bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2021–11–28
23 papers selected by
Connor Rogerson, University of Cambridge, MRC Cancer Unit
  1. Recognition of acetylated histone by Yaf9 regulates metabolic cycling of transcription initiation and chromatin regulatory factors.
  2. Discrete regulatory modules instruct hematopoietic lineage commitment and differentiation.
  3. ecDNA hubs drive cooperative intermolecular oncogene expression.
  4. BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma.
  5. Reprogramming of H3K9bhb at regulatory elements is a key feature of fasting in the small intestine.
  6. A trivalent nucleosome interaction by PHIP/BRWD2 is disrupted in neurodevelopmental disorders and cancer.
  7. A 37 kb region upstream of Brachyury comprising a notochord enhancer is essential for notochord and tail development.
  8. A genome-scale TF-DNA interaction network of transcriptional regulation of Arabidopsis primary and specialized metabolism.
  9. Genome-wide cancer-specific chromatin accessibility patterns derived from archival processed xenograft tumors.
  10. Single-cell analysis identifies dynamic gene expression networks that govern B cell development and transformation.
  11. Coordinated maintenance of H3K36/K27 methylation by histone demethylases preserves germ cell identity and immortality.
  12. Epigenetic interaction between UTX and DNMT1 regulates diet-induced myogenic remodeling in brown fat.
  13. A human fetal liver-derived infant MLL-AF4 acute lymphoblastic leukemia model reveals a distinct fetal gene expression program.
  14. The interaction between S100A2 and KPNA2 mediates NFYA nuclear import and is a novel therapeutic target for colorectal cancer metastasis.
  15. MAP2K6 remodels chromatin and facilitates reprogramming by activating Gatad2b-phosphorylation dependent heterochromatin loosening.
  16. Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry.
  17. SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning.
  18. Comprehensive determination of transcription start sites derived from all RNA polymerases using ReCappable-seq.
  19. TBX2 controls a proproliferative gene expression program in melanoma.
  20. The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters.
  21. Genome-wide CRISPR-Cas9 screens identify mechanisms of BET bromodomain inhibitor sensitivity.
  22. Cascading epigenomic analysis for identifying disease genes from the regulatory landscape of GWAS variants.
  23. Mutational signatures in GATA3 transcription factor and its DNA binding domain that stimulate breast cancer and HDR syndrome.
  1. Genes Dev. 2021 Nov 24.
    Recognition of acetylated histone by Yaf9 regulates metabolic cycling of transcription initiation and chromatin regulatory factors.
    Jibo Zhang, Aakanksha Gundu, Brian D Strahl.
      How transcription programs rapidly adjust to changing metabolic and cellular cues remains poorly defined. Here, we reveal a function for the Yaf9 component of the SWR1-C and NuA4 chromatin regulatory complexes in maintaining timely transcription of metabolic genes across the yeast metabolic cycle (YMC). By reading histone acetylation during the oxidative and respiratory phase of the YMC, Yaf9 recruits SWR1-C and NuA4 complexes to deposit H2A.Z and acetylate H4, respectively. Increased H2A.Z and H4 acetylation during the oxidative phase promotes transcriptional initiation and chromatin machinery occupancy and is associated with reduced RNA polymerase II levels at genes-a pattern reversed during transition from oxidative to reductive metabolism. Prevention of Yaf9-H3 acetyl reading disrupted this pattern of transcriptional and chromatin regulator recruitment and impaired the timely transcription of metabolic genes. Together, these findings reveal that Yaf9 contributes to a dynamic chromatin and transcription initiation factor signature that is necessary for the proper regulation of metabolic gene transcription during the YMC. They also suggest that unique regulatory mechanisms of transcription exist at distinct metabolic states.
    Keywords:  H2A.Z; NuA4; SWR1-C; YEATS domain; Yaf9; chromatin regulatory complexes; histone; histone acetylation; transcription; yeast metabolic cycle
    DOI:  https://doi.org/10.1101/gad.348904.121
  2. Nat Commun. 2021 Nov 23. 12(1): 6790
    Discrete regulatory modules instruct hematopoietic lineage commitment and differentiation.
    Grigorios Georgolopoulos, Nikoletta Psatha, Mineo Iwata, Andrew Nishida, Tannishtha Som, Minas Yiangou, John A Stamatoyannopoulos, Jeff Vierstra.
      Lineage commitment and differentiation is driven by the concerted action of master transcriptional regulators at their target chromatin sites. Multiple efforts have characterized the key transcription factors (TFs) that determine the various hematopoietic lineages. However, the temporal interactions between individual TFs and their chromatin targets during differentiation and how these interactions dictate lineage commitment remains poorly understood. Here we perform dense, daily, temporal profiling of chromatin accessibility (DNase I-seq) and gene expression changes (total RNA-seq) along ex vivo human erythropoiesis to comprehensively define developmentally regulated DNase I hypersensitive sites (DHSs) and transcripts. We link both distal DHSs to their target gene promoters and individual TFs to their target DHSs, revealing that the regulatory landscape is organized in distinct sequential regulatory modules that regulate lineage restriction and maturation. Finally, direct comparison of transcriptional dynamics (bulk and single-cell) and lineage potential between erythropoiesis and megakaryopoiesis uncovers differential fate commitment dynamics between the two lineages as they exit the stem and progenitor stage. Collectively, these data provide insights into the temporally regulated synergy of the cis- and the trans-regulatory components underlying hematopoietic lineage commitment and differentiation.
    DOI:  https://doi.org/10.1038/s41467-021-27159-x
  3. Nature. 2021 Nov 24.
    ecDNA hubs drive cooperative intermolecular oncogene expression.
    King L Hung, Kathryn E Yost, Liangqi Xie, Quanming Shi, Konstantin Helmsauer, Jens Luebeck, Robert Schöpflin, Joshua T Lange, Rocío Chamorro González, Natasha E Weiser, Celine Chen, Maria E Valieva, Ivy Tsz-Lo Wong, Sihan Wu, Siavash R Dehkordi, Connor V Duffy, Katerina Kraft, Jun Tang, Julia A Belk, John C Rose, M Ryan Corces, Jeffrey M Granja, Rui Li, Utkrisht Rajkumar, Jordan Friedlein, Anindya Bagchi, Ansuman T Satpathy, Robert Tjian, Stefan Mundlos, Vineet Bafna, Anton G Henssen, Paul S Mischel, Zhe Liu, Howard Y Chang.
      Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.
    DOI:  https://doi.org/10.1038/s41586-021-04116-8
  4. Nat Commun. 2021 Nov 26. 12(1): 6924
    BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma.
    Dominik Laubscher, Berkley E Gryder, Benjamin D Sunkel, Thorkell Andresson, Marco Wachtel, Sudipto Das, Bernd Roschitzki, Witold Wolski, Xiaoli S Wu, Hsien-Chao Chou, Young K Song, Chaoyu Wang, Jun S Wei, Meng Wang, Xinyu Wen, Quy Ai Ngo, Joana G Marques, Christopher R Vakoc, Beat W Schäfer, Benjamin Z Stanton, Javed Khan.
      Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor.
    DOI:  https://doi.org/10.1038/s41467-021-27176-w
  5. Cell Rep. 2021 Nov 23. pii: S2211-1247(21)01530-8. [Epub ahead of print]37(8): 110044
    Reprogramming of H3K9bhb at regulatory elements is a key feature of fasting in the small intestine.
    Christopher J Terranova, Kristina M Stemler, Praveen Barrodia, Sabrina L Jeter-Jones, Zhongqi Ge, Marimar de la Cruz Bonilla, Ayush Raman, Chia-Wei Cheng, Kendra L Allton, Emre Arslan, Ömer H Yilmaz, Michelle C Barton, Kunal Rai, Helen Piwnica-Worms.
      β-hydroxybutyrate (β-OHB) is an essential metabolic energy source during fasting and functions as a chromatin regulator by lysine β-hydroxybutyrylation (Kbhb) modification of the core histones H3 and H4. We report that Kbhb on histone H3 (H3K9bhb) is enriched at proximal promoters of critical gene subsets associated with lipolytic and ketogenic metabolic pathways in small intestine (SI) crypts during fasting. Similar Kbhb enrichment is observed in Lgr5+ stem cell-enriched epithelial spheroids treated with β-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb is associated with active chromatin states and that fasting enriches for an H3K9bhb-H3K27ac signature at active metabolic gene promoters and distal enhancer elements. Intestinal knockout of Hmgcs2 results in marked loss of H3K9bhb-associated loci, suggesting that local production of β-OHB is responsible for chromatin reprogramming within the SI crypt. We conclude that modulation of H3K9bhb in SI crypts is a key gene regulatory event in response to fasting.
    Keywords:  chromatin states; fasting response; gene expression; histone modifications; ketogenic; small intestine crypts; β-hydroxybutyrate
    DOI:  https://doi.org/10.1016/j.celrep.2021.110044
  6. Genes Dev. 2021 Nov 24.
    A trivalent nucleosome interaction by PHIP/BRWD2 is disrupted in neurodevelopmental disorders and cancer.
    Marc A J Morgan, Irina K Popova, Anup Vaidya, Jonathan M Burg, Matthew R Marunde, Emily J Rendleman, Zachary J Dumar, Rachel Watson, Matthew J Meiners, Sarah A Howard, Natalia Khalatyan, Robert M Vaughan, Scott B Rothbart, Michael-C Keogh, Ali Shilatifard.
      Mutations in the PHIP/BRWD2 chromatin regulator cause the human neurodevelopmental disorder Chung-Jansen syndrome, while alterations in PHIP expression are linked to cancer. Precisely how PHIP functions in these contexts is not fully understood. Here we demonstrate that PHIP is a chromatin-associated CRL4 ubiquitin ligase substrate receptor and is required for CRL4 recruitment to chromatin. PHIP binds to chromatin through a trivalent reader domain consisting of a H3K4-methyl binding Tudor domain and two bromodomains (BD1 and BD2). Using semisynthetic nucleosomes with defined histone post-translational modifications, we characterize PHIPs BD1 and BD2 as respective readers of H3K14ac and H4K12ac, and identify human disease-associated mutations in each domain and the intervening linker region that likely disrupt chromatin binding. These findings provide new insight into the biological function of this enigmatic chromatin protein and set the stage for the identification of both upstream chromatin modifiers and downstream targets of PHIP in human disease.
    Keywords:  chromatin; histone; neurodevelopmental disorder; neuroepigenetics
    DOI:  https://doi.org/10.1101/gad.348766.121
  7. Development. 2021 Nov 25. pii: dev.200059. [Epub ahead of print]
    A 37 kb region upstream of Brachyury comprising a notochord enhancer is essential for notochord and tail development.
    Dennis Schifferl, Manuela Scholze-Wittler, Lars Wittler, Jesse V Veenvliet, Frederic Koch, Bernhard G Herrmann.
      The node-streak border region comprising notochord progenitor cells (NPCs) at the posterior node and neuro-mesodermal progenitor cells (NMPs) in the adjacent epiblast is the prime organizing center for axial elongation in mouse embryos. The T-box transcription factor Brachyury (T) is essential for both, formation of the notochord and maintenance of NMPs, and thus is a key regulator of trunk and tail development. The T promoter controlling T expression in NMPs and nascent mesoderm has been characterized in detail. However, control elements for T expression in the notochord have not been identified yet. We have generated a series of deletion alleles by CRISPR/Cas9 genome editing in mESCs, and analyzed their effects in mutant mouse embryos. We identified a 37 kb region upstream of T essential for notochord function and tailbud outgrowth. Within that region we discovered a T binding enhancer required for notochord cell specification and differentiation. Our data reveal a complex regulatory landscape controlling cell type-specific expression and function of T in NMP/nascent mesoderm and node/notochord allowing proper trunk and tail development.
    Keywords:  Brachyury; Development; Embryo; Enhancer; Mouse; Notochord
    DOI:  https://doi.org/10.1242/dev.200059
  8. Mol Syst Biol. 2021 Nov;17(11): e10625
    A genome-scale TF-DNA interaction network of transcriptional regulation of Arabidopsis primary and specialized metabolism.
    Michelle Tang, Baohua Li, Xue Zhou, Tayah Bolt, Jia Jie Li, Neiman Cruz, Allison Gaudinier, Richard Ngo, Caitlin Clark-Wiest, Daniel J Kliebenstein, Siobhan M Brady.
      Plant metabolism is more complex relative to individual microbes. In single-celled microbes, transcriptional regulation by single transcription factors (TFs) is sufficient to shift primary metabolism. Corresponding genome-level transcriptional regulatory maps of metabolism reveal the underlying design principles responsible for these shifts as a model in which master regulators largely coordinate specific metabolic pathways. Plant primary and specialized metabolism occur within innumerable cell types, and their reactions shift depending on internal and external cues. Given the importance of plants and their metabolites in providing humanity with food, fiber, and medicine, we set out to develop a genome-scale transcriptional regulatory map of Arabidopsis metabolic genes. A comprehensive set of protein-DNA interactions between Arabidopsis thaliana TFs and gene promoters in primary and specialized metabolic pathways were mapped. To demonstrate the utility of this resource, we identified and functionally validated regulators of the tricarboxylic acid (TCA) cycle. The resulting network suggests that plant metabolic design principles are distinct from those of microbes. Instead, metabolism appears to be transcriptionally coordinated via developmental- and stress-conditional processes that can coordinate across primary and specialized metabolism. These data represent the most comprehensive resource of interactions between TFs and metabolic genes in plants.
    Keywords:  CCPs central carbon promoters; GSL glucosinolate; TF transcription factor; Y1H yeast one-hybrid
    DOI:  https://doi.org/10.15252/msb.202110625
  9. Genome Res. 2021 Nov 23.
    Genome-wide cancer-specific chromatin accessibility patterns derived from archival processed xenograft tumors.
    Shelsa S Marcel, Austin L Quimby, Melodie P Noel, Oscar C Jaimes, Marjan Mehrab-Mohseni, Suud A Ashur, Brian Velasco, James K Tsuruta, Sandeep K Kasoji, Charlene M Santos, Paul A Dayton, Joel S Parker, Ian J Davis, Samantha G Pattenden.
      Chromatin accessibility states that influence gene expression and other nuclear processes can be altered in disease. The constellation of transcription factors and chromatin regulatory complexes in cells results in characteristic patterns of chromatin accessibility. The study of these patterns in tissues has been limited because existing chromatin accessibility assays are ineffective for archival formalin-fixed, paraffin-embedded (FFPE) tissues. We have developed a method to efficiently extract intact chromatin from archival tissue via enhanced cavitation with a nanodroplet reagent consisting of a lipid shell with a liquid perfluorocarbon core. Inclusion of nanodroplets during the extraction of chromatin from FFPE tissues enhances the recovery of intact accessible and nucleosome-bound chromatin. We show that the addition of nanodroplets to the chromatin accessibility assay formaldehyde-assisted isolation of regulatory elements (FAIRE), does not affect the accessible chromatin signal. Applying the technique to FFPE human tumor xenografts, we identified tumor-relevant regions of accessible chromatin shared with those identified in primary tumors. Further, we deconvoluted non-tumor signal to identify cellular components of the tumor microenvironment. Incorporation of this method of enhanced cavitation into FAIRE offers the potential for extending chromatin accessibility to clinical diagnosis and personalized medicine, while also enabling the exploration of gene regulatory mechanisms in archival samples.
    DOI:  https://doi.org/10.1101/gr.275219.121
  10. Nat Commun. 2021 Nov 25. 12(1): 6843
    Single-cell analysis identifies dynamic gene expression networks that govern B cell development and transformation.
    Robin D Lee, Sarah A Munro, Todd P Knutson, Rebecca S LaRue, Lynn M Heltemes-Harris, Michael A Farrar.
      Integration of external signals and B-lymphoid transcription factor activities organise B cell lineage commitment through alternating cycles of proliferation and differentiation, producing a diverse repertoire of mature B cells. We use single-cell transcriptomics/proteomics to identify differentially expressed gene networks across B cell development and correlate these networks with subtypes of B cell leukemia. Here we show unique transcriptional signatures that refine the pre-B cell expansion stages into pre-BCR-dependent and pre-BCR-independent proliferative phases. These changes correlate with reciprocal changes in expression of the transcription factor EBF1 and the RNA binding protein YBX3, that are defining features of the pre-BCR-dependent stage. Using pseudotime analysis, we further characterize the expression kinetics of different biological modalities across B cell development, including transcription factors, cytokines, chemokines, and their associated receptors. Our findings demonstrate the underlying heterogeneity of developing B cells and characterise developmental nodes linked to B cell transformation.
    DOI:  https://doi.org/10.1038/s41467-021-27232-5
  11. Cell Rep. 2021 Nov 23. pii: S2211-1247(21)01536-9. [Epub ahead of print]37(8): 110050
    Coordinated maintenance of H3K36/K27 methylation by histone demethylases preserves germ cell identity and immortality.
    Nico Zaghet, Katrine Madsen, Federico Rossi, Daniel Fernandez Perez, Pier Giorgio Amendola, Samuel Demharter, Ulrich Pfisterer, Konstantin Khodosevich, Diego Pasini, Anna Elisabetta Salcini.
      Germ cells have evolved unique mechanisms to ensure the transmission of genetically and nongenetically encoded information, whose alteration compromises germ cell immortality. Chromatin factors play fundamental roles in these mechanisms. H3K36 and H3K27 methyltransferases shape and propagate a pattern of histone methylation essential for C. elegans germ cell maintenance, but the role of respective histone demethylases remains unexplored. Here, we show that jmjd-5 regulates H3K36me2 and H3K27me3 levels, preserves germline immortality, and protects germ cell identity by controlling gene expression. The transcriptional and biological effects of jmjd-5 loss can be hindered by the removal of H3K27demethylases, indicating that H3K36/K27 demethylases act in a transcriptional framework and promote the balance between H3K36 and H3K27 methylation required for germ cell immortality. Furthermore, we find that in wild-type, but not in jmjd-5 mutants, alterations of H3K36 methylation and transcription occur at high temperature, suggesting a role for jmjd-5 in adaptation to environmental changes.
    Keywords:  H3K27 methylation; H3K36 methylation; germ cell identity; germ cell immortality; histone demethylases; temperature
    DOI:  https://doi.org/10.1016/j.celrep.2021.110050
  12. Nat Commun. 2021 Nov 25. 12(1): 6838
    Epigenetic interaction between UTX and DNMT1 regulates diet-induced myogenic remodeling in brown fat.
    Fenfen Li, Jia Jing, Miranda Movahed, Xin Cui, Qiang Cao, Rui Wu, Ziyue Chen, Liqing Yu, Yi Pan, Huidong Shi, Hang Shi, Bingzhong Xue.
      Brown adipocytes share the same developmental origin with skeletal muscle. Here we find that a brown adipocyte-to-myocyte remodeling also exists in mature brown adipocytes, and is induced by prolonged high fat diet (HFD) feeding, leading to brown fat dysfunction. This process is regulated by the interaction of epigenetic pathways involving histone and DNA methylation. In mature brown adipocytes, the histone demethylase UTX maintains persistent demethylation of the repressive mark H3K27me3 at Prdm16 promoter, leading to high Prdm16 expression. PRDM16 then recruits DNA methyltransferase DNMT1 to Myod1 promoter, causing Myod1 promoter hypermethylation and suppressing its expression. The interaction between PRDM16 and DNMT1 coordinately serves to maintain brown adipocyte identity while repressing myogenic remodeling in mature brown adipocytes, thus promoting their active brown adipocyte thermogenic function. Suppressing this interaction by HFD feeding induces brown adipocyte-to-myocyte remodeling, which limits brown adipocyte thermogenic capacity and compromises diet-induced thermogenesis, leading to the development of obesity.
    DOI:  https://doi.org/10.1038/s41467-021-27141-7
  13. Nat Commun. 2021 Nov 25. 12(1): 6905
    A human fetal liver-derived infant MLL-AF4 acute lymphoblastic leukemia model reveals a distinct fetal gene expression program.
    Siobhan Rice, Thomas Jackson, Nicholas T Crump, Nicholas Fordham, Natalina Elliott, Sorcha O'Byrne, Maria Del Mar Lara Fanego, Dilys Addy, Trisevgeni Crabb, Carryl Dryden, Sarah Inglott, Dariusz Ladon, Gary Wright, Jack Bartram, Philip Ancliff, Adam J Mead, Christina Halsey, Irene Roberts, Thomas A Milne, Anindita Roy.
      Although 90% of children with acute lymphoblastic leukemia (ALL) are now cured, the prognosis for infant-ALL remains dismal. Infant-ALL is usually caused by a single genetic hit that arises in utero: an MLL/KMT2A gene rearrangement (MLL-r). This is sufficient to induce a uniquely aggressive and treatment-refractory leukemia compared to older children. The reasons for disparate outcomes in patients of different ages with identical driver mutations are unknown. Using the most common MLL-r in infant-ALL, MLL-AF4, as a disease model, we show that fetal-specific gene expression programs are maintained in MLL-AF4 infant-ALL but not in MLL-AF4 childhood-ALL. We use CRISPR-Cas9 gene editing of primary human fetal liver hematopoietic cells to produce a t(4;11)/MLL-AF4 translocation, which replicates the clinical features of infant-ALL and drives infant-ALL-specific and fetal-specific gene expression programs. These data support the hypothesis that fetal-specific gene expression programs cooperate with MLL-AF4 to initiate and maintain the distinct biology of infant-ALL.
    DOI:  https://doi.org/10.1038/s41467-021-27270-z
  14. Oncogene. 2021 Nov 20.
    The interaction between S100A2 and KPNA2 mediates NFYA nuclear import and is a novel therapeutic target for colorectal cancer metastasis.
    Fengyan Han, Lei Zhang, Shaoxia Liao, Yanmin Zhang, Lili Qian, Feijun Hou, Jingwen Gong, Maode Lai, Honghe Zhang.
      Nucleocytoplasmic transport of proteins is disrupted and dysregulated in cancer cells. Nuclear pore complexes and cargo proteins are two main transportation regulators. However, the mechanism regulating nucleocytoplasmic transport in cancer remains elusive. Here, we identified a S100A2/KPNA2 cotransport complex that transports the tumor-associated transcription factor NFYA in colorectal cancer (CRC). Through the S100A2/KNPA2 complex, depending on its interaction with S100A2, NFYA is transported to the nucleus and inhibits the transcriptional activity of E-cadherin, which in turn promotes CRC metastasis. Targeting the S100A2/KPNA2 binding sites with the specific inhibitor delanzomib is a potential therapeutic approach for CRC.
    DOI:  https://doi.org/10.1038/s41388-021-02116-6
  15. Cell Death Differ. 2021 Nov 24.
    MAP2K6 remodels chromatin and facilitates reprogramming by activating Gatad2b-phosphorylation dependent heterochromatin loosening.
    Guangsuo Xing, Zichao Liu, Luyuan Huang, Danyun Zhao, Tao Wang, Hao Yuan, Yi Wu, Linpeng Li, Qi Long, Yanshuang Zhou, Zhihong Hao, Yang Liu, Jianghuan Lu, Shiting Li, Jieying Zhu, Bo Wang, Junwei Wang, Jing Liu, Jiekai Chen, Duanqing Pei, Xingguo Liu, Keshi Chen.
      Somatic cell reprogramming is an ideal model for studying epigenetic regulation as it undergoes dramatic chromatin remodeling. However, a role for phosphorylation signaling in chromatin protein modifications for reprogramming remains unclear. Here, we identified mitogen-activated protein kinase kinase 6 (Mkk6) as a chromatin relaxer and found that it could significantly enhance reprogramming. The function of Mkk6 in heterochromatin loosening and reprogramming requires its kinase activity but does not depend on its best-known target, P38. We identified Gatad2b as a novel target of Mkk6 phosphorylation that acts downstream to elevate histone acetylation levels and loosen heterochromatin. As a result, Mkk6 over-expression facilitates binding of Sox2 and Klf4 to their targets and promotes pluripotency gene expression during reprogramming. Our studies not only reveal an Mkk phosphorylation mediated modulation of chromatin status in reprogramming, but also provide new rationales to further investigate and improve the cell fate determination processes.
    DOI:  https://doi.org/10.1038/s41418-021-00902-z
  16. Nucleic Acids Res. 2021 Nov 19. pii: gkab1068. [Epub ahead of print]
    Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry.
    Yoko Hayashi-Takanaka, Yuichiro Hayashi, Yasuhiro Hirano, Atsuko Miyawaki-Kuwakado, Yasuyuki Ohkawa, Chikashi Obuse, Hiroshi Kimura, Tokuko Haraguchi, Yasushi Hiraoka.
      DNA replication is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase onto DNA replication origins during the G1 phase is essential for initiating DNA replication. Here, we examined MCM hexamer states during the cell cycle in human hTERT-RPE1 cells using multicolor immunofluorescence-based, single-cell plot analysis, and biochemical size fractionation. Experiments involving cell-cycle arrest at the G1 phase and release from the arrest revealed that a double MCM hexamer was formed via a single hexamer during G1 progression. A single MCM hexamer was recruited to chromatin in the early G1 phase. Another single hexamer was recruited to form a double hexamer in the late G1 phase. We further examined relationship between the MCM hexamer states and the methylation levels at lysine 20 of histone H4 (H4K20) and found that the double MCM hexamer state was correlated with di/trimethyl-H4K20 (H4K20me2/3). Inhibiting the conversion from monomethyl-H4K20 (H4K20me1) to H4K20me2/3 retained the cells in the single MCM hexamer state. Non-proliferative cells, including confluent cells or Cdk4/6 inhibitor-treated cells, also remained halted in the single MCM hexamer state. We propose that the single MCM hexamer state is a halting step in the determination of cell cycle progression.
    DOI:  https://doi.org/10.1093/nar/gkab1068
  17. Development. 2021 Nov 25. pii: dev.200182. [Epub ahead of print]
    SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning.
    Julie Gamart, Iros Barozzi, Frédéric Laurent, Robert Reinhardt, Laurène Ramos Martins, Thomas Oberholzer, Axel Visel, Rolf Zeller, Aimée Zuniga.
      SMAD4 regulates gene expression in response to BMP and TGFβ signal transduction and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we identify the SMAD4 target genes in mouse limb buds using an epitope-tagged Smad4 allele for ChIP-seq analysis in combination with transcription profiling. This analysis shows that SMAD4 predominantly mediates BMP signal-transduction during early limb bud development. Unexpectedly, the expression of cholesterol biosynthesis enzymes is precociously down-regulated and intracellular cholesterol levels are reduced in Smad4-deficient limb bud mesenchymal progenitors. Most importantly, our analysis reveals a predominant function of SMAD4 in up-regulating target genes in the anterior limb bud mesenchyme. Analysis of differentially expressed genes shared between Smad4- and Shh-deficient limb buds corroborates this function of SMAD4 and also reveals the repressive effect of SMAD4 on posterior genes that are up-regulated in response to SHH signaling. This analysis uncovers opposing trans-regulatory inputs from SHH and SMAD4-mediated BMP signal transduction on anterior and posterior gene expression during the digit patterning and outgrowth in early limb buds.
    Keywords:  Anterior; BMP; ChIP-seq; Cistrome; Limb development; Mouse; RNA-seq; SHH; SMAD4
    DOI:  https://doi.org/10.1242/dev.200182
  18. Genome Res. 2021 Nov 23. pii: gr.275784.121. [Epub ahead of print]
    Comprehensive determination of transcription start sites derived from all RNA polymerases using ReCappable-seq.
    Bo Yan, George Tzertzinis, Ira Schildkraut, Laurence Ettwiller.
      Determination of eukaryotic Transcription Start Sites (TSS) has been based on methods that require the cap structure at the 5-prime end of transcripts derived from Pol-II RNA polymerase. Consequently, these methods do not reveal TSS derived from the other RNA polymerases which also play critical roles in various cell functions. To address this limitation, we developed ReCappable-seq which comprehensively identifies TSS for both Pol-lI and non-Pol-II transcripts at single-nucleotide resolution. The method relies on specific enzymatic exchange of 5-prime m7G caps and 5-prime triphosphates with a selectable tag. When applied to human transcriptomes, ReCappable-seq identifies Pol-II TSS that are in agreement with orthogonal methods such as CAGE. Additionally, ReCappable-seq reveals a rich landscape of TSS associated with Pol-III transcripts which have not previously been amenable to study at genome-wide scale. Novel TSS from non-Pol-II transcription can be located in the nuclear and mitochondrial genomes. ReCappable-seq interrogates the regulatory landscape of coding and noncoding RNA concurrently and enables the classification of epigenetic profiles associated with Pol-lI and non-Pol-II TSS.
    DOI:  https://doi.org/10.1101/gr.275784.121
  19. Genes Dev. 2021 Nov 24.
    TBX2 controls a proproliferative gene expression program in melanoma.
    Sizhu Lu, Pakavarin Louphrasitthiphol, Nishit Goradia, Jean-Philippe Lambert, Johannes Schmidt, Jagat Chauhan, Milap G Rughani, Lionel Larue, Matthias Wilmanns, Colin R Goding.
      Senescence shapes embryonic development, plays a key role in aging, and is a critical barrier to cancer initiation, yet how senescence is regulated remains incompletely understood. TBX2 is an antisenescence T-box family transcription repressor implicated in embryonic development and cancer. However, the repertoire of TBX2 target genes, its cooperating partners, and how TBX2 promotes proliferation and senescence bypass are poorly understood. Here, using melanoma as a model, we show that TBX2 lies downstream from PI3K signaling and that TBX2 binds and is required for expression of E2F1, a key antisenescence cell cycle regulator. Remarkably, TBX2 binding in vivo is associated with CACGTG E-boxes, present in genes down-regulated by TBX2 depletion, more frequently than the consensus T-element DNA binding motif that is restricted to Tbx2 repressed genes. TBX2 is revealed to interact with a wide range of transcription factors and cofactors, including key components of the BCOR/PRC1.1 complex that are recruited by TBX2 to the E2F1 locus. Our results provide key insights into how PI3K signaling modulates TBX2 function in cancer to drive proliferation.
    Keywords:  DNA binding; DNA binding specificity; TBX2; cell cycle; proliferation; senescence
    DOI:  https://doi.org/10.1101/gad.348746.121
  20. PLoS Genet. 2021 Nov 22. 17(11): e1009668
    The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters.
    Jelly H M Soffers, Sergio G-M Alcantara, Xuanying Li, Wanqing Shao, Christopher W Seidel, Hua Li, Julia Zeitlinger, Susan M Abmayr, Jerry L Workman.
      The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene expression is not well understood. During Drosophila oogenesis, the enzymatic functions are not equally required, which may indicate that different genes require different enzymatic functions. An analogy for this phenomenon is the handyman principle: while a handyman has many tools, which tool he uses depends on what requires maintenance. Here we analyzed the role of the non-enzymatic core module during Drosophila oogenesis, which interacts with TBP. We show that depletion of SAGA-specific core subunits blocked egg chamber development at earlier stages than depletion of enzymatic subunits. These results, as well as additional genetic analyses, point to an interaction with TBP and suggest a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments, and the complex was not specifically associated with distinct promoter types in the ovary. The high-resolution genomic binding profiles were congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. Our data illustrate that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present and suggests that the biological context defines which module functions are critical.
    DOI:  https://doi.org/10.1371/journal.pgen.1009668
  21. iScience. 2021 Nov 19. 24(11): 103323
    Genome-wide CRISPR-Cas9 screens identify mechanisms of BET bromodomain inhibitor sensitivity.
    David Estoppey, Gabi Schutzius, Christian Kolter, Adrian Salathe, Tiffany Wunderlin, Amandine Meyer, Florian Nigsch, Tewis Bouwmeester, Dominic Hoepfner, Susan Kirkland.
      BET bromodomain inhibitors hold promise as therapeutic agents in diverse indications, but their clinical progression has been challenging and none have received regulatory approval. Early clinical trials in cancer have shown heterogeneous clinical responses, development of resistance, and adverse events. Increased understanding of their mechanism(s) of action and identification of biomarkers are needed to identify appropriate indication(s) and achieve efficacious dosing. Using genome-wide CRISPR-Cas9 screens at different concentrations, we report molecular mechanisms defining cellular responses to BET inhibitors, some of which appear specific to a single compound concentration. We identify multiple transcriptional regulators and mTOR pathway members as key determinants of JQ1 sensitivity and two Ca2+/Mn2+ transporters, ATP2C1 and TMEM165, as key determinants of JQ1 resistance. Our study reveals new molecular mediators of BET bromodomain inhibitor effects, suggests the involvement of manganese, and provides a rich resource for discovery of biomarkers and targets for combination therapies.
    Keywords:  Cell biology; Chemogenomics; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2021.103323
  22. PLoS Genet. 2021 Nov 22. 17(11): e1009918
    Cascading epigenomic analysis for identifying disease genes from the regulatory landscape of GWAS variants.
    Bernard Ng, William Casazza, Nam Hee Kim, Chendi Wang, Farnush Farhadi, Shinya Tasaki, David A Bennett, Philip L De Jager, Christopher Gaiteri, Sara Mostafavi.
      The majority of genetic variants detected in genome wide association studies (GWAS) exert their effects on phenotypes through gene regulation. Motivated by this observation, we propose a multi-omic integration method that models the cascading effects of genetic variants from epigenome to transcriptome and eventually to the phenome in identifying target genes influenced by risk alleles. This cascading epigenomic analysis for GWAS, which we refer to as CEWAS, comprises two types of models: one for linking cis genetic effects to epigenomic variation and another for linking cis epigenomic variation to gene expression. Applying these models in cascade to GWAS summary statistics generates gene level statistics that reflect genetically-driven epigenomic effects. We show on sixteen brain-related GWAS that CEWAS provides higher gene detection rate than related methods, and finds disease relevant genes and gene sets that point toward less explored biological processes. CEWAS thus presents a novel means for exploring the regulatory landscape of GWAS variants in uncovering disease mechanisms.
    DOI:  https://doi.org/10.1371/journal.pgen.1009918
  23. Sci Rep. 2021 Nov 23. 11(1): 22762
    Mutational signatures in GATA3 transcription factor and its DNA binding domain that stimulate breast cancer and HDR syndrome.
    Atlal El-Assaad, Zaher Dawy, Athar Khalil, Georges Nemer.
      Transcription factors (TFs) play important roles in many biochemical processes. Many human genetic disorders have been associated with mutations in the genes encoding these transcription factors, and so those mutations became targets for medications and drug design. In parallel, since many transcription factors act either as tumor suppressors or oncogenes, their mutations are mostly associated with cancer. In this perspective, we studied the GATA3 transcription factor when bound to DNA in a crystal structure and assessed the effect of different mutations encountered in patients with different diseases and phenotypes. We generated all missense mutants of GATA3 protein and DNA within the adjacent and the opposite GATA3:DNA complex models. We mutated every amino acid and studied the new binding of the complex after each mutation. Similarly, we did for every DNA base. We applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations. After analyzing our data, we identified amino acids and DNA bases keys for binding. Furthermore, we validated those findings against experimental genetic data. Our results are the first to propose in silico modeling for GATA:DNA bound complexes that could be used to score effects of missense mutations in other classes of transcription factors involved in common and genetic diseases.
    DOI:  https://doi.org/10.1038/s41598-021-01832-z
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