliferating at the nonpermissive temperature, suggesting that loss of HCF-1 chromatin association is the primary cause of the temperature-induced tsBN67 cell proliferation arrest. We propose that the role of HCF-1 in cell proliferation is to regulate gene transcription by associating with a multiplicity of DNA-bound transcription factors through its VP16 interaction domain.
View details for Web of Science ID 000168706600019
View details for PubMedID 11340173
Developmental and cell-cycle regulation of Caenorhabditis elegans HCF phosphorylation
2001; 40 (19): 5786-5794
HCF-1 is a mammalian protein required for cell proliferation. It is also involved in transcriptional activation of herpes-simplex-virus immediate-early gene transcription in association with the viral transactivator VP16. HCF-1 and a related protein called HCF-2 possess a homologue in Caenorhabditis elegans that can associate with and activate VP16. Here, we demonstrate developmental regulation of C. elegans HCF (CeHCF) phosphorylation: a hyperphosphorylated form of CeHCF is present in embryos, whereas a hypophosphorylated form is present in L1 larvae. The phosphorylation patterns of endogenous CeHCF in worms and ectopically synthesized CeHCF in mammalian cells are remarkably similar, suggesting that the way CeHCF can be recognized by kinases is conserved in animals. Phosphorylation-site mapping of endogenous CeHCF, however, revealed that phosphorylation occurs at four clustered sites in the region of the protein that is not highly conserved among HCF proteins and is not required for VP16-induced complex formation. Indeed, phosphorylation of either CeHCF or human HCF-1 appears to be dispensable for association with VP16. All four CeHCF phosphorylation sites match the consensus recognition site for the cell-cycle kinases CDC2 and CDK2. Consistent with this similarity and with the developmental phosphorylation of CeHCF in C. elegans embryos, CeHCF phosphorylation is cell-cycle-regulated in mammalian cells.
View details for Web of Science ID 000168635900023
View details for PubMedID 11341844