bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2022–02–27
24 papers selected by
Connor Rogerson, University of Cambridge, MRC Cancer Unit
  1. Sequence determinants of human gene regulatory elements.
  2. H3K27ac nucleosomes facilitate HMGN localization at regulatory sites to modulate chromatin binding of transcription factors.
  3. The landscape of pioneer factor activity reveals the mechanisms of chromatin reprogramming and genome activation.
  4. HiCAR is a robust and sensitive method to analyze open-chromatin-associated genome organization.
  5. CpG island reconfiguration for the establishment and synchronization of polycomb functions upon exit from naive pluripotency.
  6. Epigenetic reprogramming by TET enzymes impacts co-transcriptional R-loops.
  7. Application of ATAC-Seq for genome-wide analysis of the chromatin state at single myofiber resolution.
  8. KDM6B interacts with TFDP1 to activate P53 signalling in regulating mouse palatogenesis.
  9. Transcriptional repression of estrogen receptor alpha by YAP reveals the Hippo pathway as therapeutic target for ER+ breast cancer.
  10. monaLisa: an R/Bioconductor package for identifying regulatory motifs.
  11. A genome-scale CRISPR screen reveals PRMT1 as a critical regulator of androgen receptor signaling in prostate cancer.
  12. EBF1 nuclear repositioning instructs chromatin refolding to promote therapy resistance in T leukemic cells.
  13. SEAseq: a portable and cloud-based chromatin occupancy analysis suite.
  14. A fast Myosin super enhancer dictates muscle fiber phenotype through competitive interactions with Myosin genes.
  15. NOXA expression drives synthetic lethality to RUNX1 inhibition in pancreatic cancer.
  16. A global timing mechanism regulates cell-type-specific wiring programmes.
  17. TRmir: A Comprehensive Resource for Human Transcriptional Regulatory Information of MiRNAs.
  18. H3K36 methylation and DNA-binding both promote Ioc4 recruitment and Isw1b remodeler function.
  19. High performance single-cell gene regulatory network inference at scale: The Inferelator 3.0.
  20. 8C-like cells capture the human zygotic genome activation program in vitro.
  21. HyDrop enables droplet based single-cell ATAC-seq and single-cell RNA-seq using dissolvable hydrogel beads.
  22. EZH2 noncanonically binds cMyc and p300 through a cryptic transactivation domain to mediate gene activation and promote oncogenesis.
  23. Transcription factor protein interactomes reveal genetic determinants in heart disease.
  24. ERK signaling dissolves ERF repression condensates in living embryos.
  1. Nat Genet. 2022 Feb 21.
    Sequence determinants of human gene regulatory elements.
    Biswajyoti Sahu, Tuomo Hartonen, Päivi Pihlajamaa, Bei Wei, Kashyap Dave, Fangjie Zhu, Eevi Kaasinen, Katja Lidschreiber, Michael Lidschreiber, Carsten O Daub, Patrick Cramer, Teemu Kivioja, Jussi Taipale.
      DNA can determine where and when genes are expressed, but the full set of sequence determinants that control gene expression is unknown. Here, we measured the transcriptional activity of DNA sequences that represent an ~100 times larger sequence space than the human genome using massively parallel reporter assays (MPRAs). Machine learning models revealed that transcription factors (TFs) generally act in an additive manner with weak grammar and that most enhancers increase expression from a promoter by a mechanism that does not appear to involve specific TF-TF interactions. The enhancers themselves can be classified into three types: classical, closed chromatin and chromatin dependent. We also show that few TFs are strongly active in a cell, with most activities being similar between cell types. Individual TFs can have multiple gene regulatory activities, including chromatin opening and enhancing, promoting and determining transcription start site (TSS) activity, consistent with the view that the TF binding motif is the key atomic unit of gene expression.
    DOI:  https://doi.org/10.1038/s41588-021-01009-4
  2. Commun Biol. 2022 02 23. 5(1): 159
    H3K27ac nucleosomes facilitate HMGN localization at regulatory sites to modulate chromatin binding of transcription factors.
    Shaofei Zhang, Yuri Postnikov, Alexei Lobanov, Takashi Furusawa, Tao Deng, Michael Bustin.
      Nucleosomes containing acetylated H3K27 are a major epigenetic mark of active chromatin and identify cell-type specific chromatin regulatory regions which serve as binding sites for transcription factors. Here we show that the ubiquitous nucleosome binding proteins HMGN1 and HMGN2 bind preferentially to H3K27ac nucleosomes at cell-type specific chromatin regulatory regions. HMGNs bind directly to the acetylated nucleosome; the H3K27ac residue and linker DNA facilitate the preferential binding of HMGNs to the modified nucleosomes. Loss of HMGNs increases the levels of H3K27me3 and the histone H1 occupancy at enhancers and promoters and alters the interaction of transcription factors with chromatin. These experiments indicate that the H3K27ac epigenetic mark enhances the interaction of architectural protein with chromatin regulatory sites and identify determinants that facilitate the localization of HMGN proteins at regulatory sites to modulate cell-type specific gene expression.
    DOI:  https://doi.org/10.1038/s42003-022-03099-0
  3. Mol Cell. 2022 Feb 15. pii: S1097-2765(22)00100-9. [Epub ahead of print]
    The landscape of pioneer factor activity reveals the mechanisms of chromatin reprogramming and genome activation.
    Liyun Miao, Yin Tang, Ashley R Bonneau, Shun Hang Chan, Mina L Kojima, Mark E Pownall, Charles E Vejnar, Feng Gao, Smita Krishnaswamy, Caroline E Hendry, Antonio J Giraldez.
      Upon fertilization, embryos undergo chromatin reprogramming and genome activation; however, the mechanisms that regulate these processes are poorly understood. Here, we generated a triple mutant for Nanog, Pou5f3, and Sox19b (NPS) in zebrafish and found that NPS pioneer chromatin opening at >50% of active enhancers. NPS regulate acetylation across core histones at enhancers and promoters, and their function in gene activation can be bypassed by recruiting histone acetyltransferase to individual genes. NPS pioneer chromatin opening individually, redundantly, or additively depending on sequence context, and we show that high nucleosome occupancy facilitates NPS pioneering activity. Nucleosome position varies based on the input of different transcription factors (TFs), providing a flexible platform to modulate pioneering activity. Altogether, our results illuminate the sequence of events during genome activation and offer a conceptual framework to understand how pioneer factors interpret the genome and integrate different TF inputs across cell types and developmental transitions.
    Keywords:  chromatin accessibility; chromatin reprogramming; genome activation; histone acetylation; nucleosome occupancy; pioneer transcription factors
    DOI:  https://doi.org/10.1016/j.molcel.2022.01.024
  4. Mol Cell. 2022 Feb 12. pii: S1097-2765(22)00098-3. [Epub ahead of print]
    HiCAR is a robust and sensitive method to analyze open-chromatin-associated genome organization.
    Xiaolin Wei, Yu Xiang, Derek T Peters, Choiselle Marius, Tongyu Sun, Ruocheng Shan, Jianhong Ou, Xin Lin, Feng Yue, Wei Li, Kevin W Southerland, Yarui Diao.
      The long-range interactions of cis-regulatory elements (cREs) play a central role in gene regulation. cREs can be characterized as accessible chromatin sequences. However, it remains technically challenging to comprehensively identify their spatial interactions. Here, we report a new method HiCAR (Hi-C on accessible regulatory DNA), which utilizes Tn5 transposase and chromatin proximity ligation, for the analysis of open-chromatin-anchored interactions with low-input cells. By applying HiCAR in human embryonic stem cells and lymphoblastoid cells, we demonstrate that HiCAR identifies high-resolution chromatin contacts with an efficiency comparable with that of in situ Hi-C over all distance ranges. Interestingly, we found that the "poised" gene promoters exhibit silencer-like function to repress the expression of distal genes via promoter-promoter interactions. Lastly, we applied HiCAR to 30,000 primary human muscle stem cells and demonstrated that HiCAR is capable of analyzing chromatin accessibility and looping using low-input primary cells and clinical samples.
    Keywords:  HiCAR; Silencer-like promoter; chromatin accessibility; chromatin organization; low-input multi-omic
    DOI:  https://doi.org/10.1016/j.molcel.2022.01.023
  5. Mol Cell. 2022 Feb 15. pii: S1097-2765(22)00103-4. [Epub ahead of print]
    CpG island reconfiguration for the establishment and synchronization of polycomb functions upon exit from naive pluripotency.
    Dawei Huo, Zhaowei Yu, Rui Li, Meihan Gong, Simone Sidoli, Xukun Lu, Yuying Hou, Zhongye Dai, Yu Kong, Guifen Liu, Ole Jensen, Wei Xie, Kristian Helin, Chaoyang Xiong, Guohong Li, Yong Zhang, Xudong Wu.
      Polycomb group (PcG) proteins are essential for post-implantation development by depositing repressive histone modifications at promoters, mainly CpG islands (CGIs), of developmental regulator genes. However, promoter PcG marks are erased after fertilization and de novo established in peri-implantation embryos, coinciding with the transition from naive to primed pluripotency. Nevertheless, the molecular basis for this establishment remains unknown. In this study, we show that the expression of the long KDM2B isoform (KDM2BLF), which contains the demethylase domain, is specifically induced at peri-implantation and that its H3K36me2 demethylase activity is required for PcG enrichment at CGIs. Moreover, KDM2BLF interacts with BRG1/BRM-associated factor (BAF) and stabilizes BAF occupancy at CGIs for subsequent gain of accessibility, which precedes PcG enrichment. Consistently, KDM2BLF inactivation results in significantly delayed post-implantation development. In summary, our data unveil dynamic chromatin configuration of CGIs during exit from naive pluripotency and provide a conceptual framework for the spatiotemporal establishment of PcG functions.
    Keywords:  BRG1; CpG islands; H2AK119ub; H3K27me3; H3K36me2; SWI/SNF; accessibility; demethylation; polycomb; post-implantation development
    DOI:  https://doi.org/10.1016/j.molcel.2022.01.027
  6. Elife. 2022 Feb 22. pii: e69476. [Epub ahead of print]11
    Epigenetic reprogramming by TET enzymes impacts co-transcriptional R-loops.
    João C Sabino, Madalena R de Almeida, Patrícia L Abreu, Ana M Ferreira, Paulo Caldas, Marco M Domingues, Nuno C Santos, Claus M Azzalin, Ana Rita Grosso, Sérgio Fernandes de Almeida.
      DNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) initiates developmental and cell-type-specific transcriptional programs through mechanisms that include changes in the chromatin structure. Here, we show that the presence of 5hmC in the transcribed gene promotes the annealing of the nascent RNA to the template DNA strand, leading to the formation of an R-loop. Depletion of TET enzymes reduced global R-loops in absence of gene expression changes, whereas CRISPR-mediated tethering of TET to an active gene promoted the formation of R-loops. The genome-wide distribution of 5hmC and R-loops show a positive correlation in mouse and human stem cells and overlap in half of all active genes. Moreover, R-loop resolution leads to differential expression of a subset of genes that are involved in crucial events during stem cell proliferation. Altogether, our data reveal that epigenetic reprogramming via TET activity promotes co-transcriptional R-loop formation, disclosing new mechanisms of gene expression regulation.
    Keywords:  chromosomes; gene expression; human; mouse
    DOI:  https://doi.org/10.7554/eLife.69476
  7. Elife. 2022 Feb 21. pii: e72792. [Epub ahead of print]11
    Application of ATAC-Seq for genome-wide analysis of the chromatin state at single myofiber resolution.
    Korin Sahinyan, Darren M Blackburn, Marie-Michelle Simon, Felicia Lazure, Tony Kwan, Guillaume Bourque, Vahab D Soleimani.
      Myofibers are the main components of skeletal muscle, which is the largest tissue in the body. Myofibers are highly adaptive and can be altered under different biological and disease conditions. Therefore, transcriptional and epigenetic studies on myofibers are crucial to discover how chromatin alterations occur in the skeletal muscle under different conditions. However, due to the heterogenous nature of skeletal muscle, studying myofibers in isolation proves to be a challenging task. Single cell sequencing has permitted the study of the epigenome of isolated myonuclei. While this provides sequencing with high dimensionality, the sequencing depth is lacking, which makes comparisons between different biological conditions difficult. Here we report the first implementation of single myofiber ATAC-Seq, which allows for the sequencing of an individual myofiber at a depth sufficient for peak calling and for comparative analysis of chromatin accessibility under various physiological and disease conditions. Application of this technique revealed significant differences in chromatin accessibility between resting and regenerating myofibers, as well as between myofibers from a mouse model of Duchenne Muscular Dystrophy (mdx) and wild type (WT) counterparts. This technique can lead to a wide application in the identification of chromatin regulatory elements and epigenetic mechanisms in muscle fibers during development and in muscle-wasting diseases.
    Keywords:  genetics; genomics; mouse
    DOI:  https://doi.org/10.7554/eLife.72792
  8. Elife. 2022 Feb 25. pii: e74595. [Epub ahead of print]11
    KDM6B interacts with TFDP1 to activate P53 signalling in regulating mouse palatogenesis.
    Tingwei Guo, Xia Han, Jinzhi He, Jifan Feng, Junjun Jing, Eva Janečková, Jie Lei, Thach-Vu Ho, Jian Xu, Yang Chai.
      Epigenetic regulation plays extensive roles in diseases and development. Disruption of epigenetic regulation not only increases the risk of cancer, but can also cause various developmental defects. However, the question of how epigenetic changes lead to tissue-specific responses during neural crest fate determination and differentiation remains understudied. Using palatogenesis as a model, we reveal the functional significance of Kdm6b, a H3K27me3 demethylase, in regulating mouse embryonic development. Our study shows that Kdm6b plays an essential role in cranial neural crest development, and loss of Kdm6b disturbs P53 pathway-mediated activity, leading to complete cleft palate along with cell proliferation and differentiation defects in mice. Furthermore, activity of H3K27me3 on the promoter of Trp53 is antagonistically controlled by Kdm6b, and Ezh2 in cranial neural crest cells. More importantly, without Kdm6b, the transcription factor TFDP1, which normally binds to the promoter of Trp53, cannot activate Trp53 expression in palatal mesenchymal cells. Furthermore, the function of Kdm6b in activating Trp53 in these cells cannot be compensated for by the closely related histone demethylase Kdm6a. Collectively, our results highlight the important role of the epigenetic regulator KDM6B and how it specifically interacts with TFDP1 to achieve its functional specificity in regulating Trp53 expression, and further provide mechanistic insights into the epigenetic regulatory network during organogenesis.
    Keywords:  developmental biology; mouse
    DOI:  https://doi.org/10.7554/eLife.74595
  9. Nat Commun. 2022 Feb 25. 13(1): 1061
    Transcriptional repression of estrogen receptor alpha by YAP reveals the Hippo pathway as therapeutic target for ER+ breast cancer.
    Shenghong Ma, Tracy Tang, Gary Probst, Andrei Konradi, Chunyu Jin, Fulong Li, J Silvio Gutkind, Xiang-Dong Fu, Kun-Liang Guan.
      Extensive knowledge has been gained on the transcription network controlled by ERα, however, the mechanism underlying ESR1 (encoding ERα) expression is less understood. We recently discovered that the Hippo pathway is required for the proper expression of ESR1. YAP/TAZ are transcription coactivators that are phosphorylated and inhibited by the Hippo pathway kinase LATS. Here we delineated the molecular mechanisms underlying ESR1 transcription repression by the Hippo pathway. Mechanistically, YAP binds to TEAD to increase local chromatin accessibility to stimulate transcription of nearby genes. Among the YAP target genes, Vestigial-Like Protein 3 (VGLL3) competes with YAP/TAZ for binding to TEAD transcription factor and recruits the NCOR2/SMRT repressor to the super-enhancer of ESR1 gene, leading to epigenetic alteration and transcriptional silencing. We developed a potent LATS inhibitor VT02956. Targeting the Hippo pathway by VT02956 represses ESR1 expression and inhibits the growth of ER+ breast cancer cells as well as patient-derived tumour organoids. Moreover, histone deacetylase inhibitors, such as Entinostat, induce VGLL3 expression to inhibit ER+ breast cancer cells. Our study suggests LATS as unexpected cancer therapeutic targets, especially for endocrine-resistant breast cancers.
    DOI:  https://doi.org/10.1038/s41467-022-28691-0
  10. Bioinformatics. 2022 Feb 23. pii: btac102. [Epub ahead of print]
    monaLisa: an R/Bioconductor package for identifying regulatory motifs.
    Dania Machlab, Lukas Burger, Charlotte Soneson, Filippo M Rijli, Dirk Schübeler, Michael B Stadler.
        : Proteins binding to specific nucleotide sequences, such as transcription factors, play key roles in the regulation of gene expression. Their binding can be indirectly observed via associated changes in transcription, chromatin accessibility, DNA methylation and histone modifications. Identifying candidate factors that are responsible for these observed experimental changes is critical to understand the underlying biological processes. Here we present monaLisa, an R/Bioconductor package that implements approaches to identify relevant transcription factors from experimental data. The package can be easily integrated with other Bioconductor packages and enables seamless motif analyses without any software dependencies outside of R.
    AVAILABILITY: monaLisa is implemented in R and available on Bioconductor at https://bioconductor.org/packages/monaLisa with the development version hosted on GitHub at https://github.com/fmicompbio/monaLisa.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btac102
  11. Cell Rep. 2022 Feb 22. pii: S2211-1247(22)00141-3. [Epub ahead of print]38(8): 110417
    A genome-scale CRISPR screen reveals PRMT1 as a critical regulator of androgen receptor signaling in prostate cancer.
    Stephen Tang, Vidyalakshmi Sethunath, Nebiyou Y Metaferia, Marina F Nogueira, Daniel S Gallant, Emma R Garner, Lauren A Lairson, Christopher M Penney, Jiao Li, Maya K Gelbard, Sarah Abou Alaiwi, Ji-Heui Seo, Justin H Hwang, Craig A Strathdee, Sylvan C Baca, Shatha AbuHammad, Xiaoyang Zhang, John G Doench, William C Hahn, David Y Takeda, Matthew L Freedman, Peter S Choi, Srinivas R Viswanathan.
      Androgen receptor (AR) signaling is the central driver of prostate cancer across disease states. While androgen deprivation therapy (ADT) is effective in the initial treatment of prostate cancer, resistance to ADT or to next-generation androgen pathway inhibitors invariably arises, most commonly through the re-activation of the AR axis. Thus, orthogonal approaches to inhibit AR signaling in advanced prostate cancer are essential. Here, via genome-scale CRISPR-Cas9 screening, we identify protein arginine methyltransferase 1 (PRMT1) as a critical mediator of AR expression and signaling. PRMT1 regulates the recruitment of AR to genomic target sites and the inhibition of PRMT1 impairs AR binding at lineage-specific enhancers, leading to decreased expression of key oncogenes, including AR itself. In addition, AR-driven prostate cancer cells are uniquely susceptible to combined AR and PRMT1 inhibition. Our findings implicate PRMT1 as a key regulator of AR output and provide a preclinical framework for co-targeting of AR and PRMT1 in advanced prostate cancer.
    Keywords:  CRISPR screen; PRMT1; androgen receptor; prostate cancer; splicing; superenhancer; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2022.110417
  12. Mol Cell. 2022 Feb 09. pii: S1097-2765(22)00058-2. [Epub ahead of print]
    EBF1 nuclear repositioning instructs chromatin refolding to promote therapy resistance in T leukemic cells.
    Yeqiao Zhou, Jelena Petrovic, Jingru Zhao, Wu Zhang, Ashkan Bigdeli, Zhen Zhang, Shelley L Berger, Warren S Pear, Robert B Faryabi.
      Chromatin misfolding has been implicated in cancer pathogenesis; yet, its role in therapy resistance remains unclear. Here, we systematically integrated sequencing and imaging data to examine the spatial and linear chromatin structures in targeted therapy-sensitive and -resistant human T cell acute lymphoblastic leukemia (T-ALL). We found widespread alterations in successive layers of chromatin organization including spatial compartments, contact domain boundaries, and enhancer positioning upon the emergence of targeted therapy resistance. The reorganization of genome folding structures closely coincides with the restructuring of chromatin activity and redistribution of architectural proteins. Mechanistically, the derepression and repositioning of the B-lineage-determining transcription factor EBF1 from the heterochromatic nuclear envelope to the euchromatic interior instructs widespread genome refolding and promotes therapy resistance in leukemic T cells. Together, our findings suggest that lineage-determining transcription factors can instruct changes in genome topology as a driving force for epigenetic adaptations in targeted therapy resistance.
    Keywords:  EBF1; T cell leukemia; TCF1; chromatin folding; drug resistance; epigenetic adaptation; genome topology; lineage-determining transcription factors; pioneer transcription factors; targeted therapy
    DOI:  https://doi.org/10.1016/j.molcel.2022.01.015
  13. BMC Bioinformatics. 2022 Feb 23. 23(1): 77
    SEAseq: a portable and cloud-based chromatin occupancy analysis suite.
    Modupeore O Adetunji, Brian J Abraham.
       BACKGROUND: Genome-wide protein-DNA binding is popularly assessed using specific antibody pulldown in Chromatin Immunoprecipitation Sequencing (ChIP-Seq) or Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing experiments. These technologies generate high-throughput sequencing data that necessitate the use of multiple sophisticated, computationally intensive genomic tools to make discoveries, but these genomic tools often have a high barrier to use because of computational resource constraints.
    RESULTS: We present a comprehensive, infrastructure-independent, computational pipeline called SEAseq, which leverages field-standard, open-source tools for processing and analyzing ChIP-Seq/CUT&RUN data. SEAseq performs extensive analyses from the raw output of the experiment, including alignment, peak calling, motif analysis, promoters and metagene coverage profiling, peak annotation distribution, clustered/stitched peaks (e.g. super-enhancer) identification, and multiple relevant quality assessment metrics, as well as automatic interfacing with data in GEO/SRA. SEAseq enables rapid and cost-effective resource for analysis of both new and publicly available datasets as demonstrated in our comparative case studies.
    CONCLUSIONS: The easy-to-use and versatile design of SEAseq makes it a reliable and efficient resource for ensuring high quality analysis. Its cloud implementation enables a broad suite of analyses in environments with constrained computational resources. SEAseq is platform-independent and is aimed to be usable by everyone with or without programming skills. It is available on the cloud at https://platform.stjude.cloud/workflows/seaseq and can be locally installed from the repository at https://github.com/stjude/seaseq .
    Keywords:  Analysis pipeline; CUT&RUN; ChIP sequencing; Cloud; Computational genomics; Data analysis; GEO; Motif analysis; Peak calling; Platform independent; SRA
    DOI:  https://doi.org/10.1186/s12859-022-04588-z
  14. Nat Commun. 2022 Feb 24. 13(1): 1039
    A fast Myosin super enhancer dictates muscle fiber phenotype through competitive interactions with Myosin genes.
    Matthieu Dos Santos, Stéphanie Backer, Frédéric Auradé, Matthew Man-Kin Wong, Maud Wurmser, Rémi Pierre, Francina Langa, Marcio Do Cruzeiro, Alain Schmitt, Jean-Paul Concordet, Athanassia Sotiropoulos, F Jeffrey Dilworth, Daan Noordermeer, Frédéric Relaix, Iori Sakakibara, Pascal Maire.
      The contractile properties of adult myofibers are shaped by their Myosin heavy chain isoform content. Here, we identify by snATAC-seq a 42 kb super-enhancer at the locus regrouping the fast Myosin genes. By 4C-seq we show that active fast Myosin promoters interact with this super-enhancer by DNA looping, leading to the activation of a single promoter per nucleus. A rainbow mouse transgenic model of the locus including the super-enhancer recapitulates the endogenous spatio-temporal expression of adult fast Myosin genes. In situ deletion of the super-enhancer by CRISPR/Cas9 editing demonstrates its major role in the control of associated fast Myosin genes, and deletion of two fast Myosin genes at the locus reveals an active competition of the promoters for the shared super-enhancer. Last, by disrupting the organization of fast Myosin, we uncover positional heterogeneity within limb skeletal muscles that may underlie selective muscle susceptibility to damage in certain myopathies.
    DOI:  https://doi.org/10.1038/s41467-022-28666-1
  15. Proc Natl Acad Sci U S A. 2022 Mar 01. pii: e2105691119. [Epub ahead of print]119(9):
    NOXA expression drives synthetic lethality to RUNX1 inhibition in pancreatic cancer.
    Josefina Doffo, Stefanos A Bamopoulos, Hazal Köse, Felix Orben, Chuanbing Zang, Miriam Pons, Alexander T den Dekker, Rutger W W Brouwer, Apoorva Baluapuri, Stefan Habringer, Maximillian Reichert, Anuradha Illendula, Oliver H Krämer, Markus Schick, Elmar Wolf, Wilfred F J van IJcken, Irene Esposito, Ulrich Keller, Günter Schneider, Matthias Wirth.
      Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The proapoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models, we identified an inhibitor of the transcription factor heterodimer CBFβ/RUNX1. By genetic gain and loss of function experiments, we validated that the mode of action depends on RUNX1 and NOXA. Of note is that RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome-wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a way to target a therapy-resistant PDAC, an unmet clinical need.
    Keywords:  NOXA; PDAC; RUNX1; apoptosis; pancreatic cancer
    DOI:  https://doi.org/10.1073/pnas.2105691119
  16. Nature. 2022 Feb 23.
    A global timing mechanism regulates cell-type-specific wiring programmes.
    Saumya Jain, Ying Lin, Yerbol Z Kurmangaliyev, Javier Valdes-Aleman, Samuel A LoCascio, Parmis Mirshahidi, Brianna Parrington, S Lawrence Zipursky.
      The assembly of neural circuits is dependent on precise spatiotemporal expression of cell recognition molecules1-5. Factors controlling cell type specificity have been identified6-8, but how timing is determined remains unknown. Here we describe induction of a cascade of transcription factors by a steroid hormone (ecdysone) in all fly visual system neurons spanning target recognition and synaptogenesis. We demonstrate through single-cell sequencing that the ecdysone pathway regulates the expression of a common set of targets required for synaptic maturation and cell-type-specific targets enriched for cell-surface proteins regulating wiring specificity. Transcription factors in the cascade regulate the expression of the same wiring genes in complex ways, including activation in one cell type and repression in another. We show that disruption of the ecdysone pathway generates specific defects in dendritic and axonal processes and synaptic connectivity, with the order of transcription factor expression correlating with sequential steps in wiring. We also identify shared targets of a cell-type-specific transcription factor and the ecdysone pathway that regulate specificity. We propose that neurons integrate a global temporal transcriptional module with cell-type-specific transcription factors to generate different cell-type-specific patterns of cell recognition molecules regulating wiring.
    DOI:  https://doi.org/10.1038/s41586-022-04418-5
  17. Front Genet. 2022 ;13 808950
    TRmir: A Comprehensive Resource for Human Transcriptional Regulatory Information of MiRNAs.
    Yu Gao, Chenchen Feng, Yuexin Zhang, Chao Song, Jiaxin Chen, Yanyu Li, Ling Wei, Fengcui Qian, Bo Ai, Yuejuan Liu, Jiang Zhu, Xiaojie Su, Chunquan Li, Qiuyu Wang.
      MicroRNAs (miRNAs) are small non-coding RNAs, which play important roles in regulating various biological functions. Many available miRNA databases have provided a large number of valuable resources for miRNA investigation. However, not all existing databases provide comprehensive information regarding the transcriptional regulatory regions of miRNAs, especially typical enhancer, super-enhancer (SE), and chromatin accessibility regions. An increasing number of studies have shown that the transcriptional regulatory regions of miRNAs, as well as related single-nucleotide polymorphisms (SNPs) and transcription factors (TFs) have a strong influence on human diseases and biological processes. Here, we developed a comprehensive database for the human transcriptional regulation of miRNAs (TRmir), which is focused on providing a wealth of available resources regarding the transcriptional regulatory regions of miRNAs and annotating their potential roles in the regulation of miRNAs. TRmir contained a total of 5,754,414 typical enhancers/SEs and 1,733,966 chromatin accessibility regions associated with 1,684 human miRNAs. These regions were identified from over 900 human H3K27ac ChIP-seq, ATAC-seq, and DNase-seq samples. Furthermore, TRmir provided detailed (epi)genetic information about the transcriptional regulatory regions of miRNAs, including TFs, common SNPs, risk SNPs, linkage disequilibrium (LD) SNPs, expression quantitative trait loci (eQTLs), 3D chromatin interactions, and methylation sites, especially supporting the display of TF binding sites in the regulatory regions of over 7,000 TF ChIP-seq samples. In addition, TRmir integrated miRNA expression and related disease information, supporting extensive pathway analysis. TRmir is a powerful platform that offers comprehensive information about the transcriptional regulation of miRNAs for users and provides detailed annotations of regulatory regions. TRmir is free for academic users and can be accessed at http://bio.liclab.net/trmir/index.html.
    Keywords:  chromatin accessibility; genetics and epigenetics; microRNA; super-enhancer/typical enhancer; transcriptional regulation
    DOI:  https://doi.org/10.3389/fgene.2022.808950
  18. Nucleic Acids Res. 2022 Feb 21. pii: gkac077. [Epub ahead of print]
    H3K36 methylation and DNA-binding both promote Ioc4 recruitment and Isw1b remodeler function.
    Jian Li, Lena Bergmann, Andreia Rafael de Almeida, Kimberly M Webb, Madelaine M Gogol, Philipp Voigt, Yingfang Liu, Huanhuan Liang, Michaela M Smolle.
      The Isw1b chromatin-remodeling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II. Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain. Here, we present the crystal structure of the Ioc4-PWWP domain, including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeler. The Ioc4-PWWP domain preferentially binds H3K36me3-containing nucleosomes. Its ability to bind DNA is required for nucleosome binding. It is also furthered by the unique insertion motif present in Ioc4-PWWP. The ability to bind H3K36me3 and DNA promotes the interaction of full-length Ioc4 with nucleosomes in vitro and they are necessary for its recruitment to gene bodies in vivo. Furthermore, a fully functional Ioc4-PWWP domain promotes efficient remodeling by Isw1b and the maintenance of ordered chromatin in vivo, thereby preventing the production of non-coding RNAs.
    DOI:  https://doi.org/10.1093/nar/gkac077
  19. Bioinformatics. 2022 Feb 21. pii: btac117. [Epub ahead of print]
    High performance single-cell gene regulatory network inference at scale: The Inferelator 3.0.
    Claudia Skok Gibbs, Christopher A Jackson, Giuseppe-Antonio Saldi, Andreas Tjärnberg, Aashna Shah, Aaron Watters, Nicholas De Veaux, Konstantine Tchourine, Ren Yi, Tymor Hamamsy, Dayanne M Castro, Nicholas Carriero, Bram L Gorissen, David Gresham, Emily R Miraldi, Richard Bonneau.
       MOTIVATION: Gene regulatory networks define regulatory relationships between transcription factors and target genes within a biological system, and reconstructing them is essential for understanding cellular growth and function. Methods for inferring and reconstructing networks from genomics data have evolved rapidly over the last decade in response to advances in sequencing technology and machine learning. The scale of data collection has increased dramatically; the largest genome-wide gene expression datasets have grown from thousands of measurements to millions of single cells, and new technologies are on the horizon to increase to tens of millions of cells and above.
    RESULTS: In this work, we present the Inferelator 3.0, which has been significantly updated to integrate data from distinct cell types to learn context-specific regulatory networks and aggregate them into a shared regulatory network, while retaining the functionality of the previous versions. The Inferelator is able to integrate the largest single-cell datasets and learn cell-type specific gene regulatory networks. Compared to other network inference methods, the Inferelator learns new and informative Saccharomyces cerevisiae networks from single-cell gene expression data, measured by recovery of a known gold standard. We demonstrate its scaling capabilities by learning networks for multiple distinct neuronal and glial cell types in the developing Mus musculus brain at E18 from a large (1.3 million) single-cell gene expression dataset with paired single-cell chromatin accessibility data.
    AVAILABILITY: The inferelator software is available on GitHub (https://github.com/flatironinstitute/inferelator) under the MIT license and has been released as python packages with associated documentation (https://inferelator.readthedocs.io/).
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btac117
  20. Cell Stem Cell. 2022 Feb 15. pii: S1934-5909(22)00050-9. [Epub ahead of print]
    8C-like cells capture the human zygotic genome activation program in vitro.
    Jasmin Taubenschmid-Stowers, Maria Rostovskaya, Fátima Santos, Sebastian Ljung, Ricard Argelaguet, Felix Krueger, Jennifer Nichols, Wolf Reik.
      The activation of the embryonic genome marks the first major wave of transcription in the developing organism. Zygotic genome activation (ZGA) in mouse 2-cell embryos and 8-cell embryos in humans is crucial for development. Here, we report the discovery of human 8-cell-like cells (8CLCs) among naive embryonic stem cells, which transcriptionally resemble the 8-cell human embryo. They express ZGA markers, including ZSCAN4 and LEUTX, and transposable elements, such as HERVL and MLT2A1. 8CLCs show reduced SOX2 levels and can be identified using TPRX1 and H3.Y marker proteins in vitro. Overexpression of the transcription factor DUX4 and spliceosome inhibition increase human ZGA-like transcription. Excitingly, the 8CLC markers TPRX1 and H3.Y are also expressed in ZGA-stage 8-cell human embryos and may thus be relevant in vivo. 8CLCs provide a unique opportunity to characterize human ZGA-like transcription and might provide critical insights into early events in embryogenesis in humans.
    Keywords:  8CLCs; human pre-implantation development; in vitro model system; naive pluripotent stem cells; zygotic genome activation
    DOI:  https://doi.org/10.1016/j.stem.2022.01.014
  21. Elife. 2022 Feb 23. pii: e73971. [Epub ahead of print]11
    HyDrop enables droplet based single-cell ATAC-seq and single-cell RNA-seq using dissolvable hydrogel beads.
    Florian De Rop, Joy N Ismail, Carmen Bravo González-Blas, Gert J Hulselmans, Christopher Campbell Flerin, Jasper Janssens, Koen Theunis, Valerie M Christiaens, Jasper Wouters, Gabriele Marcassa, Joris de Wit, Suresh Poovathingal, Stein Aerts.
      Single-cell RNA-seq and single-cell ATAC-seq technologies are used extensively to create cell type atlases for a wide range of organisms, tissues, and disease processes. To increase the scale of these atlases, lower the cost, and pave the way for more specialized multi-ome assays, custom droplet microfluidics may provide solutions complementary to commercial setups. We developed HyDrop, a flexible and open-source droplet microfluidic platform encompassing three protocols. The first protocol involves creating dissolvable hydrogel beads with custom oligos that can be released in the droplets. In the second protocol, we demonstrate the use of these beads for HyDrop-ATAC, a low-cost non-commercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, we applied it to flash-frozen mouse cortex and generated 7,996 high-quality single-cell chromatin accessibility profiles in a single run. In the third protocol, we adapt both the reaction chemistry and the capture sequence of the barcoded hydrogel bead to capture mRNA, and demonstrate a significant improvement in throughput and sensitivity compared to previous open-source droplet-based scRNA-seq assays (Drop-seq and inDrop). Similarly, we applied HyDrop-RNA to flash-frozen mouse cortex and generated 9,508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, we leveraged HyDrop-RNA's high capture rate to analyse a small population of FAC-sorted neurons from the Drosophila brain, confirming the protocol's applicability to low-input samples and small cells. HyDrop is currently capable of generating single-cell data in high throughput and at a reduced cost compared to commercial methods, and we envision that HyDrop can be further developed to be compatible with novel (multi-) omics protocols.
    Keywords:  D. melanogaster; chromosomes; gene expression; genetics; genomics; human; mouse
    DOI:  https://doi.org/10.7554/eLife.73971
  22. Nat Cell Biol. 2022 Feb 24.
    EZH2 noncanonically binds cMyc and p300 through a cryptic transactivation domain to mediate gene activation and promote oncogenesis.
    Jun Wang, Xufen Yu, Weida Gong, Xijuan Liu, Kwang-Su Park, Anqi Ma, Yi-Hsuan Tsai, Yudao Shen, Takashi Onikubo, Wen-Chieh Pi, David F Allison, Jing Liu, Wei-Yi Chen, Ling Cai, Robert G Roeder, Jian Jin, Gang Greg Wang.
      Canonically, EZH2 serves as the catalytic subunit of PRC2, which mediates H3K27me3 deposition and transcriptional repression. Here, we report that in acute leukaemias, EZH2 has additional noncanonical functions by binding cMyc at non-PRC2 targets and uses a hidden transactivation domain (TAD) for (co)activator recruitment and gene activation. Both canonical (EZH2-PRC2) and noncanonical (EZH2-TAD-cMyc-coactivators) activities of EZH2 promote oncogenesis, which explains the slow and ineffective antitumour effect of inhibitors of the catalytic function of EZH2. To suppress the multifaceted activities of EZH2, we used proteolysis-targeting chimera (PROTAC) to develop a degrader, MS177, which achieved effective, on-target depletion of EZH2 and interacting partners (that is, both canonical EZH2-PRC2 and noncanonical EZH2-cMyc complexes). Compared with inhibitors of the enzymatic function of EZH2, MS177 is fast-acting and more potent in suppressing cancer growth. This study reveals noncanonical oncogenic roles of EZH2, reports a PROTAC for targeting the multifaceted tumorigenic functions of EZH2 and presents an attractive strategy for treating EZH2-dependent cancers.
    DOI:  https://doi.org/10.1038/s41556-022-00850-x
  23. Cell. 2022 Feb 15. pii: S0092-8674(22)00079-4. [Epub ahead of print]
    Transcription factor protein interactomes reveal genetic determinants in heart disease.
    Barbara Gonzalez-Teran, Maureen Pittman, Franco Felix, Reuben Thomas, Desmond Richmond-Buccola, Ruth Hüttenhain, Krishna Choudhary, Elisabetta Moroni, Mauro W Costa, Yu Huang, Arun Padmanabhan, Michael Alexanian, Clara Youngna Lee, Bonnie E J Maven, Kaitlen Samse-Knapp, Sarah U Morton, Michael McGregor, Casey A Gifford, J G Seidman, Christine E Seidman, Bruce D Gelb, Giorgio Colombo, Bruce R Conklin, Brian L Black, Benoit G Bruneau, Nevan J Krogan, Katherine S Pollard, Deepak Srivastava.
      Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains challenging. We hypothesized that genetic determinants for CHDs may lie in the protein interactomes of transcription factors whose mutations cause CHDs. Defining the interactomes of two transcription factors haplo-insufficient in CHD, GATA4 and TBX5, within human cardiac progenitors, and integrating the results with nearly 9,000 exomes from proband-parent trios revealed an enrichment of de novo missense variants associated with CHD within the interactomes. Scoring variants of interactome members based on residue, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes, and the identified GLYR1 missense variant disrupted interaction with GATA4, impairing in vitro and in vivo function in mice. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating genetic variants in heart disease.
    Keywords:  GATA4; GLYR1; NPAC; TBX5; congenital heart disease; de novo variants; disease variants; genetics; protein interactome networks
    DOI:  https://doi.org/10.1016/j.cell.2022.01.021
  24. Proc Natl Acad Sci U S A. 2022 Mar 01. pii: e2119187119. [Epub ahead of print]119(9):
    ERK signaling dissolves ERF repression condensates in living embryos.
    Claire J Weaver, Aleena L Patel, Stanislav Y Shvartsman, Michael S Levine, Nicholas Treen.
      Phase separation underlies the organization of the nucleus, including the biogenesis of nucleoli and the packaging of heterochromatin. Here we explore the regulation of transcription factor condensates involved in gene repression by ERK signaling in gastrulating embryos of a simple proto-vertebrate (Ciona). ERK signaling induces nuclear export of the transcriptional repressor Ets-2 repressive factor (ERF), which has been linked to various human developmental disorders. Using high-resolution imaging, we show that ERF is localized within discrete nuclear condensates that dissolve upon ERK activation. Interestingly, we observe dynamic pulses of assembly and dissociation during interphase, providing visualization of a nuclear phase separation process regulated by cell signaling. We discuss the implications of these observations for producing sharp on/off switches in gene activity and suppressing noise in cell-cell signaling events.
    Keywords:  developmental biology; phase separation; transcriptional repression
    DOI:  https://doi.org/10.1073/pnas.2119187119
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