bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2023–05–21
24 papers selected by
Connor Rogerson, University of Cambridge
  1. SEanalysis 2.0: a comprehensive super-enhancer regulatory network analysis tool for human and mouse.
  2. Shadow enhancers mediate trade-offs between transcriptional noise and fidelity.
  3. Functional coordination between transcription factor clustering and gene activity.
  4. Single-cell chromatin accessibility of developing murine pancreas identifies cell state-specific gene regulatory programs.
  5. DNA supercoiling restricts the transcriptional bursting of neighboring eukaryotic genes.
  6. Regulatory architecture of housekeeping genes is driven by promoter assemblies.
  7. Nr2f1a maintains atrial nkx2.5 expression to repress pacemaker identity within venous atrial cardiomyocytes of zebrafish.
  8. Hi-TrAC detects active sub-TADs and reveals internal organizations of super-enhancers.
  9. Epigenetic plasticity safeguards heterochromatin configuration in mammals.
  10. An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability.
  11. Integrating single-cell transcriptomes, chromatin accessibility, and multiomics analysis of mesoderm-induced embryonic stem cells.
  12. Epigenetic landscape reveals MECOM as an endothelial lineage regulator.
  13. The NELF pausing checkpoint mediates the functional divergence of Cdk9.
  14. Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases.
  15. LKB1 controls inflammatory potential through CRTC2-dependent histone acetylation.
  16. Methylated histones on mitotic chromosomes promote topoisomerase IIα function for high fidelity chromosome segregation.
  17. The NuRD complex cooperates with SALL4 to orchestrate reprogramming.
  18. DNA binding specificity of all four Saccharomyces cerevisiae forkhead transcription factors.
  19. High-throughput single nucleus total RNA sequencing of formalin-fixed paraffin-embedded tissues by snRandom-seq.
  20. An unbiased AAV-STARR-seq screen revealing the enhancer activity map of genomic regions in the mouse brain in vivo.
  21. RNA polymerase drives ribonucleotide excision DNA repair in E. coli.
  22. Single-nucleus multi-omics of human stem cell-derived islets identifies deficiencies in lineage specification.
  23. Differential cell-cycle control by oscillatory versus sustained Hes1 expression via p21.
  24. ERα-associated translocations underlie oncogene amplifications in breast cancer.
  1. Nucleic Acids Res. 2023 May 17. pii: gkad408. [Epub ahead of print]
    SEanalysis 2.0: a comprehensive super-enhancer regulatory network analysis tool for human and mouse.
    Feng-Cui Qian, Li-Wei Zhou, Yan-Yu Li, Zheng-Min Yu, Li-Dong Li, Yue-Zhu Wang, Ming-Cong Xu, Qiu-Yu Wang, Chun-Quan Li.
      Super-enhancers (SEs) play an essential regulatory role in various biological processes and diseases through their specific interaction with transcription factors (TFs). Here, we present the release of SEanalysis 2.0 (http://licpathway.net/SEanalysis), an updated version of the SEanalysis web server for the comprehensive analyses of transcriptional regulatory networks formed by SEs, pathways, TFs, and genes. The current version added mouse SEs and further expanded the scale of human SEs, documenting 1 167 518 human SEs from 1739 samples and 550 226 mouse SEs from 931 samples. The SE-related samples in SEanalysis 2.0 were more than five times that in version 1.0, which significantly improved the ability of original SE-related network analyses ('pathway downstream analysis', 'upstream regulatory analysis' and 'genomic region annotation') for understanding context-specific gene regulation. Furthermore, we designed two novel analysis models, 'TF regulatory analysis' and 'Sample comparative analysis' for supporting more comprehensive analyses of SE regulatory networks driven by TFs. Further, the risk SNPs were annotated to the SE regions to provide potential SE-related disease/trait information. Hence, we believe that SEanalysis 2.0 has significantly expanded the data and analytical capabilities of SEs, which helps researchers in an in-depth understanding of the regulatory mechanisms of SEs.
    DOI:  https://doi.org/10.1093/nar/gkad408
  2. PLoS Comput Biol. 2023 May 19. 19(5): e1011071
    Shadow enhancers mediate trade-offs between transcriptional noise and fidelity.
    Alvaro Fletcher, Zeba Wunderlich, German Enciso.
      Enhancers are stretches of regulatory DNA that bind transcription factors (TFs) and regulate the expression of a target gene. Shadow enhancers are two or more enhancers that regulate the same target gene in space and time and are associated with most animal developmental genes. These multi-enhancer systems can drive more consistent transcription than single enhancer systems. Nevertheless, it remains unclear why shadow enhancer TF binding sites are distributed across multiple enhancers rather than within a single large enhancer. Here, we use a computational approach to study systems with varying numbers of TF binding sites and enhancers. We employ chemical reaction networks with stochastic dynamics to determine the trends in transcriptional noise and fidelity, two key performance objectives of enhancers. This reveals that while additive shadow enhancers do not differ in noise and fidelity from their single enhancer counterparts, sub- and superadditive shadow enhancers have noise and fidelity trade-offs not available to single enhancers. We also use our computational approach to compare the duplication and splitting of a single enhancer as mechanisms for the generation of shadow enhancers and find that the duplication of enhancers can decrease noise and increase fidelity, although at the metabolic cost of increased RNA production. A saturation mechanism for enhancer interactions similarly improves on both of these metrics. Taken together, this work highlights that shadow enhancer systems may exist for several reasons: genetic drift or the tuning of key functions of enhancers, including transcription fidelity, noise and output.
    DOI:  https://doi.org/10.1371/journal.pcbi.1011071
  3. Mol Cell. 2023 May 18. pii: S1097-2765(23)00289-7. [Epub ahead of print]83(10): 1605-1622.e9
    Functional coordination between transcription factor clustering and gene activity.
    Koji Kawasaki, Takashi Fukaya.
      The prevailing view of metazoan gene regulation is that transcription is facilitated through the formation of static activator complexes at distal regulatory regions. Here, we employed quantitative single-cell live-imaging and computational analysis to provide evidence that the dynamic assembly and disassembly process of transcription factor (TF) clusters at enhancers is a major source of transcriptional bursting in developing Drosophila embryos. We further show that the regulatory connectivity between TF clustering and burst induction is highly regulated through intrinsically disordered regions (IDRs). Addition of a poly-glutamine tract to the maternal morphogen Bicoid demonstrated that extended IDR length leads to ectopic TF clustering and burst induction from its endogenous target genes, resulting in defects in body segmentation during embryogenesis. Moreover, we successfully visualized the presence of "shared" TF clusters during the co-activation of two distant genes, which provides a concrete molecular explanation for the newly proposed "topological operon" hypothesis in metazoan gene regulation.
    Keywords:  Drosophila embryo; enhancer; live imaging; transcription factor; transcriptional burst
    DOI:  https://doi.org/10.1016/j.molcel.2023.04.018
  4. Mol Metab. 2023 May 11. pii: S2212-8778(23)00069-8. [Epub ahead of print] 101735
    Single-cell chromatin accessibility of developing murine pancreas identifies cell state-specific gene regulatory programs.
    Sean de la O, Xinkai Yao, Sean Chang, Zhe Liu, Julie B Sneddon.
      Numerous studies have characterized the existence of cell subtypes, along with their corresponding transcriptional profiles, within the developing mouse pancreas. The upstream mechanisms that initiate and maintain gene expression programs across cell states, however, remain largely unknown. Here, we generate single-nucleus ATAC-Sequencing data of developing murine pancreas and perform an integrated, multi-omic analysis of both chromatin accessibility and RNA expression to describe the chromatin landscape of the developing pancreas at both E14.5 and E17.5 at single-cell resolution. We identify candidate transcription factors regulating cell fate and construct gene regulatory networks of active transcription factor binding to regulatory regions of downstream target genes. This work serves as a valuable resource for the field of pancreatic biology in general and contributes to our understanding of lineage plasticity among endocrine cell types. In addition, these data identify which epigenetic states should be represented in the differentiation of stem cells to the pancreatic beta cell fate in order to best recapitulate in vitro the gene regulatory networks that are critical for progression along the beta cell lineage in vivo.
    Keywords:  ATAC-Sequencing; Chromatin accessibility; Endocrine differentiation; Gene regulatory networks; Multi-omic analysis; Pancreatic development; Single-cell RNA-Sequencing
    DOI:  https://doi.org/10.1016/j.molmet.2023.101735
  5. Mol Cell. 2023 May 18. pii: S1097-2765(23)00286-1. [Epub ahead of print]83(10): 1573-1587.e8
    DNA supercoiling restricts the transcriptional bursting of neighboring eukaryotic genes.
    Heta P Patel, Stefano Coppola, Wim Pomp, Umberto Aiello, Ineke Brouwer, Domenico Libri, Tineke L Lenstra.
      DNA supercoiling has emerged as a major contributor to gene regulation in bacteria, but how DNA supercoiling impacts transcription dynamics in eukaryotes is unclear. Here, using single-molecule dual-color nascent transcription imaging in budding yeast, we show that transcriptional bursting of divergent and tandem GAL genes is coupled. Temporal coupling of neighboring genes requires rapid release of DNA supercoils by topoisomerases. When DNA supercoils accumulate, transcription of one gene inhibits transcription at its adjacent genes. Transcription inhibition of the GAL genes results from destabilized binding of the transcription factor Gal4. Moreover, wild-type yeast minimizes supercoiling-mediated inhibition by maintaining sufficient levels of topoisomerases. Overall, we discover fundamental differences in transcriptional control by DNA supercoiling between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes.
    Keywords:  DNA supercoiling; budding yeast; single-molecule live-cell imaging; topoisomerases; transcriptional bursting
    DOI:  https://doi.org/10.1016/j.molcel.2023.04.015
  6. Cell Rep. 2023 May 12. pii: S2211-1247(23)00516-8. [Epub ahead of print]42(5): 112505
    Regulatory architecture of housekeeping genes is driven by promoter assemblies.
    Marion Dejosez, Alessandra Dall'Agnese, Mahesh Ramamoorthy, Jesse Platt, Xing Yin, Megan Hogan, Ran Brosh, Abraham S Weintraub, Denes Hnisz, Brian J Abraham, Richard A Young, Thomas P Zwaka.
      Genes that are key to cell identity are generally regulated by cell-type-specific enhancer elements bound by transcription factors, some of which facilitate looping to distant gene promoters. In contrast, genes that encode housekeeping functions, whose regulation is essential for normal cell metabolism and growth, generally lack interactions with distal enhancers. We find that Ronin (Thap11) assembles multiple promoters of housekeeping and metabolic genes to regulate gene expression. This behavior is analogous to how enhancers are brought together with promoters to regulate cell identity genes. Thus, Ronin-dependent promoter assemblies provide a mechanism to explain why housekeeping genes can forgo distal enhancer elements and why Ronin is important for cellular metabolism and growth control. We propose that clustering of regulatory elements is a mechanism common to cell identity and housekeeping genes but is accomplished by different factors binding distinct control elements to establish enhancer-promoter or promoter-promoter interactions, respectively.
    Keywords:  CP: Molecular biology; DNA looping; P element; domestication; embryonic stem cells; genome organization; promoter-promoter interaction; ronin; transcriptional regulation; transposon
    DOI:  https://doi.org/10.1016/j.celrep.2023.112505
  7. Elife. 2023 May 15. pii: e77408. [Epub ahead of print]12
    Nr2f1a maintains atrial nkx2.5 expression to repress pacemaker identity within venous atrial cardiomyocytes of zebrafish.
    Kendall E Martin, Padmapriyadarshini Ravisankar, Manu Beerens, Calum A MacRae, Joshua S Waxman.
      Maintenance of cardiomyocyte identity is vital for normal heart development and function. However, our understanding of cardiomyocyte plasticity remains incomplete. Here, we show that sustained expression of the zebrafish transcription factor Nr2f1a prevents the progressive acquisition of ventricular cardiomyocyte (VC) and pacemaker cardiomyocyte (PC) identities within distinct regions of the atrium. Transcriptomic analysis of flow-sorted atrial cardiomyocytes (ACs) from nr2f1a mutant zebrafish embryos showed increased VC marker gene expression and altered expression of core PC regulatory genes, including decreased expression of nkx2.5, a critical repressor of PC differentiation. At the arterial (outflow) pole of the atrium in nr2f1a mutants, cardiomyocytes resolve to VC identity within the expanded atrioventricular canal. However, at the venous (inflow) pole of the atrium, there is a progressive wave of AC transdifferentiation into PCs across the atrium toward the arterial pole. Restoring Nkx2.5 is sufficient to repress PC marker identity in nr2f1a mutant atria and analysis of chromatin accessibility identified an Nr2f1a-dependent nkx2.5 enhancer expressed in the atrial myocardium directly adjacent to PCs. CRISPR/Cas9-mediated deletion of the putative nkx2.5 enhancer leads to a loss of Nkx2.5-expressing ACs and expansion of a PC reporter, supporting that Nr2f1a limits PC differentiation within venous ACs via maintaining nkx2.5 expression. The Nr2f-dependent maintenance of AC identity within discrete atrial compartments may provide insights into the molecular etiology of concurrent structural congenital heart defects and associated arrhythmias.
    Keywords:  Nkx2.5 transcription factor; Nr2f transcription factor; atrial cardiomyocytes; cell biology; cellular plasticity; developmental biology; heart development; pacemaker cardiomyocyte; zebrafish
    DOI:  https://doi.org/10.7554/eLife.77408
  8. Nucleic Acids Res. 2023 May 13. pii: gkad378. [Epub ahead of print]
    Hi-TrAC detects active sub-TADs and reveals internal organizations of super-enhancers.
    Yaqiang Cao, Shuai Liu, Kairong Cui, Qingsong Tang, Keji Zhao.
      The spatial folding of eukaryotic genome plays a key role in genome function. We report here that our recently developed method, Hi-TrAC, which specializes in detecting chromatin loops among accessible genomic regions, can detect active sub-TADs with a median size of 100 kb, most of which harbor one or two cell specifically expressed genes and regulatory elements such as super-enhancers organized into nested interaction domains. These active sub-TADs are characterized by highly enriched histone mark H3K4me1 and chromatin-binding proteins, including Cohesin complex. Deletion of selected sub-TAD boundaries have different impacts, such as decreased chromatin interaction and gene expression within the sub-TADs or compromised insulation between the sub-TADs, depending on the specific chromatin environment. We show that knocking down core subunit of the Cohesin complex using shRNAs in human cells or decreasing the H3K4me1 modification by deleting the H3K4 methyltransferase Mll4 gene in mouse Th17 cells disrupted the sub-TADs structure. Our data also suggest that super-enhancers exist as an equilibrium globule structure, while inaccessible chromatin regions exist as a fractal globule structure. In summary, Hi-TrAC serves as a highly sensitive and inexpensive approach to study dynamic changes of active sub-TADs, providing more explicit insights into delicate genome structures and functions.
    DOI:  https://doi.org/10.1093/nar/gkad378
  9. Nucleic Acids Res. 2023 May 13. pii: gkad387. [Epub ahead of print]
    Epigenetic plasticity safeguards heterochromatin configuration in mammals.
    Kei Fukuda, Takeshi Shimi, Chikako Shimura, Takao Ono, Takehiro Suzuki, Kenta Onoue, Satoko Okayama, Hisashi Miura, Ichiro Hiratani, Kazuho Ikeda, Yasushi Okada, Naoshi Dohmae, Shigenobu Yonemura, Azusa Inoue, Hiroshi Kimura, Yoichi Shinkai.
      Heterochromatin is a key architectural feature of eukaryotic chromosomes critical for cell type-specific gene expression and genome stability. In the mammalian nucleus, heterochromatin segregates from transcriptionally active genomic regions and exists in large, condensed, and inactive nuclear compartments. However, the mechanisms underlying the spatial organization of heterochromatin need to be better understood. Histone H3 lysine 9 trimethylation (H3K9me3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that enrich constitutive and facultative heterochromatin, respectively. Mammals have at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization using a combination of mutant cells for five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We showed that H3K27me3, which is normally segregated from H3K9me3, was redistributed to regions targeted by H3K9me3 after the loss of H3K9 methylation and that the loss of both H3K9 and H3K27 methylation resulted in impaired condensation and spatial organization of heterochromatin. Our data demonstrate that the H3K27me3 pathway safeguards heterochromatin organization after the loss of H3K9 methylation in mammalian cells.
    DOI:  https://doi.org/10.1093/nar/gkad387
  10. Elife. 2023 May 19. pii: e82596. [Epub ahead of print]12
    An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability.
    Kanishk Jain, Matthew R Marunde, Jonathan M Burg, Susan L Gloor, Faith M Joseph, Karl F Poncha, Zachary B Gillespie, Keli L Rodriguez, Irina K Popova, Nathan W Hall, Anup Vaidya, Sarah A Howard, Hailey F Taylor, Laylo Mukhsinova, Ugochi C Onuoha, Emily F Patteson, Spencer W Cooke, Bethany C Taylor, Ellen N Weinzapfel, Marcus A Cheek, Matthew J Meiners, Geoffrey C Fox, Kevin E W Namitz, Martis W Cowles, Krzysztof Krajewski, Zu-Wen Sun, Michael S Cosgrove, Nicolas Young, Michael-C Keogh, Brian D Strahl.
      In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g., K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has broader extension. Here we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on the cis H3 tail, as determined with fully-defined heterotypic nucleosomes. In vivo, H3 tail acetylation is directly and dynamically coupled with cis H3K4 methylation levels. Together, these observations reveal an acetylation 'chromatin switch' on the H3 tail that modulates read-write accessibility in nucleosomes and resolve the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.
    Keywords:  biochemistry; chemical biology; chromosomes; gene expression; human
    DOI:  https://doi.org/10.7554/eLife.82596
  11. STAR Protoc. 2023 May 15. pii: S2666-1667(23)00274-5. [Epub ahead of print]4(2): 102307
    Integrating single-cell transcriptomes, chromatin accessibility, and multiomics analysis of mesoderm-induced embryonic stem cells.
    Kyung Dae Ko, Kan Jiang, Stefania Dell'Orso, Vittorio Sartorelli.
      Here, we present workflows for integrating independent transcriptomic and chromatin accessibility datasets and analyzing multiomics. First, we describe steps for integrating independent transcriptomic and chromatin accessibility measurements. Next, we detail multimodal analysis of transcriptomes and chromatin accessibility performed in the same sample. We demonstrate their use by analyzing datasets obtained from mouse embryonic stem cells induced to differentiate toward mesoderm-like, myogenic, or neurogenic phenotypes. For complete details on the use and execution of this protocol, please refer to Khateb et al.1.
    Keywords:  Bioinformatics; Computer sciences
    DOI:  https://doi.org/10.1016/j.xpro.2023.102307
  12. Nat Commun. 2023 Apr 25. 14(1): 2390
    Epigenetic landscape reveals MECOM as an endothelial lineage regulator.
    Jie Lv, Shu Meng, Qilin Gu, Rongbin Zheng, Xinlei Gao, Jun-Dae Kim, Min Chen, Bo Xia, Yihan Zuo, Sen Zhu, Dongyu Zhao, Yanqiang Li, Guangyu Wang, Xin Wang, Qingshu Meng, Qi Cao, John P Cooke, Longhou Fang, Kaifu Chen, Lili Zhang.
      A comprehensive understanding of endothelial cell lineage specification will advance cardiovascular regenerative medicine. Recent studies found that unique epigenetic signatures preferentially regulate cell identity genes. We thus systematically investigate the epigenetic landscape of endothelial cell lineage and identify MECOM to be the leading candidate as an endothelial cell lineage regulator. Single-cell RNA-Seq analysis verifies that MECOM-positive cells are exclusively enriched in the cell cluster of bona fide endothelial cells derived from induced pluripotent stem cells. Our experiments demonstrate that MECOM depletion impairs human endothelial cell differentiation, functions, and Zebrafish angiogenesis. Through integrative analysis of Hi-C, DNase-Seq, ChIP-Seq, and RNA-Seq data, we find MECOM binds enhancers that form chromatin loops to regulate endothelial cell identity genes. Further, we identify and verify the VEGF signaling pathway to be a key target of MECOM. Our work provides important insights into epigenetic regulation of cell identity and uncovered MECOM as an endothelial cell lineage regulator.
    DOI:  https://doi.org/10.1038/s41467-023-38002-w
  13. Nat Commun. 2023 May 13. 14(1): 2762
    The NELF pausing checkpoint mediates the functional divergence of Cdk9.
    Michael DeBerardine, Gregory T Booth, Philip P Versluis, John T Lis.
      Promoter-proximal pausing by RNA Pol II is a rate-determining step in gene transcription that is hypothesized to be a prominent point at which regulatory factors act. The pausing factor NELF is known to induce and stabilize pausing, but not all kinds of pausing are NELF-mediated. Here, we find that NELF-depleted Drosophila melanogaster cells functionally recapitulate the NELF-independent pausing we previously observed in fission yeast (which lack NELF). Critically, only NELF-mediated pausing establishes a strict requirement for Cdk9 kinase activity for the release of paused Pol II into productive elongation. Upon inhibition of Cdk9, cells with NELF efficiently shutdown gene transcription, while in NELF-depleted cells, defective, non-productive transcription continues unabated. By introducing a strict checkpoint for Cdk9, the evolution of NELF was likely critical to enable increased regulation of Cdk9 in higher eukaryotes, as Cdk9 availability can be restricted to limit gene transcription without inducing wasteful, non-productive transcription.
    DOI:  https://doi.org/10.1038/s41467-023-38359-y
  14. Nat Commun. 2023 May 13. 14(1): 2758
    Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases.
    Jina Yu, Chunli Yan, Thomas Dodd, Chi-Lin Tsai, John A Tainer, Susan E Tsutakawa, Ivaylo Ivanov.
      Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH's global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.
    DOI:  https://doi.org/10.1038/s41467-023-38416-6
  15. Mol Cell. 2023 May 02. pii: S1097-2765(23)00288-5. [Epub ahead of print]
    LKB1 controls inflammatory potential through CRTC2-dependent histone acetylation.
    Shelby E Compton, Susan M Kitchen-Goosen, Lisa M DeCamp, Kin H Lau, Batsirai Mabvakure, Matthew Vos, Kelsey S Williams, Kwok-Kin Wong, Xiaobing Shi, Scott B Rothbart, Connie M Krawczyk, Russell G Jones.
      Deregulated inflammation is a critical feature driving the progression of tumors harboring mutations in the liver kinase B1 (LKB1), yet the mechanisms linking LKB1 mutations to deregulated inflammation remain undefined. Here, we identify deregulated signaling by CREB-regulated transcription coactivator 2 (CRTC2) as an epigenetic driver of inflammatory potential downstream of LKB1 loss. We demonstrate that LKB1 mutations sensitize both transformed and non-transformed cells to diverse inflammatory stimuli, promoting heightened cytokine and chemokine production. LKB1 loss triggers elevated CRTC2-CREB signaling downstream of the salt-inducible kinases (SIKs), increasing inflammatory gene expression in LKB1-deficient cells. Mechanistically, CRTC2 cooperates with the histone acetyltransferases CBP/p300 to deposit histone acetylation marks associated with active transcription (i.e., H3K27ac) at inflammatory gene loci, promoting cytokine expression. Together, our data reveal a previously undefined anti-inflammatory program, regulated by LKB1 and reinforced through CRTC2-dependent histone modification signaling, that links metabolic and epigenetic states to cell-intrinsic inflammatory potential.
    Keywords:  CREB; CREB-regulated transcription coactivator 2; CRTC2; H3K27; IL-1β; IL-6; LIF; LKB1; SIKs; cAMP response element binding protein; histone acetylation; inflammation; interleukin-6; leukemia inhibitory factor; liver kinase B1; salt-inducible kinases
    DOI:  https://doi.org/10.1016/j.molcel.2023.04.017
  16. iScience. 2023 May 19. 26(5): 106743
    Methylated histones on mitotic chromosomes promote topoisomerase IIα function for high fidelity chromosome segregation.
    Sanjana Sundararajan, Hyewon Park, Shinji Kawano, Marnie Johansson, Bunu Lama, Tomoko Saito-Fujita, Noriko Saitoh, Alexei Arnaoutov, Mary Dasso, Zhengqiang Wang, Daniel Keifenheim, Duncan J Clarke, Yoshiaki Azuma.
      DNA Topoisomerase IIα (TopoIIα) decatenates sister chromatids, allowing their segregation in mitosis. Without the TopoIIα Strand Passage Reaction (SPR), chromosome bridges and ultra-fine DNA bridges (UFBs) arise in anaphase. The TopoIIα C-terminal domain is dispensable for the SPR in vitro but essential for mitotic functions in vivo. Here, we present evidence that the Chromatin Tether (ChT) within the CTD interacts with specific methylated nucleosomes and is crucial for high-fidelity chromosome segregation. Mutation of individual αChT residues disrupts αChT-nucleosome interaction, induces loss of segregation fidelity and reduces association of TopoIIα with chromosomes. Specific methyltransferase inhibitors reducing histone H3 or H4 methylation decreased TopoIIα at centromeres and increased segregation errors. Methyltransferase inhibition did not further increase aberrant anaphases in the ChT mutants, indicating a functional connection. The evidence reveals novel cellular regulation whereby TopoIIα specifically interacts with methylated nucleosomes via the αChT to ensure high-fidelity chromosome segregation.
    Keywords:  Biological sciences; Chromosome organization; Molecular biology; Molecular mechanism of gene regulation
    DOI:  https://doi.org/10.1016/j.isci.2023.106743
  17. Nat Commun. 2023 May 18. 14(1): 2846
    The NuRD complex cooperates with SALL4 to orchestrate reprogramming.
    Bo Wang, Chen Li, Jin Ming, Linlin Wu, Shicai Fang, Yi Huang, Lihui Lin, He Liu, Junqi Kuang, Chengchen Zhao, Xingnan Huang, Huijian Feng, Jing Guo, Xuejie Yang, Liman Guo, Xiaofei Zhang, Jiekai Chen, Jing Liu, Ping Zhu, Duanqing Pei.
      Cell fate decision involves rewiring of the genome, but remains poorly understood at the chromatin level. Here, we report that chromatin remodeling complex NuRD participates in closing open chromatin in the early phase of somatic reprogramming. Sall4, Jdp2, Glis1 and Esrrb can reprogram MEFs to iPSCs efficiently, but only Sall4 is indispensable capable of recruiting endogenous components of NuRD. Yet knocking down NuRD components only reduces reprogramming modestly, in contrast to disrupting the known Sall4-NuRD interaction by mutating or deleting the NuRD interacting motif at its N-terminus that renders Sall4 inept to reprogram. Remarkably, these defects can be partially rescured by grafting NuRD interacting motif onto Jdp2. Further analysis of chromatin accessibility dynamics demonstrates that the Sall4-NuRD axis plays a critical role in closing the open chromatin in the early phase of reprogramming. Among the chromatin loci closed by Sall4-NuRD encode genes resistant to reprogramming. These results identify a previously unrecognized role of NuRD in reprogramming, and may further illuminate chromatin closing as a critical step in cell fate control.
    DOI:  https://doi.org/10.1038/s41467-023-38543-0
  18. Nucleic Acids Res. 2023 May 13. pii: gkad372. [Epub ahead of print]
    DNA binding specificity of all four Saccharomyces cerevisiae forkhead transcription factors.
    Brendon H Cooper, Ana Carolina Dantas Machado, Yan Gan, Oscar M Aparicio, Remo Rohs.
      Quantifying the nucleotide preferences of DNA binding proteins is essential to understanding how transcription factors (TFs) interact with their targets in the genome. High-throughput in vitro binding assays have been used to identify the inherent DNA binding preferences of TFs in a controlled environment isolated from confounding factors such as genome accessibility, DNA methylation, and TF binding cooperativity. Unfortunately, many of the most common approaches for measuring binding preferences are not sensitive enough for the study of moderate-to-low affinity binding sites, and are unable to detect small-scale differences between closely related homologs. The Forkhead box (FOX) family of TFs is known to play a crucial role in regulating a variety of key processes from proliferation and development to tumor suppression and aging. By using the high-sequencing depth SELEX-seq approach to study all four FOX homologs in Saccharomyces cerevisiae, we have been able to precisely quantify the contribution and importance of nucleotide positions all along an extended binding site. Essential to this process was the alignment of our SELEX-seq reads to a set of candidate core sequences determined using a recently developed tool for the alignment of enriched k-mers and a newly developed approach for the reprioritization of candidate cores.
    DOI:  https://doi.org/10.1093/nar/gkad372
  19. Nat Commun. 2023 May 12. 14(1): 2734
    High-throughput single nucleus total RNA sequencing of formalin-fixed paraffin-embedded tissues by snRandom-seq.
    Ziye Xu, Tianyu Zhang, Hongyu Chen, Yuyi Zhu, Yuexiao Lv, Shunji Zhang, Jiaye Chen, Haide Chen, Lili Yang, Weiqin Jiang, Shengyu Ni, Fangru Lu, Zhaolun Wang, Hao Yang, Ling Dong, Feng Chen, Hong Zhang, Yu Chen, Jiong Liu, Dandan Zhang, Longjiang Fan, Guoji Guo, Yongcheng Wang.
      Formalin-fixed paraffin-embedded (FFPE) tissues constitute a vast and valuable patient material bank for clinical history and follow-up data. It is still challenging to achieve single cell/nucleus RNA (sc/snRNA) profile in FFPE tissues. Here, we develop a droplet-based snRNA sequencing technology (snRandom-seq) for FFPE tissues by capturing full-length total RNAs with random primers. snRandom-seq shows a minor doublet rate (0.3%), a much higher RNA coverage, and detects more non-coding RNAs and nascent RNAs, compared with state-of-art high-throughput scRNA-seq technologies. snRandom-seq detects a median of >3000 genes per nucleus and identifies 25 typical cell types. Moreover, we apply snRandom-seq on a clinical FFPE human liver cancer specimen and reveal an interesting subpopulation of nuclei with high proliferative activity. Our method provides a powerful snRNA-seq platform for clinical FFPE specimens and promises enormous applications in biomedical research.
    DOI:  https://doi.org/10.1038/s41467-023-38409-5
  20. Sci Rep. 2023 Apr 25. 13(1): 6745
    An unbiased AAV-STARR-seq screen revealing the enhancer activity map of genomic regions in the mouse brain in vivo.
    Ya-Chien Chan, Eike Kienle, Martin Oti, Antonella Di Liddo, Maria Mendez-Lago, Dominik F Aschauer, Manuel Peter, Michaela Pagani, Cosmas Arnold, Andreas Vonderheit, Christian Schön, Sebastian Kreuz, Alexander Stark, Simon Rumpel.
      Enhancers are important cis-regulatory elements controlling cell-type specific expression patterns of genes. Furthermore, combinations of enhancers and minimal promoters are utilized to construct small, artificial promoters for gene delivery vectors. Large-scale functional screening methodology to construct genomic maps of enhancer activities has been successfully established in cultured cell lines, however, not yet applied to terminally differentiated cells and tissues in a living animal. Here, we transposed the Self-Transcribing Active Regulatory Region Sequencing (STARR-seq) technique to the mouse brain using adeno-associated-viruses (AAV) for the delivery of a highly complex screening library tiling entire genomic regions and covering in total 3 Mb of the mouse genome. We identified 483 sequences with enhancer activity, including sequences that were not predicted by DNA accessibility or histone marks. Characterizing the expression patterns of fluorescent reporters controlled by nine candidate sequences, we observed differential expression patterns also in sparse cell types. Together, our study provides an entry point for the unbiased study of enhancer activities in organisms during health and disease.
    DOI:  https://doi.org/10.1038/s41598-023-33448-w
  21. Cell. 2023 May 10. pii: S0092-8674(23)00458-0. [Epub ahead of print]
    RNA polymerase drives ribonucleotide excision DNA repair in E. coli.
    Zhitai Hao, Manjunath Gowder, Sergey Proshkin, Binod K Bharati, Vitaly Epshtein, Vladimir Svetlov, Ilya Shamovsky, Evgeny Nudler.
      Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside monophosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence demonstrating that ribonucleotide excision repair (RER) is directly coupled to transcription. Affinity pull-downs and mass-spectrometry-assisted mapping of in cellulo inter-protein cross-linking reveal the majority of RNaseHII molecules interacting with RNA polymerase (RNAP) in E. coli. Cryoelectron microscopy structures of RNaseHII bound to RNAP during elongation, with and without the target rNMP substrate, show specific protein-protein interactions that define the transcription-coupled RER (TC-RER) complex in engaged and unengaged states. The weakening of RNAP-RNaseHII interactions compromises RER in vivo. The structure-functional data support a model where RNaseHII scans DNA in one dimension in search for rNMPs while "riding" the RNAP. We further demonstrate that TC-RER accounts for a significant fraction of repair events, thereby establishing RNAP as a surveillance "vehicle" for detecting the most frequently occurring replication errors.
    Keywords:  DNA repair; RNA polymerase; RNaseHII; cryo-EM; transcription elongation
    DOI:  https://doi.org/10.1016/j.cell.2023.04.029
  22. Nat Cell Biol. 2023 May 15.
    Single-nucleus multi-omics of human stem cell-derived islets identifies deficiencies in lineage specification.
    Punn Augsornworawat, Nathaniel J Hogrebe, Matthew Ishahak, Mason D Schmidt, Erica Marquez, Marlie M Maestas, Daniel A Veronese-Paniagua, Sarah E Gale, Julia R Miller, Leonardo Velazco-Cruz, Jeffrey R Millman.
      Insulin-producing β cells created from human pluripotent stem cells have potential as a therapy for insulin-dependent diabetes, but human pluripotent stem cell-derived islets (SC-islets) still differ from their in vivo counterparts. To better understand the state of cell types within SC-islets and identify lineage specification deficiencies, we used single-nucleus multi-omic sequencing to analyse chromatin accessibility and transcriptional profiles of SC-islets and primary human islets. Here we provide an analysis that enabled the derivation of gene lists and activity for identifying each SC-islet cell type compared with primary islets. Within SC-islets, we found that the difference between β cells and awry enterochromaffin-like cells is a gradient of cell states rather than a stark difference in identity. Furthermore, transplantation of SC-islets in vivo improved cellular identities overtime, while long-term in vitro culture did not. Collectively, our results highlight the importance of chromatin and transcriptional landscapes during islet cell specification and maturation.
    DOI:  https://doi.org/10.1038/s41556-023-01150-8
  23. Cell Rep. 2023 May 17. pii: S2211-1247(23)00531-4. [Epub ahead of print]42(5): 112520
    Differential cell-cycle control by oscillatory versus sustained Hes1 expression via p21.
    Yuki Maeda, Akihiro Isomura, Taimu Masaki, Ryoichiro Kageyama.
      Oscillatory Hes1 expression activates cell proliferation, while high and sustained Hes1 expression induces quiescence, but the mechanism by which Hes1 differentially controls cell proliferation depending on its expression dynamics is unclear. Here, we show that oscillatory Hes1 expression down-regulates the expression of the cyclin-dependent kinase inhibitor p21 (Cdkn1a), which delays cell-cycle progression, and thereby activates the proliferation of mouse neural stem cells (NSCs). By contrast, sustained Hes1 overexpression up-regulates p21 expression and inhibits NSC proliferation, although it initially down-regulates p21 expression. Compared with Hes1 oscillation, sustained Hes1 overexpression represses Dusp7, a phosphatase for phosphorylated Erk (p-Erk), and increases the levels of p-Erk, which can up-regulate p21 expression. These results indicate that p21 expression is directly repressed by oscillatory Hes1 expression, but indirectly up-regulated by sustained Hes1 overexpression, suggesting that depending on its expression dynamics, Hes1 differentially controls NSC proliferation via p21.
    Keywords:  CP: Cell biology; Hes1; neural stem cell; oscillation; p21; proliferation
    DOI:  https://doi.org/10.1016/j.celrep.2023.112520
  24. Nature. 2023 May 17.
    ERα-associated translocations underlie oncogene amplifications in breast cancer.
    Jake June-Koo Lee, Youngsook Lucy Jung, Taek-Chin Cheong, Jose Espejo Valle-Inclan, Chong Chu, Doga C Gulhan, Viktor Ljungström, Hu Jin, Vinayak V Viswanadham, Emma V Watson, Isidro Cortés-Ciriano, Stephen J Elledge, Roberto Chiarle, David Pellman, Peter J Park.
      Focal copy-number amplification is an oncogenic event. Although recent studies have revealed the complex structure1-3 and the evolutionary trajectories4 of oncogene amplicons, their origin remains poorly understood. Here we show that focal amplifications in breast cancer frequently derive from a mechanism-which we term translocation-bridge amplification-involving inter-chromosomal translocations that lead to dicentric chromosome bridge formation and breakage. In 780 breast cancer genomes, we observe that focal amplifications are frequently connected to each other by inter-chromosomal translocations at their boundaries. Subsequent analysis indicates the following model: the oncogene neighbourhood is translocated in G1 creating a dicentric chromosome, the dicentric chromosome is replicated, and as dicentric sister chromosomes segregate during mitosis, a chromosome bridge is formed and then broken, with fragments often being circularized in extrachromosomal DNAs. This model explains the amplifications of key oncogenes, including ERBB2 and CCND1. Recurrent amplification boundaries and rearrangement hotspots correlate with oestrogen receptor binding in breast cancer cells. Experimentally, oestrogen treatment induces DNA double-strand breaks in the oestrogen receptor target regions that are repaired by translocations, suggesting a role of oestrogen in generating the initial translocations. A pan-cancer analysis reveals tissue-specific biases in mechanisms initiating focal amplifications, with the breakage-fusion-bridge cycle prevalent in some and the translocation-bridge amplification in others, probably owing to the different timing of DNA break repair. Our results identify a common mode of oncogene amplification and propose oestrogen as its mechanistic origin in breast cancer.
    DOI:  https://doi.org/10.1038/s41586-023-06057-w
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