bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2021–06–13
24 papers selected by
Connor Rogerson, University of Cambridge, MRC Cancer Unit
  1. High throughput screening identifies SOX2 as a super pioneer factor that inhibits DNA methylation maintenance at its binding sites.
  2. The global and promoter-centric 3D genome organization temporally resolved during a circadian cycle.
  3. The H3K36me2 methyltransferase NSD1 modulates H3K27ac at active enhancers to safeguard gene expression.
  4. Defining genome architecture at base-pair resolution.
  5. Genome-wide detection of enhancer-hijacking events from chromatin interaction data in rearranged genomes.
  6. E2F6 initiates stable epigenetic silencing of germline genes during embryonic development.
  7. Deep learning connects DNA traces to transcription to reveal predictive features beyond enhancer-promoter contact.
  8. The regulatory landscape of Arabidopsis thaliana roots at single-cell resolution.
  9. Methylation-eQTL Analysis in Cancer Research.
  10. Enhancer hijacking drives oncogenic BCL11B expression in lineage ambiguous stem cell leukemia.
  11. Transcription shapes DNA replication initiation to preserve genome integrity.
  12. The Dickkopf1 and FOXM1 positive feedback loop promotes tumor growth in pancreatic and esophageal cancers.
  13. HP1α is a chromatin crosslinker that controls nuclear and mitotic chromosome mechanics.
  14. The Fkh1 Forkhead associated domain promotes ORC binding to a subset of DNA replication origins in budding yeast.
  15. HOXA13 in etiology and oncogenic potential of Barrett's esophagus.
  16. Differential chromatin accessibility landscape of gain-of-function mutant p53 tumours.
  17. mSWI/SNF promotes Polycomb repression both directly and through genome-wide redistribution.
  18. Site-specific ubiquitylation acts as a regulator of linker histone H1.
  19. Hybrid stomach-intestinal chromatin states underlie human Barrett's metaplasia.
  20. Functional in vivo characterization of sox10 enhancers in neural crest and melanoma development.
  21. Gene set enrichment analysis for genome-wide DNA methylation data.
  22. HIRA stabilizes skeletal muscle lineage identity.
  23. Hoxa10 mediates positional memory to govern stem cell function in adult skeletal muscle.
  24. Chromatin architectural proteins regulate flowering time by precluding gene looping.
  1. Nat Commun. 2021 06 07. 12(1): 3337
    High throughput screening identifies SOX2 as a super pioneer factor that inhibits DNA methylation maintenance at its binding sites.
    Ludovica Vanzan, Hadrien Soldati, Victor Ythier, Santosh Anand, Simon M G Braun, Nicole Francis, Rabih Murr.
      Binding of mammalian transcription factors (TFs) to regulatory regions is hindered by chromatin compaction and DNA methylation of their binding sites. Nevertheless, pioneer transcription factors (PFs), a distinct class of TFs, have the ability to access nucleosomal DNA, leading to nucleosome remodelling and enhanced chromatin accessibility. Whether PFs can bind to methylated sites and induce DNA demethylation is largely unknown. Using a highly parallelized approach to investigate PF ability to bind methylated DNA and induce DNA demethylation, we show that the interdependence between DNA methylation and TF binding is more complex than previously thought, even within a select group of TFs displaying pioneering activity; while some PFs do not affect the methylation status of their binding sites, we identified PFs that can protect DNA from methylation and others that can induce DNA demethylation at methylated binding sites. We call the latter super pioneer transcription factors (SPFs), as they are seemingly able to overcome several types of repressive epigenetic marks. Finally, while most SPFs induce TET-dependent active DNA demethylation, SOX2 binding leads to passive demethylation, an activity enhanced by the co-binding of OCT4. This finding suggests that SPFs could interfere with epigenetic memory during DNA replication.
    DOI:  https://doi.org/10.1038/s41467-021-23630-x
  2. Genome Biol. 2021 Jun 08. 22(1): 162
    The global and promoter-centric 3D genome organization temporally resolved during a circadian cycle.
    Mayra Furlan-Magaril, Masami Ando-Kuri, Rodrigo G Arzate-Mejía, Jörg Morf, Jonathan Cairns, Abraham Román-Figueroa, Luis Tenorio-Hernández, A César Poot-Hernández, Simon Andrews, Csilla Várnai, Boo Virk, Steven W Wingett, Peter Fraser.
       BACKGROUND: Circadian gene expression is essential for organisms to adjust their physiology and anticipate daily changes in the environment. The molecular mechanisms controlling circadian gene transcription are still under investigation. In particular, how chromatin conformation at different genomic scales and regulatory elements impact rhythmic gene expression has been poorly characterized.
    RESULTS: Here we measure changes in the spatial chromatin conformation in mouse liver using genome-wide and promoter-capture Hi-C alongside daily oscillations in gene transcription. We find topologically associating domains harboring circadian genes that switch assignments between the transcriptionally active and inactive compartment at different hours of the day, while their boundaries stably maintain their structure over time. To study chromatin contacts of promoters at high resolution over time, we apply promoter capture Hi-C. We find circadian gene promoters displayed a maximal number of chromatin contacts at the time of their peak transcriptional output. Furthermore, circadian genes, as well as contacted and transcribed regulatory elements, reach maximal expression at the same timepoints. Anchor sites of circadian gene promoter loops are enriched in DNA binding sites for liver nuclear receptors and other transcription factors, some exclusively present in either rhythmic or stable contacts. Finally, by comparing the interaction profiles between core clock and output circadian genes, we show that core clock interactomes are more dynamic compared to output circadian genes.
    CONCLUSION: Our results identify chromatin conformation dynamics at different scales that parallel oscillatory gene expression and characterize the repertoire of regulatory elements that control circadian gene transcription through rhythmic or stable chromatin configurations.
    Keywords:  Chromatin compartments; Circadian gene expression; Circadian rhythms; Enhancers; Genome 3D organization; Promoter interactions; TADs; Transcription regulation
    DOI:  https://doi.org/10.1186/s13059-021-02374-3
  3. Nucleic Acids Res. 2021 Jun 09. pii: gkab473. [Epub ahead of print]
    The H3K36me2 methyltransferase NSD1 modulates H3K27ac at active enhancers to safeguard gene expression.
    Yuan Fang, Yin Tang, Yanjun Zhang, Yixin Pan, Junqi Jia, Zhongxing Sun, Weiwu Zeng, Jiaqi Chen, Ying Yuan, Dong Fang.
      Epigenetics, especially histone marks, functions beyond the DNA sequences to regulate gene expression. Depletion of NSD1, which catalyzes H3K36me2, leads to both up- and down-regulation of gene expression, indicating NSD1 is associated with both active and repressed gene expression. It's known that NSD1 regulates the deposition and expansion of H3K27me3, a repressive mark for gene expression, to keep active gene transcription. However, how NSD1 functions to repress gene expression is largely unknown. Here, we find that, when NSD1 is knocked out in mouse embryonic stem cells (mESCs), H3K27ac increases correlatively with the decrease of H3K36me2 at active enhancers, which is associated with mesoderm differentiation genes, leading to elevated gene expression. Mechanistically, NSD1 recruits HDAC1, the deacetylase of H3K27ac, to chromatin. Moreover, HDAC1 knockout (KO) recapitulates the increase of H3K27ac at active enhancers as the NSD1 depletion. Together, we propose that NSD1 deposits H3K36me2 and recruits HDAC1 at active enhancers to serve as a 'safeguard', preventing further activation of active enhancer-associated genes.
    DOI:  https://doi.org/10.1093/nar/gkab473
  4. Nature. 2021 Jun 09.
    Defining genome architecture at base-pair resolution.
    Peng Hua, Mohsin Badat, Lars L P Hanssen, Lance D Hentges, Nicholas Crump, Damien J Downes, Danuta M Jeziorska, A Marieke Oudelaar, Ron Schwessinger, Stephen Taylor, Thomas A Milne, Jim R Hughes, Doug R Higgs, James O J Davies.
      In higher eukaryotes, many genes are regulated by enhancers that are 104-106 base pairs (bp) away from the promoter. Enhancers contain transcription-factor-binding sites (which are typically around 7-22 bp), and physical contact between the promoters and enhancers is thought to be required to modulate gene expression. Although chromatin architecture has been mapped extensively at resolutions of 1 kilobase and above; it has not been possible to define physical contacts at the scale of the proteins that determine gene expression. Here we define these interactions in detail using a chromosome conformation capture method (Micro-Capture-C) that enables the physical contacts between different classes of regulatory elements to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters. Our data show that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. This supports a model in which chromatin loop extrusion1 is dependent on cohesin loading at active promoters and enhancers, which explains the formation of tissue-specific chromatin domains without changes in CTCF binding.
    DOI:  https://doi.org/10.1038/s41586-021-03639-4
  5. Nat Methods. 2021 Jun;18(6): 661-668
    Genome-wide detection of enhancer-hijacking events from chromatin interaction data in rearranged genomes.
    Xiaotao Wang, Jie Xu, Baozhen Zhang, Ye Hou, Fan Song, Huijue Lyu, Feng Yue.
      Recent efforts have shown that structural variations (SVs) can disrupt three-dimensional genome organization and induce enhancer hijacking, yet no computational tools exist to identify such events from chromatin interaction data. Here, we develop NeoLoopFinder, a computational framework to identify the chromatin interactions induced by SVs, including interchromosomal translocations, large deletions and inversions. Our framework can automatically resolve complex SVs, reconstruct local Hi-C maps surrounding the breakpoints, normalize copy number variation and allele effects and predict chromatin loops induced by SVs. We applied NeoLoopFinder in Hi-C data from 50 cancer cell lines and primary tumors and identified tens of recurrent genes associated with enhancer hijacking. To experimentally validate NeoLoopFinder, we deleted the hijacked enhancers in prostate adenocarcinoma cells using CRISPR-Cas9, which significantly reduced expression of the target oncogene. In summary, NeoLoopFinder enables identification of critical oncogenic regulatory elements that can potentially reveal therapeutic targets.
    DOI:  https://doi.org/10.1038/s41592-021-01164-w
  6. Nat Commun. 2021 Jun 11. 12(1): 3582
    E2F6 initiates stable epigenetic silencing of germline genes during embryonic development.
    Thomas Dahlet, Matthias Truss, Ute Frede, Hala Al Adhami, Anaïs F Bardet, Michael Dumas, Judith Vallet, Johana Chicher, Philippe Hammann, Sarah Kottnik, Peter Hansen, Uschi Luz, Gonzalo Alvarez, Ghislain Auclair, Jochen Hecht, Peter N Robinson, Christian Hagemeier, Michael Weber.
      In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes; however, the molecular mechanisms of this specificity remain unclear. Here, we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in embryos, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads to a failure to deposit CpG island DNA methylation at these genes during implantation. Furthermore, E2F6 is required to initiate epigenetic silencing in early embryonic cells but becomes dispensable for the maintenance in differentiated cells. Our findings elucidate the mechanisms of epigenetic targeting of germline genes and provide a paradigm for how transient repression signals by DNA-binding factors in early embryonic cells are translated into long-term epigenetic silencing during mouse development.
    DOI:  https://doi.org/10.1038/s41467-021-23596-w
  7. Nat Commun. 2021 06 08. 12(1): 3423
    Deep learning connects DNA traces to transcription to reveal predictive features beyond enhancer-promoter contact.
    Aparna R Rajpurkar, Leslie J Mateo, Sedona E Murphy, Alistair N Boettiger.
      Chromatin architecture plays an important role in gene regulation. Recent advances in super-resolution microscopy have made it possible to measure chromatin 3D structure and transcription in thousands of single cells. However, leveraging these complex data sets with a computationally unbiased method has been challenging. Here, we present a deep learning-based approach to better understand to what degree chromatin structure relates to transcriptional state of individual cells. Furthermore, we explore methods to "unpack the black box" to determine in an unbiased manner which structural features of chromatin regulation are most important for gene expression state. We apply this approach to an Optical Reconstruction of Chromatin Architecture dataset of the Bithorax gene cluster in Drosophila and show it outperforms previous contact-focused methods in predicting expression state from 3D structure. We find the structural information is distributed across the domain, overlapping and extending beyond domains identified by prior genetic analyses. Individual enhancer-promoter interactions are a minor contributor to predictions of activity.
    DOI:  https://doi.org/10.1038/s41467-021-23831-4
  8. Nat Commun. 2021 06 07. 12(1): 3334
    The regulatory landscape of Arabidopsis thaliana roots at single-cell resolution.
    Michael W Dorrity, Cristina M Alexandre, Morgan O Hamm, Anna-Lena Vigil, Stanley Fields, Christine Queitsch, Josh T Cuperus.
      The scarcity of accessible sites that are dynamic or cell type-specific in plants may be due in part to tissue heterogeneity in bulk studies. To assess the effects of tissue heterogeneity, we apply single-cell ATAC-seq to Arabidopsis thaliana roots and identify thousands of differentially accessible sites, sufficient to resolve all major cell types of the root. We find that the entirety of a cell's regulatory landscape and its transcriptome independently capture cell type identity. We leverage this shared information on cell identity to integrate accessibility and transcriptome data to characterize developmental progression, endoreduplication and cell division. We further use the combined data to characterize cell type-specific motif enrichments of transcription factor families and link the expression of family members to changing accessibility at specific loci, resolving direct and indirect effects that shape expression. Our approach provides an analytical framework to infer the gene regulatory networks that execute plant development.
    DOI:  https://doi.org/10.1038/s41467-021-23675-y
  9. Bioinformatics. 2021 Jun 12. pii: btab443. [Epub ahead of print]
    Methylation-eQTL Analysis in Cancer Research.
    Yusha Liu, Keith A Baggerly, Elias Orouji, Ganiraju Manyam, Huiqin Chen, Michael Lam, Jennifer S Davis, Michael S Lee, Bradley M Broom, David G Menter, Kunal Rai, Scott Kopetz, Jeffrey S Morris.
       MOTIVATION: DNA methylation is a key epigenetic factor regulating gene expression. While promoter methylation has been well studied, recent publications have revealed that functionally important methylation also occurs in intergenic and distal regions, and varies across genes and tissue types. Given the growing importance of inter-platform integrative genomic analyses, there is an urgent need to develop methods to discover and characterize gene-level relationships between methylation and expression.
    RESULTS: We introduce a novel sequential penalized regression approach to identify methylation-expression quantitative trait loci (methyl-eQTLs), a term that we have coined to represent, for each gene and tissue type, a sparse set of CpG loci best explaining gene expression and accompanying weights indicating direction and strength of association. Using TCGA and MD Anderson colorectal cohorts to build and validate our models, we demonstrate our strategy better explains expression variability than current commonly used gene-level methylation summaries. The methyl-eQTLs identified by our approach can be used to construct gene-level methylation summaries that are maximally correlated with gene expression for use in integrative models, and produce a tissue-specific summary of which genes appear to be strongly regulated by methylation. Our results introduce an important resource to the biomedical community for integrative genomics analyses involving DNA methylation.
    AVAILABILITY AND IMPLEMENTATION: We produce an R Shiny app (https://rstudio-prd-c1.pmacs.upenn.edu/methyl-eQTL/) that interactively presents methyl-eQTL results for colorectal, breast, and pancreatic cancer. The source R code for this work is provided in the supplement.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btab443
  10. Cancer Discov. 2021 Jun 08. pii: candisc.0145.2021. [Epub ahead of print]
    Enhancer hijacking drives oncogenic BCL11B expression in lineage ambiguous stem cell leukemia.
    Lindsey E Montefiori, Sonja Bendig, Zhaohui Gu, Xiaolong Chen, Petri Polonen, Xiaotu Ma, Alex Murison, Andy Zeng, Laura Garcia-Prat, Kirsten Dickerson, Ilaria Iacobucci, Sherif Abdelhamed, Ryan Hiltenbrand, Paul E Mead, Cyrus M Mehr, Beisi Xu, Zhongshan Cheng, Ti-Cheng Chang, Tamara Westover, Jing Ma, Anna Stengel, Shunsuke Kimura, Chunxu Qu, Marcus B Valentine, Marissa Rashkovan, Selina Luger, Mark R Litzow, Jacob M Rowe, Monique L den Boer, Victoria Wang, Jun Yin, Steven M Kornblau, Stephen P Hunger, Mignon L Loh, Ching-Hon Pui, Wenjian Yang, Kristine R Crews, Kathryn G Roberts, Jun J Yang, Mary V Relling, William E Evans, Wendy Stock, Elisabeth M Paietta, Adolfo A Ferrando, Jinghui Zhang, Wolfgang Kern, Torsten Haferlach, Gang Wu, John E Dick, Jeffery M Klco, Claudia Haferlach, Charles G Mullighan.
      Lineage ambiguous leukemias are high-risk malignancies of poorly understood genetic basis. Here, we describe a distinct subgroup of acute leukemia with expression of myeloid, T lymphoid and stem cell markers driven by aberrant allele-specific deregulation of BCL11B, a master transcription factor responsible for thymic T-lineage commitment and specification. Mechanistically, this deregulation was driven by chromosomal rearrangements that juxtapose BCL11B to super-enhancers active in hematopoietic progenitors, or focal amplifications that generate a super-enhancer from a non-coding element distal to BCL11B. Chromatin conformation analyses demonstrate long range interactions of rearranged enhancers with the expressed BCL11B allele, and association of BCL11B with activated hematopoietic progenitor cell cis-regulatory elements, suggesting BCL11B is aberrantly co-opted into a gene regulatory network that drives transformation by maintaining a progenitor state. These data support a role for ectopic BCL11B expression in primitive hematopoietic cells mediated by enhancer hijacking as an oncogenic driver of human lineage ambiguous leukemia.
    DOI:  https://doi.org/10.1158/2159-8290.CD-21-0145
  11. Genome Biol. 2021 Jun 09. 22(1): 176
    Transcription shapes DNA replication initiation to preserve genome integrity.
    Yang Liu, Chen Ai, Tingting Gan, Jinchun Wu, Yongpeng Jiang, Xuhao Liu, Rusen Lu, Ning Gao, Qing Li, Xiong Ji, Jiazhi Hu.
       BACKGROUND: Early DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage.
    RESULTS: We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage.
    CONCLUSION: RNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.
    Keywords:  DNA damage; DNA replication initiation; Genome instability; MCM redistribution; Transcription; Transcription-replication initiation collision
    DOI:  https://doi.org/10.1186/s13059-021-02390-3
  12. Oncogene. 2021 Jun 11.
    The Dickkopf1 and FOXM1 positive feedback loop promotes tumor growth in pancreatic and esophageal cancers.
    Hirokazu Kimura, Ryota Sada, Naoki Takada, Akikazu Harada, Yuichiro Doki, Hidetoshi Eguchi, Hideki Yamamoto, Akira Kikuchi.
      Dickkopf1 (DKK1) is overexpressed in various cancers and promotes cancer cell proliferation by binding to cytoskeleton-associated protein 4 (CKAP4). However, the mechanisms underlying DKK1 expression are poorly understood. RNA sequence analysis revealed that expression of the transcription factor forkhead box M1 (FOXM1) and its target genes concordantly fluctuated with expression of DKK1 in pancreatic ductal adenocarcinoma (PDAC) cells. DKK1 knockdown decreased FOXM1 expression and vice versa in PDAC and esophageal squamous cell carcinoma (ESCC) cells. Inhibition of either the DKK1-CKAP4-AKT pathway or the ERK pathway suppressed FOXM1 expression, and simultaneous inhibition of both pathways showed synergistic effects. A FOXM1 binding site was identified in the 5'-untranslated region of the DKK1 gene, and its depletion decreased DKK1 expression and cancer cell proliferation. Clinicopathological and database analysis revealed that PDAC and ESCC patients who simultaneously express DKK1 and FOXM1 have a poorer prognosis. Multivariate analysis demonstrated that expression of both DKK1 and FOXM1 is the independent prognostic factor in ESCC patients. Although it has been reported that FOXM1 enhances Wnt signaling, FOXM1 induced DKK1 expression independently of Wnt signaling in PDAC and ESCC cells. These results suggest that DKK1 and FOXM1 create a positive feedback loop to promote cancer cell proliferation.
    DOI:  https://doi.org/10.1038/s41388-021-01860-z
  13. Elife. 2021 Jun 09. pii: e63972. [Epub ahead of print]10
    HP1α is a chromatin crosslinker that controls nuclear and mitotic chromosome mechanics.
    Amy R Strom, Ronald J Biggs, Edward J Banigan, Xiaotao Wang, Katherine Chiu, Cameron Herman, Jimena Collado, Feng Yue, Joan C Ritland Politz, Leah J Tait, David Scalzo, Agnes Telling, Mark Groudine, Clifford P Brangwynne, John F Marko, Andrew D Stephens.
      Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165E indicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.
    Keywords:  cell biology; chromosomes; gene expression; human
    DOI:  https://doi.org/10.7554/eLife.63972
  14. Nucleic Acids Res. 2021 Jun 07. pii: gkab450. [Epub ahead of print]
    The Fkh1 Forkhead associated domain promotes ORC binding to a subset of DNA replication origins in budding yeast.
    Timothy Hoggard, Allison J Hollatz, Rachel E Cherney, Melissa R Seman, Catherine A Fox.
      The pioneer event in eukaryotic DNA replication is binding of chromosomal DNA by the origin recognitioncomplex (ORC). The ORC-DNA complex directs the formation of origins, the specific chromosomal regions where DNA synthesis initiates. In all eukaryotes, incompletely understood features of chromatin promote ORC-DNA binding. Here, we uncover a role for the Fkh1 (Forkhead homolog) protein and its forkhead associated (FHA) domain in promoting ORC-origin binding and origin activity at a subset of origins in Saccharomyces cerevisiae. Several of the FHA-dependent origins examined required a distinct Fkh1 binding site located 5' of and proximal to their ORC sites (5'-FKH-T site). Genetic and molecular experiments provided evidence that the Fkh1-FHA domain promoted origin activity directly through Fkh1 binding to this 5' FKH-T site. Nucleotide substitutions within two relevant origins that enhanced their ORC-DNA affinity bypassed the requirement for their 5' FKH-T sites and for the Fkh1-FHA domain. Significantly, assessment of ORC-origin binding by ChIPSeq provided evidence that this mechanism was relevant at ∼25% of yeast origins. Thus, the FHA domain of the conserved cell-cycle transcription factor Fkh1 enhanced origin selection in yeast at the level of ORC-origin binding.
    DOI:  https://doi.org/10.1093/nar/gkab450
  15. Nat Commun. 2021 06 07. 12(1): 3354
    HOXA13 in etiology and oncogenic potential of Barrett's esophagus.
    Vincent T Janmaat, Kateryna Nesteruk, Manon C W Spaander, Auke P Verhaar, Bingting Yu, Rodrigo A Silva, Wayne A Phillips, Marcin Magierowski, Anouk van de Winkel, H Scott Stadler, Tatiana Sandoval-Guzmán, Luc J W van der Laan, Ernst J Kuipers, Ron Smits, Marco J Bruno, Gwenny M Fuhler, Nicholas J Clemons, Maikel P Peppelenbosch.
      Barrett's esophagus in gastrointestinal reflux patients constitutes a columnar epithelium with distal characteristics, prone to progress to esophageal adenocarcinoma. HOX genes are known mediators of position-dependent morphology. Here we show HOX collinearity in the adult gut while Barrett's esophagus shows high HOXA13 expression in stem cells and their progeny. HOXA13 overexpression appears sufficient to explain both the phenotype (through downregulation of the epidermal differentiation complex) and the oncogenic potential of Barrett's esophagus. Intriguingly, employing a mouse model that contains a reporter coupled to the HOXA13 promotor we identify single HOXA13-positive cells distally from the physiological esophagus, which is mirrored in human physiology, but increased in Barrett's esophagus. Additionally, we observe that HOXA13 expression confers a competitive advantage to cells. We thus propose that Barrett's esophagus and associated esophageal adenocarcinoma is the consequence of expansion of this gastro-esophageal HOXA13-expressing compartment following epithelial injury.
    DOI:  https://doi.org/10.1038/s41467-021-23641-8
  16. BMC Cancer. 2021 Jun 05. 21(1): 669
    Differential chromatin accessibility landscape of gain-of-function mutant p53 tumours.
    Bhavya Dhaka, Radhakrishnan Sabarinathan.
       BACKGROUND: Mutations in TP53 not only affect its tumour suppressor activity but also exerts oncogenic gain-of-function activity. While the genome-wide mutant p53 binding sites have been identified in cancer cell lines, the chromatin accessibility landscape driven by mutant p53 in primary tumours is unknown. Here, we leveraged the chromatin accessibility data of primary tumours from The Cancer Genome Atlas (TCGA) to identify differentially accessible regions in mutant p53 tumours compared to wild-type p53 tumours, especially in breast and colon cancers.
    RESULTS: We identified 1587 lost and 984 gained accessible chromatin regions in breast, and 1143 lost and 640 gained regions in colon cancers. However, only less than half of those regions in both cancer types contain sequence motifs for wild-type or mutant p53 binding. Whereas, the remaining showed enrichment for master transcriptional regulators, such as FOX-Family TFs and NF-kB in lost and SMAD and KLF TFs in gained regions of breast. In colon, ATF3 and FOS/JUN TFs were enriched in lost, and CDX family TFs and HNF4A in gained regions. By integrating the gene expression data, we identified known and novel target genes regulated by the mutant p53.
    CONCLUSION: This study reveals the direct and indirect mechanisms by which gain-of-function mutant p53 targets the chromatin and subsequent gene expression patterns in a tumour-type specific manner. This furthers our understanding of the impact of mutant p53 in cancer development.
    Keywords:  Chromatin accessibility; Gain-of-function; Gene regulation; Mutant p53; TP53; Transcription factors
    DOI:  https://doi.org/10.1186/s12885-021-08362-x
  17. Nat Struct Mol Biol. 2021 Jun;28(6): 501-511
    mSWI/SNF promotes Polycomb repression both directly and through genome-wide redistribution.
    Christopher M Weber, Antonina Hafner, Jacob G Kirkland, Simon M G Braun, Benjamin Z Stanton, Alistair N Boettiger, Gerald R Crabtree.
      The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.
    DOI:  https://doi.org/10.1038/s41594-021-00604-7
  18. Nat Commun. 2021 06 09. 12(1): 3497
    Site-specific ubiquitylation acts as a regulator of linker histone H1.
    Eva Höllmüller, Simon Geigges, Marie L Niedermeier, Kai-Michael Kammer, Simon M Kienle, Daniel Rösner, Martin Scheffner, Andreas Marx, Florian Stengel.
      Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. This is well studied for PTMs of core histones but not for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the chemical synthesis of site-specifically mono-ubiquitylated H1.2 and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that site-specific ubiquitylation of H1 at position K64 modulates interactions with deubiquitylating enzymes and the deacetylase SIRT1. Moreover, it affects H1-dependent chromatosome assembly and phase separation resulting in a more open chromatosome conformation generally associated with a transcriptionally active chromatin state. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1.
    DOI:  https://doi.org/10.1038/s41467-021-23636-5
  19. Gastroenterology. 2021 Jun 03. pii: S0016-5085(21)03084-5. [Epub ahead of print]
    Hybrid stomach-intestinal chromatin states underlie human Barrett's metaplasia.
    Harshabad Singh, Kyungsik Ha, Jason L Hornick, Shariq Madha, Paloma Cejas, Kunal Jajoo, Pratik Singh, Paz Polak, Hwajin Lee, Ramesh A Shivdasani.
       BACKGROUND & AIMS: Tissue metaplasia is uncommon in adults because established cis-element programs resist rewiring. In Barrett's esophagus, the distal esophageal mucosa acquires predominantly intestinal character, with notable gastric features, and is predisposed to develop invasive cancers. We sought to understand the chromatin underpinnings of Barrett's metaplasia and why it commonly displays simultaneous gastric and intestinal properties.
    METHODS: We profiled cis-regulatory elements with active histone modifications in primary human biopsy materials using chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). Mutations in Barrett's esophagus were examined in relation to tissue-specific enhancer landscapes using a random-forest machine learning algorithm. We also profiled open chromatin at single-cell resolution in primary Barrett's biopsy specimens using the assay for transposase-accessible chromatin (ATAC-seq). We used one- and two-color immunohistochemistry to examine protein expression of tissue-restricted genes.
    RESULTS: Barrett's esophagus bears epigenome fingerprints of human stomach and intestinal columnar, but not esophageal squamous, epithelia. Mutational patterns were best explained as arising on the epigenome background of active gastric cis-elements, supporting the view that adjoining stomach epithelium is a likely tissue source. Individual cells in Barrett's metaplasia co-express gastric and intestinal genes, reflecting concomitant chromatin access at enhancers ordinarily restricted to one or the other epithelium. Protein expression of stomach-specific mucins, CLDN18, and a novel gastric marker, ANXA10, revealed extensive tissue and sub-clonal heterogeneity of dual stomach-intestinal cell states.
    CONCLUSIONS: These findings reveal mixed and dynamic tissue-restricted chromatin states and phenotypic heterogeneity in Barrett's esophagus. Pervasive intra-gland variation argues against stem-cell governance of this phenotype.
    Keywords:  Intestinal metaplasia; epigenetics; tissue-specific epigenomes
    DOI:  https://doi.org/10.1053/j.gastro.2021.05.057
  20. Commun Biol. 2021 Jun 07. 4(1): 695
    Functional in vivo characterization of sox10 enhancers in neural crest and melanoma development.
    Rebecca L Cunningham, Eva T Kramer, Sophia K DeGeorgia, Paula M Godoy, Anna P Zarov, Shayana Seneviratne, Vadim Grigura, Charles K Kaufman.
      The role of a neural crest developmental transcriptional program, which critically involves Sox10 upregulation, is a key conserved aspect of melanoma initiation in both humans and zebrafish, yet transcriptional regulation of sox10 expression is incompletely understood. Here we used ATAC-Seq analysis of multiple zebrafish melanoma tumors to identify recurrently open chromatin domains as putative melanoma-specific sox10 enhancers. Screening in vivo with EGFP reporter constructs revealed 9 of 11 putative sox10 enhancers with embryonic activity in zebrafish. Focusing on the most active enhancer region in melanoma, we identified a region 23 kilobases upstream of sox10, termed peak5, that drives EGFP reporter expression in a subset of neural crest cells, Kolmer-Agduhr neurons, and early melanoma patches and tumors with high specificity. A ~200 base pair region, conserved in Cyprinidae, within peak5 is required for transgenic reporter activity in neural crest and melanoma. This region contains dimeric SoxE/Sox10 dimeric binding sites essential for peak5 neural crest and melanoma activity. We show that deletion of the endogenous peak5 conserved genomic locus decreases embryonic sox10 expression and disrupts adult stripe patterning in our melanoma model background. Our work demonstrates the power of linking developmental and cancer models to better understand neural crest identity in melanoma.
    DOI:  https://doi.org/10.1038/s42003-021-02211-0
  21. Genome Biol. 2021 Jun 08. 22(1): 173
    Gene set enrichment analysis for genome-wide DNA methylation data.
    Jovana Maksimovic, Alicia Oshlack, Belinda Phipson.
      DNA methylation is one of the most commonly studied epigenetic marks, due to its role in disease and development. Illumina methylation arrays have been extensively used to measure methylation across the human genome. Methylation array analysis has primarily focused on preprocessing, normalization, and identification of differentially methylated CpGs and regions. GOmeth and GOregion are new methods for performing unbiased gene set testing following differential methylation analysis. Benchmarking analyses demonstrate GOmeth outperforms other approaches, and GOregion is the first method for gene set testing of differentially methylated regions. Both methods are publicly available in the missMethyl Bioconductor R package.
    Keywords:  DNA methylation; Differential methylation; Gene set analysis; Statistical analysis
    DOI:  https://doi.org/10.1186/s13059-021-02388-x
  22. Nat Commun. 2021 06 08. 12(1): 3450
    HIRA stabilizes skeletal muscle lineage identity.
    Joana Esteves de Lima, Reem Bou Akar, Léo Machado, Yuefeng Li, Bernadette Drayton-Libotte, F Jeffrey Dilworth, Frédéric Relaix.
      The epigenetic mechanisms coordinating the maintenance of adult cellular lineages and the inhibition of alternative cell fates remain poorly understood. Here we show that targeted ablation of the histone chaperone HIRA in myogenic cells leads to extensive transcriptional modifications, consistent with a role in maintaining skeletal muscle cellular identity. We demonstrate that conditional ablation of HIRA in muscle stem cells of adult mice compromises their capacity to regenerate and self-renew, leading to tissue repair failure. Chromatin analysis of Hira-deficient cells show a significant reduction of histone variant H3.3 deposition and H3K27ac modification at regulatory regions of muscle genes. Additionally, we find that genes from alternative lineages are ectopically expressed in Hira-mutant cells via MLL1/MLL2-mediated increase of H3K4me3 mark at silent promoter regions. Therefore, we conclude that HIRA sustains the chromatin landscape governing muscle cell lineage identity via incorporation of H3.3 at muscle gene regulatory regions, while preventing the expression of alternative lineage genes.
    DOI:  https://doi.org/10.1038/s41467-021-23775-9
  23. Sci Adv. 2021 Jun;pii: eabd7924. [Epub ahead of print]7(24):
    Hoxa10 mediates positional memory to govern stem cell function in adult skeletal muscle.
    Kiyoshi Yoshioka, Hiroshi Nagahisa, Fumihito Miura, Hiromitsu Araki, Yasutomi Kamei, Yasuo Kitajima, Daiki Seko, Jumpei Nogami, Yoshifumi Tsuchiya, Narihiro Okazaki, Akihiko Yonekura, Seigo Ohba, Yoshinori Sumita, Ko Chiba, Kosei Ito, Izumi Asahina, Yoshihiro Ogawa, Takashi Ito, Yasuyuki Ohkawa, Yusuke Ono.
      Muscle stem cells (satellite cells) are distributed throughout the body and have heterogeneous properties among muscles. However, functional topographical genes in satellite cells of adult muscle remain unidentified. Here, we show that expression of Homeobox-A (Hox-A) cluster genes accompanied with DNA hypermethylation of the Hox-A locus was robustly maintained in both somite-derived muscles and their associated satellite cells in adult mice, which recapitulates their embryonic origin. Somite-derived satellite cells were clearly separated from cells derived from cranial mesoderm in Hoxa10 expression. Hoxa10 inactivation led to genomic instability and mitotic catastrophe in somite-derived satellite cells in mice and human. Satellite cell-specific Hoxa10 ablation in mice resulted in a decline in the regenerative ability of somite-derived muscles, which were unobserved in cranial mesoderm-derived muscles. Thus, our results show that Hox gene expression profiles instill the embryonic history in satellite cells as positional memory, potentially modulating region-specific pathophysiology in adult muscles.
    DOI:  https://doi.org/10.1126/sciadv.abd7924
  24. Sci Adv. 2021 Jun;pii: eabg3097. [Epub ahead of print]7(24):
    Chromatin architectural proteins regulate flowering time by precluding gene looping.
    Bo Zhao, Yanpeng Xi, Junghyun Kim, Sibum Sung.
      Chromatin structure is critical for gene expression and many other cellular processes. In Arabidopsis thaliana, the floral repressor FLC adopts a self-loop chromatin structure via bridging of its flanking regions. This local gene loop is necessary for active FLC expression. However, the molecular mechanism underlying the formation of this class of gene loops is unknown. Here, we report the characterization of a group of linker histone-like proteins, named the GH1-HMGA family in Arabidopsis, which act as chromatin architecture modulators. We demonstrate that these family members redundantly promote the floral transition through the repression of FLC A genome-wide study revealed that this family preferentially binds to the 5' and 3' ends of gene bodies. The loss of this binding increases FLC expression by stabilizing the FLC 5' to 3' gene looping. Our study provides mechanistic insights into how a family of evolutionarily conserved proteins regulates the formation of local gene loops.
    DOI:  https://doi.org/10.1126/sciadv.abg3097
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