bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2021‒05‒23
twenty papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit

  1. Nat Genet. 2021 May 17.
      Insulators play a critical role in spatiotemporal gene regulation in animals. The evolutionarily conserved CCCTC-binding factor (CTCF) is required for insulator function in mammals, but not all of its binding sites act as insulators. Here we explore the sequence requirements of CTCF-mediated transcriptional insulation using a sensitive insulator reporter in mouse embryonic stem cells. We find that insulation potency depends on the number of CTCF-binding sites in tandem. Furthermore, CTCF-mediated insulation is dependent on upstream flanking sequences at its binding sites. CTCF-binding sites at topologically associating domain boundaries are more likely to function as insulators than those outside topologically associating domain boundaries, independently of binding strength. We demonstrate that insulators form local chromatin domain boundaries and weaken enhancer-promoter contacts. Taken together, our results provide genetic, molecular and structural evidence connecting chromatin topology to the action of insulators in the mammalian genome.
  2. Nucleic Acids Res. 2021 May 17. pii: gkab337. [Epub ahead of print]
      Chromatin accessibility, as a powerful marker of active DNA regulatory elements, provides valuable information for understanding regulatory mechanisms. The revolution in high-throughput methods has accumulated massive chromatin accessibility profiles in public repositories. Nevertheless, utilization of these data is hampered by cumbersome collection, time-consuming processing, and manual chromatin accessibility (openness) annotation of genomic regions. To fill this gap, we developed OpenAnnotate ( as the first web server for efficiently annotating openness of massive genomic regions across various biosample types, tissues, and biological systems. In addition to the annotation resource from 2729 comprehensive profiles of 614 biosample types of human and mouse, OpenAnnotate provides user-friendly functionalities, ultra-efficient calculation, real-time browsing, intuitive visualization, and elaborate application notebooks. We show its unique advantages compared to existing databases and toolkits by effectively revealing cell type-specificity, identifying regulatory elements and 3D chromatin contacts, deciphering gene functional relationships, inferring functions of transcription factors, and unprecedentedly promoting single-cell data analyses. We anticipate OpenAnnotate will provide a promising avenue for researchers to construct a more holistic perspective to understand regulatory mechanisms.
  3. Development. 2020 Jan 01. pii: dev.188516. [Epub ahead of print]
      Heterochromatin, which is a densely packed chromatin state that is transcriptionally silent, is a critical regulator of gene expression. However, it is unclear how the repressive histone modification, H4K20me3, or the histone methyltransferase, SUV420H2, regulate embryonic stem (ES) cell fate by patterning the epigenetic landscape. Here, we report that depletion of SUV420H2 leads to a near complete loss of H4K20me3 genome-wide, dysregulated gene expression, and delayed ES cell differentiation. SUV420H2-bound regions are enriched with repetitive DNA elements, which are de-repressed in SUV420H2 knockout ES cells. Moreover, SUV420H2 regulation of H4K20me3-marked heterochromatin controls chromatin architecture, including fine-scale chromatin interactions in pluripotent ES cells. SUV420H2 plays a critical role in stabilizing the three-dimensional (3D) chromatin landscape of ES cells, where loss of SUV420H2 results in A/B compartment switching, perturbed chromatin insulation, and altered chromatin interactions of pericentric heterochromatin and surrounding regions, indicative of localized decondensation. In addition, depletion of SUV420H2 resulted in compromised interactions between H4K20me3 and gene regulatory regions. Together, these findings describe a novel role for SUV420H2 in regulating the chromatin landscape of ES cells.
    Keywords:  3D genome; Chip-Seq; Chromatin; Chromatin architecture; Embryonic stem cells; Epigenetics; H4K20me3; Pluripotent; SUV420H2
  4. Genome Biol. 2021 May 17. 22(1): 156
      BACKGROUND: Lung adenocarcinoma (LUAD) is a highly malignant and heterogeneous tumor that involves various oncogenic genetic alterations. Epigenetic processes play important roles in lung cancer development. However, the variation in enhancer and super-enhancer landscapes of LUAD patients remains largely unknown. To provide an in-depth understanding of the epigenomic heterogeneity of LUAD, we investigate the H3K27ac histone modification profiles of tumors and adjacent normal lung tissues from 42 LUAD patients and explore the role of epigenetic alterations in LUAD progression.RESULTS: A high intertumoral epigenetic heterogeneity is observed across the LUAD H3K27ac profiles. We quantitatively model the intertumoral variability of H3K27ac levels at proximal gene promoters and distal enhancers and propose a new epigenetic classification of LUAD patients. Our classification defines two LUAD subgroups which are highly related to histological subtypes. Group II patients have significantly worse prognosis than group I, which is further confirmed in the public TCGA-LUAD cohort. Differential RNA-seq analysis between group I and group II groups reveals that those genes upregulated in group II group tend to promote cell proliferation and induce cell de-differentiation. We construct the gene co-expression networks and identify group-specific core regulators. Most of these core regulators are linked with group-specific regulatory elements, such as super-enhancers. We further show that CLU is regulated by 3 group I-specific core regulators and works as a novel tumor suppressor in LUAD.
    CONCLUSIONS: Our study systematically characterizes the epigenetic alterations during LUAD progression and provides a new classification model that is helpful for predicting patient prognosis.
    Keywords:  Classification model; Core regulators; Epigenome; Lung adenocarcinoma; Super-enhancers
  5. Development. 2020 Jan 01. pii: dev.185009. [Epub ahead of print]
      The dramatic changes in gene expression required for development necessitate the establishment of cis-regulatory modules defined by regions of accessible chromatin. Pioneer transcription factors have the unique property of binding closed chromatin and facilitating the establishment of these accessible regions. Nonetheless, much of how pioneer transcription factors coordinate changes in chromatin accessibility during development remains unknown. To determine whether pioneer-factor function is intrinsic to the protein or whether pioneering activity is developmentally modulated, we studied the highly conserved, essential transcription factor, Grainy head (Grh). Prior work established that Grh is expressed throughout Drosophila development and is a pioneer factor in the larva. We demonstrated that Grh remains bound to mitotic chromosomes, a property shared with other pioneer factors. By assaying chromatin accessibility in embryos lacking maternal and/or zygotic Grh at three stages of development, we discovered that Grh is not required for chromatin accessibility in early embryogenesis, in contrast to its essential functions later in development. Our data reveal that the pioneering activity of Grh is temporally regulated and likely influenced by additional factors expressed at a given developmental stage.
    Keywords:  Chromatin accessibility; Drosophila; Pioneer factor; Transcription factor; cis-regulatory region
  6. Development. 2020 Jan 01. pii: dev.184432. [Epub ahead of print]
      Identification of cell-type specific cis-regulatory elements (CREs) is critical for understanding development and disease, although identification of functional regulatory elements remains challenging. We hypothesized that context-specific CREs could be identified by context-specific non-coding RNA (ncRNA) profiling, based on the observation that active CREs produce ncRNAs. We applied ncRNA profiling to identify rod and cone photoreceptor CREs from wild-type and mutant mouse retinas, defined by presence or absence of the rod-specific transcription factor (TF), Nrl, respectively. Nrl-dependent ncRNA expression strongly correlated with epigenetic profiles of rod and cone photoreceptors, identified thousands of candidate rod- and cone-specific CREs, and identified motifs for rod- and cone-specific TFs. Colocalization of NRL and the retinal TF CRX correlated with rod-specific ncRNA expression, whereas CRX alone favored cone-specific ncRNA expression, providing quantitative evidence that heterotypic TF interactions distinguish cell type-specific CRE activity. We validated the activity of novel Nrl-dependent ncRNA-defined CREs in developing cones. This work supports differential ncRNA profiling as a platform for the identification of cell-type specific CREs and discovery of molecular mechanisms underlying TF-dependent CRE activity.
    Keywords:  Cis-regulatory element; Cone; Crx; Enhancer; Gene regulatory network; Non-coding RNA (ncRNA); Nrl; Photoreceptor; Rod; Transcription factor
  7. Nat Commun. 2021 05 20. 12(1): 2969
      Chromatin accessibility profiling can identify putative regulatory regions genome wide; however, pooled single-cell methods for assessing the effects of regulatory perturbations on accessibility are limited. Here, we report a modified droplet-based single-cell ATAC-seq protocol for perturbing and evaluating dynamic single-cell epigenetic states. This method (Spear-ATAC) enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at once. Spear-ATAC profiling of 104,592 cells representing 414 sgRNA knock-down populations reveals the temporal dynamics of epigenetic responses to regulatory perturbations in cancer cells and the associations between transcription factor binding profiles.
  8. Development. 2020 Jan 01. pii: dev.187922. [Epub ahead of print]
      Transcription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is necessary. We analyzed how the expression of the homeobox TF, Orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during development in the retina. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages. We conclude that retinal cells utilize a cohort of TFs with different expression patterns, and multiple CRMs with different chromatin configurations, to precisely regulate the expression of Otx2.
    Keywords:  Crx; Otx2; Sox2; Transcription factor (TFs); cis-regulatory module (CRM)
  9. Development. 2020 Jan 01. pii: dev.175141. [Epub ahead of print]
      We previously identified the cyclin dependent kinase Cdk8 as a putative silencing factor for Xist. To investigate its role in X inactivation, we engineered a Cdk8 mutation in mouse embryonic stem cells (ESCs) carrying an inducible system for studying Xist function. We find that Xist represses X-linked genes to half the expression level in Cdk8 mutant cells, whereas near complete silencing is observed in controls. Lack of Cdk8 impairs Ezh2 recruitment and establishment of histone H3 lysine 27 tri-methylation but not PRC1 recruitment by Xist. Transgenic expression of wild-type but not catalytically inactive Cdk8 restores efficient gene repression and PRC2 recruitment. Mutation of the paralogous kinase Cdk19 does not affect Xist function and combined mutations of Cdk8 and Cdk19 resemble the Cdk8 mutation. In mice a Cdk8 mutation causes post-implantation lethality. We observe that homozygous Cdk8 mutant female embryos show a greater developmental delay than males on day 10.5. Together with inefficient repression of X-linked genes in differentiating Cdk8 mutant female ESCs these data show a requirement of Cdk8 for the initiation of X inactivation.
    Keywords:  Cdk8; Cyclin dependent kinase; Gene regulation; Polycomb; X inactivation; Xist
  10. Development. 2020 Jan 01. pii: dev.189787. [Epub ahead of print]
      The Janus-kinase/Signal Transducers and Activators of Transcription (JAK/STAT) pathway regulates the anterior posterior axis of the Drosophila follicle cells. In the anterior, it activates the bone morphogenetic protein (BMP) signaling pathway through expression of the BMP ligand, decapentaplegic (dpp). In the posterior, JAK/STAT works with the epidermal growth factor receptor (EGFR) pathway to express the T-box transcription factor midline (mid). While MID is necessary in establishing the posterior fate of the egg chamber, we show that it is not sufficient to determine a posterior fate. The ETS-transcription factor pointed (pnt) is expressed in an overlapping domain to mid in the follicle cells. This study shows that pnt is upstream of mid, and it is sufficient to induce a posterior fate in the anterior end, which is characterized by the induction of mid, the prevention of the stretched cells formation, and the abrogation of border cells migration. We demonstrate that the anterior BMP signaling is abolished by PNT through dpp repression. However, ectopic DPP cannot rescue this repression, suggesting additional targets of PNT participate in the posterior fate determination.
    Keywords:  Anterior-posterior axis coordination; Cell morphogenesis; EGFR signaling; ETS-transcription factor
  11. Genome Res. 2021 May 17.
      Nucleosomes are a significant barrier to the repair of UV damage because they impede damage recognition by nucleotide excision repair (NER). The RSC and SWI/SNF chromatin remodelers function in cells to promote DNA access by moving or evicting nucleosomes, and both have been linked to NER in yeast. Here, we report genome-wide repair maps of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast cells lacking RSC or SWI/SNF activity. Our data indicate that SWI/SNF is not generally required for NER but instead promotes repair of CPD lesions at specific yeast genes. In contrast, mutation or depletion of RSC subunits causes a general defect in NER across the yeast genome. Our data indicate that RSC is required for repair not only in nucleosomal DNA but also in neighboring linker DNA and nucleosome-free regions (NFRs). Although depletion of the RSC catalytic subunit also affects base excision repair (BER) of N-methylpurine (NMP) lesions, RSC activity is less important for BER in linker DNA and NFRs. Furthermore, our data indicate that RSC plays a direct role in transcription-coupled NER (TC-NER) of transcribed DNA. These findings help to define the specific genomic and chromatin contexts in which each chromatin remodeler functions in DNA repair, and indicate that RSC plays a unique function in facilitating repair by both NER subpathways.
  12. Cell Rep. 2021 May 18. pii: S2211-1247(21)00484-8. [Epub ahead of print]35(7): 109145
      In addition to driving specific gene expression profiles, transcriptional regulators are becoming increasingly recognized for their capacity to modulate chromatin structure. GATA6 is essential for the formation of definitive endoderm; however, the molecular basis defining the importance of GATA6 to endoderm commitment is poorly understood. The members of the GATA family of transcription factors have the capacity to bind and alter the accessibility of chromatin. Using pluripotent stem cells as a model of human development, we reveal that GATA6 is integral to the establishment of the endoderm enhancer network via the induction of chromatin accessibility and histone modifications. We additionally identify the chromatin-modifying complexes that interact with GATA6, defining the putative mechanisms by which GATA6 modulates chromatin architecture. The identified GATA6-dependent processes further our knowledge of the molecular mechanisms that underpin cell-fate decisions during formative development.
    Keywords:  EOMES; FOXA2; GATA; cell fate; development; fate commitment; foregut; gene regulation; liver development; pioneer factor
  13. Mol Cell. 2021 May 20. pii: S1097-2765(21)00320-8. [Epub ahead of print]81(10): 2183-2200.e13
      To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.
    Keywords:  H3K27ac; P300/CBP; cancer; chromatin biology; epigenetics; histone acetylation; histone deacetylase; histone methylation; lysine acetylation; transcription
  14. Nat Cancer. 2021 Jan;2(1): 34-48
      Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) were designed to induce cancer cell cycle arrest. Recent studies have suggested that these agents also exert other effects, influencing cancer cell immunogenicity, apoptotic responses, and differentiation. Using cell-based and mouse models of breast cancer together with clinical specimens, we show that CDK4/6 inhibitors induce remodeling of cancer cell chromatin characterized by widespread enhancer activation, and that this explains many of these effects. The newly activated enhancers include classical super-enhancers that drive luminal differentiation and apoptotic evasion, as well as a set of enhancers overlying endogenous retroviral elements that is enriched for proximity to interferon-driven genes. Mechanistically, CDK4/6 inhibition increases the level of several Activator Protein-1 (AP-1) transcription factor proteins, which are in turn implicated in the activity of many of the new enhancers. Our findings offer insights into CDK4/6 pathway biology and should inform the future development of CDK4/6 inhibitors.
  15. Nat Commun. 2021 05 17. 12(1): 2886
      The brush border is comprised of microvilli surface protrusions on the apical surface of epithelia. This specialized structure greatly increases absorptive surface area and plays crucial roles in human health. However, transcriptional regulatory networks controlling brush border genes are not fully understood. Here, we identify that hepatocyte nuclear factor 4 (HNF4) transcription factor is a conserved and important regulator of brush border gene program in multiple organs, such as intestine, kidney and yolk sac. Compromised brush border gene signatures and impaired transport were observed in these tissues upon HNF4 loss. By ChIP-seq, we find HNF4 binds and activates brush border genes in the intestine and kidney. H3K4me3 HiChIP-seq identifies that HNF4 loss results in impaired chromatin looping between enhancers and promoters at gene loci of brush border genes, and instead enhanced chromatin looping at gene loci of stress fiber genes in the intestine. This study provides comprehensive transcriptional regulatory mechanisms and a functional demonstration of a critical role for HNF4 in brush border gene regulation across multiple murine epithelial tissues.
  16. Cancer Res. 2021 May 20. pii: canres.4010.2020. [Epub ahead of print]
      Despite extensive progress in developing anti-cancer therapies, therapy resistance remains a major challenge that promotes disease relapse. The changes that lead to therapy resistance can be intrinsically present or may be initiated during treatment. Genetic and epigenetic heterogeneity in tumors make it more challenging to deal with therapy resistance. Recent advances in genome-wide analyses have revealed that the deregulation of distal gene regulatory elements, such as enhancers, appears in several pathophysiological conditions, including cancer. Beyond the conventional function of enhancers in recruiting transcription factors to gene promoters, enhancer elements are also transcribed into noncoding RNAs known as enhancer RNAs (eRNA). Accumulating evidence suggests that uncontrolled enhancer activity with aberrant eRNA expression promotes oncogenesis. Interestingly, tissue-specific, transcribed eRNAs from active enhancers can serve as potential therapeutic targets or biomarkers in several cancer types. This review provides a comprehensive overview of the mechanisms of enhancer transcription and eRNAs as well as their potential roles in cancer and drug resistance.
  17. Sci Adv. 2021 May;pii: eabb2947. [Epub ahead of print]7(21):
      MRN-MDC1 plays a central role in the DNA damage response (DDR) and repair. Using proteomics of isolated chromatin fragments, we identified DDR factors, such as MDC1, among those highly associating with a genomic locus upon transcriptional activation. Purification of MDC1 in the absence of exogenous DNA damage revealed interactions with factors involved in gene expression and RNA processing, in addition to DDR factors. ChIP-seq showed that MRN subunits, MRE11 and NBS1, colocalized throughout the genome, notably at TSSs and bodies of actively transcribing genes, which was dependent on the RNAPII transcriptional complex rather than transcription per se. Depletion of MRN increased RNAPII abundance at MRE11/NBS1-bound genes. Prolonged MRE11 or NBS1 depletion induced single-nucleotide polymorphisms across actively transcribing MRN target genes. These data suggest that association of MRN with the transcriptional machinery constitutively scans active genes for transcription-induced DNA damage to preserve the integrity of the coding genome.
  18. Bioinformatics. 2021 May 17. pii: btab379. [Epub ahead of print]
      MOTIVATION: Next generation sequencing (NGS) enabled the fast accumulation of genomic data at public repositories. This technology also made it possible to better understand the regulation of gene expression by transcription factors and various chromatin-associated proteins through the integration of chromatin immunoprecipitation (ChIP-Seq). The Cistrome Project (Liu et al., 2011) has become one of the indispensable research portals for biologists to access and analyze data generated with thousands of ChIP-Seq experiments. Integrative motif analysis on shared binding regions among a set of experiments is not yet achievable despite a set of search and analysis tools provided by Cistrome via its web interface and the Galaxy framework (Afgan et al., 2016).RESULTS: We implemented a python command-line tool for searching binding sequences of a transcription factor common to multiple ChIP-Seq experiments. We use the peaks in the Cistrome database as identified by MACS 2.0 (Zhang et al., 2008) for each experiment and identify shared peak regions in a genomic locus of interest. We then scan these regions for binding sequences using a binding motif of a transcription factor obtained from the JASPAR database (Fornes et al., 2020). MotifGenie is developed in collaboration with molecular biologists and its findings are corroborated by laboratory experiments.
    AVAILABILITY: MotifGenie is freely available at
  19. Genome Res. 2021 May 19.
      Single-cell RNA sequencing (scRNA-seq) is a widely used method for identifying cell types and trajectories in biologically heterogeneous samples, but it is limited in its detection and quantification of lowly expressed genes. This results in missing important biological signals, such as the expression of key transcription factors (TFs) driving cellular differentiation. We show that targeted sequencing of ∼1000 TFs (scCapture-seq) in iPSC-derived neuronal cultures greatly improves the biological information garnered from scRNA-seq. Increased TF resolution enhanced cell type identification, developmental trajectories, and gene regulatory networks. This allowed us to resolve differences among neuronal populations, which were generated in two different laboratories using the same differentiation protocol. ScCapture-seq improved TF-gene regulatory network inference and thus identified divergent patterns of neurogenesis into either excitatory cortical neurons or inhibitory interneurons. Furthermore, scCapture-seq revealed a role for of retinoic acid signaling in the developmental divergence between these different neuronal populations. Our results show that TF targeting improves the characterization of human cellular models and allows identification of the essential differences between cellular populations, which would otherwise be missed in traditional scRNA-seq. scCapture-seq TF targeting represents a cost-effective enhancement of scRNA-seq, which could be broadly applied to improve scRNA-seq resolution.
  20. Cell Rep. 2021 May 18. pii: S2211-1247(21)00476-9. [Epub ahead of print]35(7): 109137
      Oncogenic histone lysine-to-methionine mutations block the methylation of their corresponding lysine residues on wild-type histones. One attractive model is that these mutations sequester histone methyltransferases, but genome-wide studies show that mutant histones and histone methyltransferases often do not colocalize. Using chromatin immunoprecipitation sequencing (ChIP-seq), here, we show that, in fission yeast, even though H3K9M-containing nucleosomes are broadly distributed across the genome, the histone H3K9 methyltransferase Clr4 is mainly sequestered at pericentric repeats. This selective sequestration of Clr4 depends not only on H3K9M but also on H3K14 ubiquitylation (H3K14ub), a modification deposited by a Clr4-associated E3 ubiquitin ligase complex. In vitro, H3K14ub synergizes with H3K9M to interact with Clr4 and potentiates the inhibitory effects of H3K9M on Clr4 enzymatic activity. Moreover, binding kinetics show that H3K14ub overcomes the Clr4 aversion to H3K9M and reduces its dissociation. The selective sequestration model reconciles previous discrepancies and demonstrates the importance of protein-interaction kinetics in regulating biological processes.