bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2023–01–29
twenty papers selected by
Connor Rogerson, University of Cambridge
  1. Cistrome Explorer: An Interactive Visual Analysis Tool for Large-Scale Epigenomic Data.
  2. Deciphering the multi-scale, quantitative cis-regulatory code.
  3. Enhancer remodeling drives MLL oncogene-dependent transcriptional dysregulation in leukemia stem cells.
  4. The adapted Activity-By-Contact model for enhancer-gene assignment and its application to single-cell data.
  5. An atlas of lamina-associated chromatin across twelve human cell types reveals an intermediate chromatin subtype.
  6. Dynamic changes in whole genome DNA methylation, chromatin and gene expression during mouse lens differentiation.
  7. The nucleosome unwrapping free energy landscape defines distinct regions of transcription factor accessibility and kinetics.
  8. β-actin mediated H3K27ac changes demonstrate the link between compartment switching and enhancer-dependent transcriptional regulation.
  9. DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
  10. Structural mechanism of BRD4-NUT and p300 bipartite interaction in propagating aberrant gene transcription in chromatin in NUT carcinoma.
  11. Mechanisms and function of de novo DNA methylation in placental development reveals an essential role for DNMT3B.
  12. Epigenomic charting and functional annotation of risk loci in renal cell carcinoma.
  13. Mapping nucleolus-associated chromatin interactions using nucleolus Hi-C reveals pattern of heterochromatin interactions.
  14. Dynamics and regulation of mitotic chromatin accessibility bookmarking at single-cell resolution.
  15. Auxin-inducible degron 2 system deciphers functions of CTCF domains in transcriptional regulation.
  16. Minimal synthetic enhancers reveal control of the probability of transcriptional engagement and its timing by a morphogen gradient.
  17. Chromatin complex dependencies reveal targeting opportunities in leukemia.
  18. Oncogenic ERRB2 signals through the AP-1 transcription factor to control mesenchymal-like properties of oesophageal adenocarcinoma.
  19. Expansion of ventral foregut is linked to changes in the enhancer landscape for organ-specific differentiation.
  20. peaksat: an R package for ChIP-seq peak saturation analysis.
  1. Bioinformatics. 2023 Jan 23. pii: btad018. [Epub ahead of print]
    Cistrome Explorer: An Interactive Visual Analysis Tool for Large-Scale Epigenomic Data.
    Sehi L'Yi, Mark S Keller, Ariaki Dandawate, Len Taing, Chen-Hao Chen, Myles Brown, Clifford A Meyer, Nils Gehlenborg.
       SUMMARY: The regulation of genes by cis-regulatory elements is complex and differs between cell types. Visual analysis of large collections of chromatin profiles across diverse cell types, integrated with computational methods, can reveal meaningful biological insights. We developed Cistrome Explorer, a web-based interactive visual analytics tool for exploring thousands of chromatin profiles in diverse cell types. Integrated with the Cistrome Data Browser database which contains thousands of ChIP-seq, DNase-seq, and ATAC-seq samples, Cistrome Explorer enables the discovery of patterns of cis-regulatory elements across cell types and the identification of transcription factor binding underlying these patterns.
    AVAILABILITY AND IMPLEMENTATION: Cistrome Explorer and its source code are available at http://cisvis.gehlenborglab.org/ and released under the MIT License. Documentation can be accessed via http://cisvis.gehlenborglab.org/docs/.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btad018
  2. Mol Cell. 2023 Jan 13. pii: S1097-2765(22)01215-1. [Epub ahead of print]
    Deciphering the multi-scale, quantitative cis-regulatory code.
    Seungsoo Kim, Joanna Wysocka.
      Uncovering the cis-regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state remains a central goal of biology. Here, we discuss major layers of regulation that influence how transcriptional outputs are encoded by DNA sequence and cellular context. We first discuss how transcription factors bind specific DNA sequences in a dosage-dependent and cooperative manner and then proceed to the cofactors that facilitate transcription factor function and mediate the activity of modular cis-regulatory elements such as enhancers, silencers, and promoters. We then consider the complex and poorly understood interplay of these diverse elements within regulatory landscapes and its relationships with chromatin states and nuclear organization. We propose that a mechanistically informed, quantitative model of transcriptional regulation that integrates these multiple regulatory layers will be the key to ultimately cracking the cis-regulatory code.
    Keywords:  RNA polymerase II; activation domain; chromatin; cis-regulatory code; cis-regulatory element; coactivator; cofactor; corepressor; enhancer; gene regulation; insulator; nucleosome; pioneer factor; promoter; regulatory domain; repression domain; silencer; topologically associating domain; transcription; transcription factor
    DOI:  https://doi.org/10.1016/j.molcel.2022.12.032
  3. Blood Adv. 2023 Jan 27. pii: bloodadvances.2022008787. [Epub ahead of print]
    Enhancer remodeling drives MLL oncogene-dependent transcriptional dysregulation in leukemia stem cells.
    Feng Pan, Masayuki Iwasaki, Wenqi Wu, Yanan Jiang, Xin Yang, Li Zhu, Zhigang Zhao, Michael L Cleary.
      Acute myeloid leukemia (AML) with MLL gene rearrangement (MLLr) comprises a cellular hierarchy in which a subpopulation of cells serves as functional leukemia stem cells (LSCs). They are maintained by a unique gene expression program and chromatin states, which are thought to reflect the actions of enhancers. Here, we delineate the active enhancer landscape and observe pervasive enhancer malfunction in LSCs. Reconstruction of regulatory networks revealed a master set of hematopoietic transcription factors. We show that EP300 is an essential transcriptional coregulator for maintaining LSC oncogenic potential, as it controls essential gene expression through modulation of H3K27 acetylation and assessments of transcription factor dependencies. Moreover, the EP300 inhibitor A-485 affects LSC growth by targeting enhancer activity via histone acetyltransferase domain inhibition. Together, these data implicate a perturbed MLLr-specific enhancer accessibility landscape, suggesting the possibility for disruption of the LSC enhancer regulatory axis as a promising therapeutic strategy in AML.
    DOI:  https://doi.org/10.1182/bloodadvances.2022008787
  4. Bioinformatics. 2023 Jan 27. pii: btad062. [Epub ahead of print]
    The adapted Activity-By-Contact model for enhancer-gene assignment and its application to single-cell data.
    Dennis Hecker, Fatemeh Behjati Ardakani, Alexander Karollus, Julien Gagneur, Marcel H Schulz.
       MOTIVATION: Identifying regulatory regions in the genome is of great interest for understanding the epigenomic landscape in cells. One fundamental challenge in this context is to find the target genes whose expression is affected by the regulatory regions. A recent successful method is the Activity-By-Contact (ABC) model (Fulco et al., 2019) which scores enhancer-gene interactions based on enhancer activity and the contact frequency of an enhancer to its target gene. However, it describes regulatory interactions entirely from a gene's perspective, and does not account for all the candidate target genes of an enhancer. In addition, the ABC-model requires two types of assays to measure enhancer activity, which limits the applicability. Moreover, there is no implementation available that could allow for an integration with transcription factor (TF) binding information nor an efficient analysis of single-cell data.
    RESULTS: We demonstrate that the ABC-score can yield a higher accuracy by adapting the enhancer activity according to the number of contacts the enhancer has to its candidate target genes and also by considering all annotated transcription start sites of a gene. Further, we show that the model is comparably accurate with only one assay to measure enhancer activity. We combined our generalised ABC-model (gABC) with TF binding information and illustrate an analysis of a single-cell ATAC-seq data set of the human heart, where we were able to characterise cell type-specific regulatory interactions and predict gene expression based on transcription factor affinities. All executed processing steps are incorporated into our new computational pipeline STARE.
    AVAILABILITY: The software is available at https://github.com/schulzlab/STARE.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btad062
  5. Genome Biol. 2023 Jan 23. 24(1): 16
    An atlas of lamina-associated chromatin across twelve human cell types reveals an intermediate chromatin subtype.
    Parisha P Shah, Kathleen C Keough, Ketrin Gjoni, Garrett T Santini, Richard J Abdill, Nadeera M Wickramasinghe, Carolyn E Dundes, Ashley Karnay, Angela Chen, Rachel E A Salomon, Patrick J Walsh, Son C Nguyen, Sean Whalen, Eric F Joyce, Kyle M Loh, Nicole Dubois, Katherine S Pollard, Rajan Jain.
       BACKGROUND: Association of chromatin with lamin proteins at the nuclear periphery has emerged as a potential mechanism to coordinate cell type-specific gene expression and maintain cellular identity via gene silencing. Unlike many histone modifications and chromatin-associated proteins, lamina-associated domains (LADs) are mapped genome-wide in relatively few genetically normal human cell types, which limits our understanding of the role peripheral chromatin plays in development and disease.
    RESULTS: To address this gap, we map LAMIN B1 occupancy across twelve human cell types encompassing pluripotent stem cells, intermediate progenitors, and differentiated cells from all three germ layers. Integrative analyses of this atlas with gene expression and repressive histone modification maps reveal that lamina-associated chromatin in all twelve cell types is organized into at least two subtypes defined by differences in LAMIN B1 occupancy, gene expression, chromatin accessibility, transposable elements, replication timing, and radial positioning. Imaging of fluorescently labeled DNA in single cells validates these subtypes and shows radial positioning of LADs with higher LAMIN B1 occupancy and heterochromatic histone modifications primarily embedded within the lamina. In contrast, the second subtype of lamina-associated chromatin is relatively gene dense, accessible, dynamic across development, and positioned adjacent to the lamina. Most genes gain or lose LAMIN B1 occupancy consistent with cell types along developmental trajectories; however, we also identify examples where the enhancer, but not the gene body and promoter, changes LAD state.
    CONCLUSIONS: Altogether, this atlas represents the largest resource to date for peripheral chromatin organization studies and reveals an intermediate chromatin subtype.
    Keywords:  3D genome; Cellular differentiation; Lamina-associated domains; Peripheral chromatin organization
    DOI:  https://doi.org/10.1186/s13059-023-02849-5
  6. Epigenetics Chromatin. 2023 Jan 25. 16(1): 4
    Dynamic changes in whole genome DNA methylation, chromatin and gene expression during mouse lens differentiation.
    William Chang, Yilin Zhao, Danielle Rayêe, Qing Xie, Masako Suzuki, Deyou Zheng, Ales Cvekl.
       BACKGROUND: Cellular differentiation is marked by temporally and spatially coordinated gene expression regulated at multiple levels. DNA methylation represents a universal mechanism to control chromatin organization and its accessibility. Cytosine methylation of CpG dinucleotides regulates binding of methylation-sensitive DNA-binding transcription factors within regulatory regions of transcription, including promoters and distal enhancers. Ocular lens differentiation represents an advantageous model system to examine these processes as lens comprises only two cell types, the proliferating lens epithelium and postmitotic lens fiber cells all originating from the epithelium.
    RESULTS: Using whole genome bisulfite sequencing (WGBS) and microdissected lenses, we investigated dynamics of DNA methylation and chromatin changes during mouse lens fiber and epithelium differentiation between embryos (E14.5) and newborns (P0.5). Histone H3.3 variant chromatin landscapes were also generated for both P0.5 lens epithelium and fibers by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Tissue-specific features of DNA methylation patterns are demonstrated via comparative studies with embryonic stem (ES) cells and neural progenitor cells (NPCs) at Nanog, Pou5f1, Sox2, Pax6 and Six3 loci. Comparisons with ATAC-seq and RNA-seq data demonstrate that reduced methylation is associated with increased expression of fiber cell abundant genes, including crystallins, intermediate filament (Bfsp1 and Bfsp2) and gap junction proteins (Gja3 and Gja8), marked by high levels of histone H3.3 within their transcribed regions. Interestingly, Pax6-binding sites exhibited predominantly DNA hypomethylation in lens chromatin. In vitro binding of Pax6 proteins showed Pax6's ability to interact with sites containing one or two methylated CpG dinucleotides.
    CONCLUSIONS: Our study has generated the first data on methylation changes between two different stages of mammalian lens development and linked these data with chromatin accessibility maps, presence of histone H3.3 and gene expression. Reduced DNA methylation correlates with expression of important genes involved in lens morphogenesis and lens fiber cell differentiation.
    Keywords:  ATAC-seq; DNA methylation; Differentiation; Gene regulation; Histone H3.3; Lens; Open chromatin; Pax6; RNA-seq
    DOI:  https://doi.org/10.1186/s13072-023-00478-7
  7. Nucleic Acids Res. 2023 Jan 23. pii: gkac1267. [Epub ahead of print]
    The nucleosome unwrapping free energy landscape defines distinct regions of transcription factor accessibility and kinetics.
    Benjamin T Donovan, Yi Luo, Zhiyuan Meng, Michael G Poirier.
      Transcription factors (TF) require access to target sites within nucleosomes to initiate transcription. The target site position within the nucleosome significantly influences TF occupancy, but how is not quantitatively understood. Using ensemble and single-molecule fluorescence measurements, we investigated the targeting and occupancy of the transcription factor, Gal4, at different positions within the nucleosome. We observe a dramatic decrease in TF occupancy to sites extending past 30 base pairs (bp) into the nucleosome which cannot be explained by changes in the TF dissociation rate or binding site orientation. Instead, the nucleosome unwrapping free energy landscape is the primary determinant of Gal4 occupancy by reducing the Gal4 binding rate. The unwrapping free energy landscape defines two distinct regions of accessibility and kinetics with a boundary at 30 bp into the nucleosome where the inner region is over 100-fold less accessible. The Gal4 binding rate in the inner region no longer depends on its concentration because it is limited by the nucleosome unwrapping rate, while the frequency of nucleosome rewrapping decreases because Gal4 exchanges multiple times before the nucleosome rewraps. Our findings highlight the importance of the nucleosome unwrapping free energy landscape on TF occupancy and dynamics that ultimately influences transcription initiation.
    DOI:  https://doi.org/10.1093/nar/gkac1267
  8. Genome Biol. 2023 Jan 25. 24(1): 18
    β-actin mediated H3K27ac changes demonstrate the link between compartment switching and enhancer-dependent transcriptional regulation.
    Syed Raza Mahmood, Nadine Hosny El Said, Kristin C Gunsalus, Piergiorgio Percipalle.
       BACKGROUND: Recent work has demonstrated that three-dimensional genome organization is directly affected by changes in the levels of nuclear cytoskeletal proteins such as β-actin. The mechanisms which translate changes in 3D genome structure into changes in transcription, however, are not fully understood. Here, we use a comprehensive genomic analysis of cells lacking nuclear β-actin to investigate the mechanistic links between compartment organization, enhancer activity, and gene expression.
    RESULTS: Using HiC-Seq, ATAC-Seq, and RNA-Seq, we first demonstrate that transcriptional and chromatin accessibility changes observed upon β-actin loss are highly enriched in compartment-switching regions. Accessibility changes within compartment switching genes, however, are mainly observed in non-promoter regions which potentially represent distal regulatory elements. Our results also show that β-actin loss induces widespread accumulation of the enhancer-specific epigenetic mark H3K27ac. Using the ABC model of enhancer annotation, we then establish that these epigenetic changes have a direct impact on enhancer activity and underlie transcriptional changes observed upon compartment switching. A complementary analysis of fibroblasts undergoing reprogramming into pluripotent stem cells further confirms that this relationship between compartment switching and enhancer-dependent transcriptional change is not specific to β-actin knockout cells but represents a general mechanism linking compartment-level genome organization to gene expression.
    CONCLUSIONS: We demonstrate that enhancer-dependent transcriptional regulation plays a crucial role in driving gene expression changes observed upon compartment-switching. Our results also reveal a novel function of nuclear β-actin in regulating enhancer function by influencing H3K27 acetylation levels.
    Keywords:  3D genome organization; Enhancer regulation; Nuclear actin; Transcriptional regulation
    DOI:  https://doi.org/10.1186/s13059-023-02853-9
  9. Nat Commun. 2023 Jan 23. 14(1): 367
    DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
    Andrea Lauria, Guohua Meng, Valentina Proserpio, Stefania Rapelli, Mara Maldotti, Isabelle Laurence Polignano, Francesca Anselmi, Danny Incarnato, Anna Krepelova, Daniela Donna, Chiara Levra Levron, Giacomo Donati, Ivan Molineris, Francesco Neri, Salvatore Oliviero.
      The correct establishment of DNA methylation patterns during mouse early development is essential for cell fate specification. However, the molecular targets as well as the mechanisms that determine the specificity of the de novo methylation machinery during differentiation are not completely elucidated. Here we show that the DNMT3B-dependent DNA methylation of key developmental regulatory regions at epiblast-like cells (EpiLCs) provides an epigenetic priming that ensures flawless commitment at later stages. Using in vitro stem cell differentiation and loss of function experiments combined with high-throughput genome-wide bisulfite-, bulk-, and single cell RNA-sequencing we dissected the specific role of DNMT3B in cell fate. We identify DNMT3B-dependent regulatory elements on the genome which, in Dnmt3b knockout (3BKO), impair the differentiation into meso-endodermal (ME) progenitors and redirect EpiLCs towards the neuro-ectodermal lineages. Moreover, ectopic expression of DNMT3B in 3BKO re-establishes the DNA methylation of the master regulator Sox2 super-enhancer, downmodulates its expression, and restores the expression of ME markers. Taken together, our data reveal that DNMT3B-dependent methylation at the epiblast stage is essential for the priming of the meso-endodermal lineages and provide functional characterization of the de novo DNMTs during EpiLCs lineage determination.
    DOI:  https://doi.org/10.1038/s41467-023-35938-x
  10. Nat Commun. 2023 Jan 24. 14(1): 378
    Structural mechanism of BRD4-NUT and p300 bipartite interaction in propagating aberrant gene transcription in chromatin in NUT carcinoma.
    Di Yu, Yingying Liang, Claudia Kim, Anbalagan Jaganathan, Donglei Ji, Xinye Han, Xuelan Yang, Yanjie Jia, Ruirui Gu, Chunyu Wang, Qiang Zhang, Ka Lung Cheung, Ming-Ming Zhou, Lei Zeng.
      BRD4-NUT, a driver fusion mutant in rare and highly aggressive NUT carcinoma, acts in aberrant transcription of anti-differentiation genes by recruiting histone acetyltransferase (HAT) p300 and promoting p300-driven histone hyperacetylation and nuclear condensation in chromatin. However, the molecular basis of how BRD4-NUT recruits and activates p300 remains elusive. Here, we report that BRD4-NUT contains two transactivation domains (TADs) in NUT that bind to the TAZ2 domain in p300. Our NMR structures reveal that NUT TADs adopt amphipathic helices when bound to the four-helical bundle TAZ2 domain. The NUT protein forms liquid-like droplets in-vitro that are enhanced by TAZ2 binding in 1:2 stoichiometry. The TAD/TAZ2 bipartite binding in BRD4-NUT/p300 triggers allosteric activation of p300 and acetylation-driven liquid-like condensation on chromatin that comprise histone H3 lysine 27 and 18 acetylation and transcription proteins BRD4L/S, CDK9, MED1, and RNA polymerase II. The BRD4-NUT/p300 chromatin condensation is key for activating transcription of pro-proliferation genes such as ALX1, resulting ALX1/Snail signaling and epithelial-to-mesenchymal transition. Our study provides a previously underappreciated structural mechanism illuminating BRD4-NUT's bipartite p300 recruitment and activation in NUT carcinoma that nucleates a feed-forward loop for propagating histone hyperacetylation and chromatin condensation to sustain aberrant anti-differentiation gene transcription and perpetual tumor cell growth.
    DOI:  https://doi.org/10.1038/s41467-023-36063-5
  11. Nat Commun. 2023 Jan 23. 14(1): 371
    Mechanisms and function of de novo DNA methylation in placental development reveals an essential role for DNMT3B.
    Simon Andrews, Christel Krueger, Maravillas Mellado-Lopez, Myriam Hemberger, Wendy Dean, Vicente Perez-Garcia, Courtney W Hanna.
      DNA methylation is a repressive epigenetic modification that is essential for development, exemplified by the embryonic and perinatal lethality observed in mice lacking de novo DNA methyltransferases (DNMTs). Here we characterise the role for DNMT3A, 3B and 3L in gene regulation and development of the mouse placenta. We find that each DNMT establishes unique aspects of the placental methylome through targeting to distinct chromatin features. Loss of Dnmt3b results in de-repression of germline genes in trophoblast lineages and impaired formation of the maternal-foetal interface in the placental labyrinth. Using Sox2-Cre to delete Dnmt3b in the embryo, leaving expression intact in placental cells, the placental phenotype was rescued and, consequently, the embryonic lethality, as Dnmt3b null embryos could now survive to birth. We conclude that de novo DNA methylation by DNMT3B during embryogenesis is principally required to regulate placental development and function, which in turn is critical for embryo survival.
    DOI:  https://doi.org/10.1038/s41467-023-36019-9
  12. Nat Commun. 2023 Jan 21. 14(1): 346
    Epigenomic charting and functional annotation of risk loci in renal cell carcinoma.
    Amin H Nassar, Sarah Abou Alaiwi, Sylvan C Baca, Elio Adib, Rosario I Corona, Ji-Heui Seo, Marcos A S Fonseca, Sandor Spisak, Talal El Zarif, Viktoria Tisza, David A Braun, Heng Du, Monica He, Abdallah Flaifel, Michel Alchoueiry, Thomas Denize, Sayed G Matar, Andres Acosta, Sachet Shukla, Yue Hou, John Steinharter, Gabrielle Bouchard, Jacob E Berchuck, Edward O'Connor, Connor Bell, Pier Vitale Nuzzo, Gwo-Shu Mary Lee, Sabina Signoretti, Michelle S Hirsch, Mark Pomerantz, Elizabeth Henske, Alexander Gusev, Kate Lawrenson, Toni K Choueiri, David J Kwiatkowski, Matthew L Freedman.
      While the mutational and transcriptional landscapes of renal cell carcinoma (RCC) are well-known, the epigenome is poorly understood. We characterize the epigenome of clear cell (ccRCC), papillary (pRCC), and chromophobe RCC (chRCC) by using ChIP-seq, ATAC-Seq, RNA-seq, and SNP arrays. We integrate 153 individual data sets from 42 patients and nominate 50 histology-specific master transcription factors (MTF) to define RCC histologic subtypes, including EPAS1 and ETS-1 in ccRCC, HNF1B in pRCC, and FOXI1 in chRCC. We confirm histology-specific MTFs via immunohistochemistry including a ccRCC-specific TF, BHLHE41. FOXI1 overexpression with knock-down of EPAS1 in the 786-O ccRCC cell line induces transcriptional upregulation of chRCC-specific genes, TFCP2L1, ATP6V0D2, KIT, and INSRR, implicating FOXI1 as a MTF for chRCC. Integrating RCC GWAS risk SNPs with H3K27ac ChIP-seq and ATAC-seq data reveals that risk-variants are significantly enriched in allelically-imbalanced peaks. This epigenomic atlas in primary human samples provides a resource for future investigation.
    DOI:  https://doi.org/10.1038/s41467-023-35833-5
  13. Nat Commun. 2023 Jan 21. 14(1): 350
    Mapping nucleolus-associated chromatin interactions using nucleolus Hi-C reveals pattern of heterochromatin interactions.
    Ting Peng, Yingping Hou, Haowei Meng, Yong Cao, Xiaotian Wang, Lumeng Jia, Qing Chen, Yang Zheng, Yujie Sun, Hebing Chen, Tingting Li, Cheng Li.
      As the largest substructures in the nucleus, nucleoli are the sites of ribosome biogenesis. Increasing evidence indicates that nucleoli play a key role in the organization of 3D genome architecture, but systematic studies of nucleolus-associated chromatin interactions are lacking. Here, we developed a nucleolus Hi-C (nHi-C) experimental technique to enrich nucleolus-associated chromatin interactions. Using the nHi-C experiment, we identify 264 high-confidence nucleolus-associated domains (hNADs) that form strong heterochromatin interactions associated with the nucleolus and consist of 24% of the whole genome in HeLa cells. Based on the global hNAD inter-chromosomal interactions, we find five nucleolar organizer region (NOR)-bearing chromosomes formed into two clusters that show different interaction patterns, which is concordant with their epigenetic states and gene expression levels. hNADs can be divided into three groups that display distinct cis/trans interaction signals, interaction frequencies associated with nucleoli, distance from the centromeres, and overlap percentage with lamina-associated domains (LADs). Nucleolus disassembly caused by Actinomycin D (ActD) significantly decreases the strength of hNADs and affects compartment/TAD strength genome-wide. In summary, our results provide a global view of heterochromatin interactions organized around nucleoli and demonstrate that nucleoli act as an inactive inter-chromosomal hub to shape both compartments and TADs.
    DOI:  https://doi.org/10.1038/s41467-023-36021-1
  14. Sci Adv. 2023 Jan 25. 9(4): eadd2175
    Dynamics and regulation of mitotic chromatin accessibility bookmarking at single-cell resolution.
    Qiaoni Yu, Xu Liu, Jingwen Fang, Huihui Wu, Chuang Guo, Wen Zhang, Nianping Liu, Chen Jiang, Qing Sha, Xiao Yuan, Zhikai Wang, Kun Qu.
      Although mitotic chromosomes are highly compacted and transcriptionally inert, some active chromatin features are retained during mitosis to ensure the proper postmitotic reestablishment of maternal transcriptional programs, a phenomenon termed "mitotic bookmarking." However, the dynamics and regulation of mitotic bookmarking have not been systemically surveyed. Using single-cell transposase-accessible chromatin sequencing (scATAC-seq), we examined 6538 mitotic L02 human liver cells of variable stages and found that chromatin accessibility remained changing throughout cell division, with a constant decrease until metaphase and a gradual increase as chromosomes segregated. In particular, a subset of chromatin regions were identified to remain open throughout mitosis, and genes associated with these bookmarked regions are primarily linked to rapid reactivation upon mitotic exit. We also demonstrated that nuclear transcription factor Y subunit α (NF-YA) preferentially occupied bookmarked regions and contributed to transcriptional reactivation after mitosis. Our study uncovers the dynamic and regulatory blueprint of mitotic bookmarking.
    DOI:  https://doi.org/10.1126/sciadv.add2175
  15. Genome Biol. 2023 Jan 26. 24(1): 14
    Auxin-inducible degron 2 system deciphers functions of CTCF domains in transcriptional regulation.
    Judith Hyle, Mohamed Nadhir Djekidel, Justin Williams, Shaela Wright, Ying Shao, Beisi Xu, Chunliang Li.
       BACKGROUND: CTCF is a well-established chromatin architectural protein that also plays various roles in transcriptional regulation. While CTCF biology has been extensively studied, how the domains of CTCF function to regulate transcription remains unknown. Additionally, the original auxin-inducible degron 1 (AID1) system has limitations in investigating the function of CTCF.
    RESULTS: We employ an improved auxin-inducible degron technology, AID2, to facilitate the study of acute depletion of CTCF while overcoming the limitations of the previous AID system. As previously observed through the AID1 system and steady-state RNA analysis, the new AID2 system combined with SLAM-seq confirms that CTCF depletion leads to modest nascent and steady-state transcript changes. A CTCF domain sgRNA library screening identifies the zinc finger (ZF) domain as the region within CTCF with the most functional relevance, including ZFs 1 and 10. Removal of ZFs 1 and 10 reveals genomic regions that independently require these ZFs for DNA binding and transcriptional regulation. Notably, loci regulated by either ZF1 or ZF10 exhibit unique CTCF binding motifs specific to each ZF.
    CONCLUSIONS: By extensively comparing the AID1 and AID2 systems for CTCF degradation in SEM cells, we confirm that AID2 degradation is superior for achieving miniAID-tagged protein degradation without the limitations of the AID1 system. The model we create that combines AID2 depletion of CTCF with exogenous overexpression of CTCF mutants allows us to demonstrate how peripheral ZFs intricately orchestrate transcriptional regulation in a cellular context for the first time.
    Keywords:  Auxin-inducible degron; CRISPR; CTCF; Transcription
    DOI:  https://doi.org/10.1186/s13059-022-02843-3
  16. Cell Syst. 2023 Jan 18. pii: S2405-4712(22)00496-3. [Epub ahead of print]
    Minimal synthetic enhancers reveal control of the probability of transcriptional engagement and its timing by a morphogen gradient.
    Simon Alamos, Armando Reimer, Clay Westrum, Meghan A Turner, Paul Talledo, Jiaxi Zhao, Emma Luu, Hernan G Garcia.
      How enhancers interpret morphogen gradients to generate gene expression patterns is a central question in developmental biology. Recent studies have proposed that enhancers can dictate whether, when, and at what rate promoters engage in transcription, but the complexity of endogenous enhancers calls for theoretical models with too many free parameters to quantitatively dissect these regulatory strategies. To overcome this limitation, we established a minimal promoter-proximal synthetic enhancer in embryos of Drosophila melanogaster. Here, a gradient of the Dorsal activator is read by a single Dorsal DNA binding site. Using live imaging to quantify transcriptional activity, we found that a single binding site can regulate whether promoters engage in transcription in a concentration-dependent manner. By modulating the binding-site affinity, we determined that a gene's decision to transcribe and its transcriptional onset time can be explained by a simple model where the promoter traverses multiple kinetic barriers before transcription can ensue.
    Keywords:  Drosophila melanogaster; biophysics; developmental biology; quantitative biology; transcriptional dynamic; transcriptional modeling; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.cels.2022.12.008
  17. Nat Commun. 2023 Jan 27. 14(1): 448
    Chromatin complex dependencies reveal targeting opportunities in leukemia.
    Fadi J Najm, Peter DeWeirdt, Molly M Moore, Samantha M Bevill, Chadi A El Farran, Kevin A Macias, Mudra Hegde, Amanda L Waterbury, Brian B Liau, Peter van Galen, John G Doench, Bradley E Bernstein.
      Chromatin regulators are frequently mutated in human cancer and are attractive drug targets. They include diverse proteins that share functional domains and assemble into related multi-subunit complexes. To investigate functional relationships among these regulators, here we apply combinatorial CRISPR knockouts (KOs) to test over 35,000 gene-gene pairings in leukemia cells, using a library of over 300,000 constructs. Top pairs that demonstrate either compensatory non-lethal interactions or synergistic lethality enrich for paralogs and targets that occupy the same protein complex. The screen highlights protein complex dependencies not apparent in single KO screens, for example MCM histone exchange, the nucleosome remodeling and deacetylase (NuRD) complex, and HBO1 (KAT7) complex. We explore two approaches to NuRD complex inactivation. Paralog and non-paralog combinations of the KAT7 complex emerge as synergistic lethal and specifically nominate the ING5 PHD domain as a potential therapeutic target when paired with other KAT7 complex member losses. These findings highlight the power of combinatorial screening to provide mechanistic insight and identify therapeutic targets within redundant networks.
    DOI:  https://doi.org/10.1038/s41467-023-36150-7
  18. NAR Cancer. 2023 Mar;5(1): zcad001
    Oncogenic ERRB2 signals through the AP-1 transcription factor to control mesenchymal-like properties of oesophageal adenocarcinoma.
    OCCAMS consortium
      Oesophageal adenocarcinoma (OAC) is a deadly disease with poor survival statistics and few targeted therapies available. One of the most common molecular aberrations in OAC is amplification or activation of the gene encoding the receptor tyrosine kinase ERBB2, and ERBB2 is targeted in the clinic for this subset of patients. However, the downstream consequences of these ERBB2 activating events are not well understood. Here we used a combination of phosphoproteomics, open chromatin profiling and transcriptome analysis on cell line models and patient-derived datasets to interrogate the molecular pathways operating downstream from ERBB2. Integrated analysis of these data sets converge on a model where dysregulated ERBB2 signalling is mediated at the transcriptional level by the transcription factor AP-1. AP-1 in turn controls cell behaviour by acting on cohorts of genes that regulate cell migration and adhesion, features often associated with EMT. Our study therefore provides a valuable resource for the cancer cell signalling community and reveals novel molecular determinants underlying the dysregulated behaviour of OAC cells.
    DOI:  https://doi.org/10.1093/narcan/zcad001
  19. Nat Cell Biol. 2023 Jan 23.
    Expansion of ventral foregut is linked to changes in the enhancer landscape for organ-specific differentiation.
    Yan Fung Wong, Yatendra Kumar, Martin Proks, Jose Alejandro Romero Herrera, Michaela Mrugala Rothová, Rita S Monteiro, Sara Pozzi, Rachel E Jennings, Neil A Hanley, Wendy A Bickmore, Joshua M Brickman.
      Cell proliferation is fundamental for almost all stages of development and differentiation that require an increase in cell number. Although cell cycle phase has been associated with differentiation, the actual process of proliferation has not been considered as having a specific role. Here we exploit human embryonic stem cell-derived endodermal progenitors that we find are an in vitro model for the ventral foregut. These cells exhibit expansion-dependent increases in differentiation efficiency to pancreatic progenitors that are linked to organ-specific enhancer priming at the level of chromatin accessibility and the decommissioning of lineage-inappropriate enhancers. Our findings suggest that cell proliferation in embryonic development is about more than tissue expansion; it is required to ensure equilibration of gene regulatory networks allowing cells to become primed for future differentiation. Expansion of lineage-specific intermediates may therefore be an important step in achieving high-fidelity in vitro differentiation.
    DOI:  https://doi.org/10.1038/s41556-022-01075-8
  20. BMC Genomics. 2023 Jan 25. 24(1): 43
    peaksat: an R package for ChIP-seq peak saturation analysis.
    Joseph R Boyd, Cong Gao, Kathleen Quinn, Andrew Fritz, Janet Stein, Gary Stein, Karen Glass, Seth Frietze.
       BACKGROUND: Epigenomic profiling assays such as ChIP-seq have been widely used to map the genome-wide enrichment profiles of chromatin-associated proteins and posttranslational histone modifications. Sequencing depth is a key parameter in experimental design and quality control. However, due to variable sequencing depth requirements across experimental conditions, it can be challenging to determine optimal sequencing depth, particularly for projects involving multiple targets or cell types.
    RESULTS: We developed the peaksat R package to provide target read depth estimates for epigenomic experiments based on the analysis of peak saturation curves. We applied peaksat to establish the distinctive read depth requirements for ChIP-seq studies of histone modifications in different cell lines. Using peaksat, we were able to estimate the target read depth required per library to obtain high-quality peak calls for downstream analysis. In addition, peaksat was applied to other sequence-enrichment methods including CUT&RUN and ATAC-seq.
    CONCLUSION: peaksat addresses a need for researchers to make informed decisions about whether their sequencing data has been generated to an adequate depth and subsequently sufficient meaningful peaks, and failing that, how many more reads would be required per library. peaksat is applicable to other sequence-based methods that include calling peaks in their analysis.
    Keywords:  ChIP-Seq; Peak saturation; Read depth estimate
    DOI:  https://doi.org/10.1186/s12864-023-09109-7
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