bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024–05–12
24 papers selected by
Connor Rogerson, University of Cambridge
  1. Cell type- and transcription-independent spatial proximity between enhancers and promoters.
  2. Estrogen receptor 1 chromatin profiling in human breast tumors reveals high inter-patient heterogeneity with enrichment of risk SNPs and enhancer activity at most-conserved regions.
  3. TFTG: A comprehensive database for human transcription factors and their targets.
  4. Scalable and unbiased sequence-informed embedding of single-cell ATAC-seq data with CellSpace.
  5. Regulation of Myc transcription by an enhancer cluster dedicated to pluripotency and early embryonic expression.
  6. Systematic epigenome editing captures the context-dependent instructive function of chromatin modifications.
  7. ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin.
  8. Genomic context- and H2AK119 ubiquitination-dependent inheritance of human Polycomb silencing.
  9. Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.
  10. The GATAD2B-NuRD complex drives DNA:RNA hybrid-dependent chromatin boundary formation upon DNA damage.
  11. Pervasive structural heterogeneity rewires glioblastoma chromosomes to sustain patient-specific transcriptional programs.
  12. Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters.
  13. Mapping genotypes to chromatin accessibility profiles in single cells.
  14. ARID1B controls transcriptional programs of axon projection in an organoid model of the human corpus callosum.
  15. Comprehensive analysis of the REST transcription factor regulatory networks in IDH mutant and IDH wild-type glioma cell lines and tumors.
  16. Integrated enhancer regulatory network by enhancer-promoter looping in gastric cancer.
  17. Malat1 affects transcription and splicing through distinct pathways in mouse embryonic stem cells.
  18. Genome-wide distribution of 5-hydroxymethyluracil and chromatin accessibility in the Breviolum minutum genome.
  19. Reduction of ZFX levels decreases histone H4 acetylation and increases Pol2 pausing at target promoters.
  20. Transcriptional repression of the oncofetal LIN28B gene by the transcription factor SOX6.
  21. Suv39h-catalyzed H3K9me3 is critical for euchromatic genome organization and the maintenance of gene transcription.
  22. Tracking single-cell evolution using clock-like chromatin accessibility loci.
  23. Oct4 redox sensitivity potentiates reprogramming and differentiation.
  24. Direct transposition of native DNA for sensitive multimodal single-molecule sequencing.
  1. Mol Biol Cell. 2024 May 08. mbcE24020082
    Cell type- and transcription-independent spatial proximity between enhancers and promoters.
    Yasmine Mian, Li Wang, Adib Keikhosravi, Konnie Guo, Tom Misteli, H Efsun Arda, Elizabeth H Finn.
      Cell type-specific enhancers are critically important for lineage specification. The mechanisms that determine cell type-specificity of enhancer activity, however, are not fully understood. Most current models for how enhancers function invoke physical proximity between enhancer elements and their target genes. Here, we use an imaging-based approach to examine the spatial relationship of cell type-specific enhancers and their target genes with single cell resolution. Using high-throughput microscopy, we measure the spatial distance from target promoters to their cell type-specific active and inactive enhancers in individual pancreatic cells derived from distinct lineages. We find increased proximity of all promoter-enhancer pairs relative to non-enhancer pairs separated by similar genomic distances. Strikingly, spatial proximity between enhancers and target genes was unrelated to tissue-specific enhancer activity. Furthermore, promoter-enhancer proximity did not correlate with the expression status of target genes. Our results suggest that promoter-enhancer pairs exist in a distinctive chromatin environment but that genome folding is not a universal driver of cell type-specificity in enhancer function.
    DOI:  https://doi.org/10.1091/mbc.E24-02-0082
  2. Genome Res. 2024 May 06.
    Estrogen receptor 1 chromatin profiling in human breast tumors reveals high inter-patient heterogeneity with enrichment of risk SNPs and enhancer activity at most-conserved regions.
    Stacey E P Joosten, Sebastian Gregoricchio, Suzan Stelloo, Elif Yapıcı, Chia-Chi Flora Huang, Kerim Yavuz, Maria Donaldson Collier, Tunç Morova, Umut Berkay Altintaş, Yongsoo Kim, Sander Canisius, Cathy B Moelans, Paul J van Diest, Gozde Korkmaz, Nathan A Lack, Michiel Vermeulen, Sabine C Linn, Wilbert Zwart.
      Estrogen Receptor 1 (ESR1; also known as ERα, encoded by ESR1 gene) is the main driver and prime drug target in luminal breast cancer. ESR1 chromatin binding is extensively studied in cell lines and a limited number of human tumors, using consensi of peaks shared among samples. However, little is known about inter-tumor heterogeneity of ESR1 chromatin action, along with its biological implications. Here, we use a large set of ESR1 ChIP-seq data from 70 ESR1+ breast cancers to explore inter-patient heterogeneity in ESR1 DNA binding to reveal a striking inter-tumor heterogeneity of ESR1 action. Of note, commonly shared ESR1 sites show the highest estrogen-driven enhancer activity and are most engaged in long-range chromatin interactions. In addition, the most commonly shared ESR1-occupied enhancers are enriched for breast cancer risk SNP loci. We experimentally confirm SNVs to impact chromatin binding potential for ESR1 and its pioneer factor FOXA1. Finally, in the TCGA breast cancer cohort, we can confirm these variations to associate with differences in expression for the target gene. Cumulatively, we reveal a natural hierarchy of ESR1-chromatin interactions in breast cancers within a highly heterogeneous inter-tumor ESR1 landscape, with the most common shared regions being most active and affected by germline functional risk SNPs for breast cancer development.
    DOI:  https://doi.org/10.1101/gr.278680.123
  3. Comput Struct Biotechnol J. 2024 Dec;23 1877-1885
    TFTG: A comprehensive database for human transcription factors and their targets.
    Xinyuan Zhou, Liwei Zhou, Fengcui Qian, Jiaxin Chen, Yuexin Zhang, Zhengmin Yu, Jian Zhang, Yongsan Yang, Yanyu Li, Chao Song, Yuezhu Wang, Desi Shang, Longlong Dong, Jiang Zhu, Chunquan Li, Qiuyu Wang.
      Transcription factors (TFs) are major contributors to gene transcription, especially in controlling cell-specific gene expression and disease occurrence and development. Uncovering the relationship between TFs and their target genes is critical to understanding the mechanism of action of TFs. With the development of high-throughput sequencing techniques, a large amount of TF-related data has accumulated, which can be used to identify their target genes. In this study, we developed TFTG (Transcription Factor and Target Genes) database (http://tf.liclab.net/TFTG), which aimed to provide a large number of available human TF-target gene resources by multiple strategies, besides performing a comprehensive functional and epigenetic annotations and regulatory analyses of TFs. We identified extensive available TF-target genes by collecting and processing TF-associated ChIP-seq datasets, perturbation RNA-seq datasets and motifs. We also obtained experimentally confirmed relationships between TF and target genes from available resources. Overall, the target genes of TFs were obtained through integrating the relevant data of various TFs as well as fourteen identification strategies. Meanwhile, TFTG was embedded with user-friendly search, analysis, browsing, downloading and visualization functions. TFTG is designed to be a convenient resource for exploring human TF-target gene regulations, which will be useful for most users in the TF and gene expression regulation research.
    Keywords:  Enrichment analysis; Epigenetic annotation; Functional annotation; Target gene; Transcription factor
    DOI:  https://doi.org/10.1016/j.csbj.2024.04.036
  4. Nat Methods. 2024 May 09.
    Scalable and unbiased sequence-informed embedding of single-cell ATAC-seq data with CellSpace.
    Zakieh Tayyebi, Allison R Pine, Christina S Leslie.
      Standard scATAC sequencing (scATAC-seq) analysis pipelines represent cells as sparse numeric vectors relative to an atlas of peaks or genomic tiles and consequently ignore genomic sequence information at accessible loci. Here we present CellSpace, an efficient and scalable sequence-informed embedding algorithm for scATAC-seq that learns a mapping of DNA k-mers and cells to the same space, to address this limitation. We show that CellSpace captures meaningful latent structure in scATAC-seq datasets, including cell subpopulations and developmental hierarchies, and can score transcription factor activities in single cells based on proximity to binding motifs embedded in the same space. Importantly, CellSpace implicitly mitigates batch effects arising from multiple samples, donors or assays, even when individual datasets are processed relative to different peak atlases. Thus, CellSpace provides a powerful tool for integrating and interpreting large-scale scATAC-seq compendia.
    DOI:  https://doi.org/10.1038/s41592-024-02274-x
  5. Nat Commun. 2024 May 10. 15(1): 3931
    Regulation of Myc transcription by an enhancer cluster dedicated to pluripotency and early embryonic expression.
    Lin Li-Bao, Covadonga Díaz-Díaz, Morena Raiola, Rocío Sierra, Susana Temiño, Francisco J Moya, Sandra Rodriguez-Perales, Elisa Santos, Giovanna Giovinazzo, Tore Bleckwehl, Álvaro Rada-Iglesias, Francois Spitz, Miguel Torres.
      MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.
    DOI:  https://doi.org/10.1038/s41467-024-48258-5
  6. Nat Genet. 2024 May 09.
    Systematic epigenome editing captures the context-dependent instructive function of chromatin modifications.
    Cristina Policarpi, Marzia Munafò, Stylianos Tsagkris, Valentina Carlini, Jamie A Hackett.
      Chromatin modifications are linked with regulating patterns of gene expression, but their causal role and context-dependent impact on transcription remains unresolved. Here we develop a modular epigenome editing platform that programs nine key chromatin modifications, or combinations thereof, to precise loci in living cells. We couple this with single-cell readouts to systematically quantitate the magnitude and heterogeneity of transcriptional responses elicited by each specific chromatin modification. Among these, we show that installing histone H3 lysine 4 trimethylation (H3K4me3) at promoters can causally instruct transcription by hierarchically remodeling the chromatin landscape. We further dissect how DNA sequence motifs influence the transcriptional impact of chromatin marks, identifying switch-like and attenuative effects within distinct cis contexts. Finally, we examine the interplay of combinatorial modifications, revealing that co-targeted H3K27 trimethylation (H3K27me3) and H2AK119 monoubiquitination (H2AK119ub) maximizes silencing penetrance across single cells. Our precision-perturbation strategy unveils the causal principles of how chromatin modification(s) influence transcription and dissects how quantitative responses are calibrated by contextual interactions.
    DOI:  https://doi.org/10.1038/s41588-024-01706-w
  7. J Cell Biol. 2024 Aug 05. pii: e202310084. [Epub ahead of print]223(8):
    ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin.
    Naoki Goto, Kazuma Suke, Nao Yonezawa, Hidenori Nishihara, Tetsuya Handa, Yuko Sato, Tomoya Kujirai, Hitoshi Kurumizaka, Kazuo Yamagata, Hiroshi Kimura.
      Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain-containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B could determine the localization of H3K36me2 in different cell types. In mouse embryos, H3K36me2 heterochromatin localization was observed at the two- to four-cell stages, suggesting its physiological relevance.
    DOI:  https://doi.org/10.1083/jcb.202310084
  8. Sci Adv. 2024 May 10. 10(19): eadl4529
    Genomic context- and H2AK119 ubiquitination-dependent inheritance of human Polycomb silencing.
    Tiasha A Shafiq, Juntao Yu, Wenzhi Feng, Yizhe Zhang, Haining Zhou, Joao A Paulo, Steven P Gygi, Danesh Moazed.
      Polycomb repressive complexes 1 and 2 (PRC1 and 2) are required for heritable repression of developmental genes. The cis- and trans-acting factors that contribute to epigenetic inheritance of mammalian Polycomb repression are not fully understood. Here, we show that, in human cells, ectopically induced Polycomb silencing at initially active developmental genes, but not near ubiquitously expressed housekeeping genes, is inherited for many cell divisions. Unexpectedly, silencing is heritable in cells with mutations in the H3K27me3 binding pocket of the Embryonic Ectoderm Development (EED) subunit of PRC2, which are known to disrupt H3K27me3 recognition and lead to loss of H3K27me3. This mode of inheritance is less stable and requires intact PRC2 and recognition of H2AK119ub1 by PRC1. Our findings suggest that maintenance of Polycomb silencing is sensitive to local genomic context and can be mediated by PRC1-dependent H2AK119ub1 and PRC2 independently of H3K27me3 recognition.
    DOI:  https://doi.org/10.1126/sciadv.adl4529
  9. Development. 2024 May 01. pii: dev202525. [Epub ahead of print]151(9):
    Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.
    Virginia Panara, Hujun Yu, Di Peng, Karin Staxäng, Monika Hodik, Beata Filipek-Gorniok, Jan Kazenwadel, Renae Skoczylas, Elizabeth Mason, Amin Allalou, Natasha L Harvey, Tatjana Haitina, Benjamin M Hogan, Katarzyna Koltowska.
      During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.
    Keywords:  Enhancers; Evolutionary conservation; Gene regulation; Lymphatic endothelial cell; Prox1; Transcription factor; Zebrafish
    DOI:  https://doi.org/10.1242/dev.202525
  10. EMBO J. 2024 May 08.
    The GATAD2B-NuRD complex drives DNA:RNA hybrid-dependent chromatin boundary formation upon DNA damage.
    Zhichao Liu, Kamal Ajit, Yupei Wu, Wei-Guo Zhu, Monika Gullerova.
      Double-strand breaks (DSBs) are the most lethal form of DNA damage. Transcriptional activity at DSBs, as well as transcriptional repression around DSBs, are both required for efficient DNA repair. The chromatin landscape defines and coordinates these two opposing events. However, how the open and condensed chromatin architecture is regulated remains unclear. Here, we show that the GATAD2B-NuRD complex associates with DSBs in a transcription- and DNA:RNA hybrid-dependent manner, to promote histone deacetylation and chromatin condensation. This activity establishes a spatio-temporal boundary between open and closed chromatin, which is necessary for the correct termination of DNA end resection. The lack of the GATAD2B-NuRD complex leads to chromatin hyperrelaxation and extended DNA end resection, resulting in homologous recombination (HR) repair failure. Our results suggest that the GATAD2B-NuRD complex is a key coordinator of the dynamic interplay between transcription and the chromatin landscape, underscoring its biological significance in the RNA-dependent DNA damage response.
    Keywords:  Chromatin Boundary; DNA Damage; DNA:RNA Hybrids; GATAD2B; NuRD Complex
    DOI:  https://doi.org/10.1038/s44318-024-00111-7
  11. Nat Commun. 2024 May 09. 15(1): 3905
    Pervasive structural heterogeneity rewires glioblastoma chromosomes to sustain patient-specific transcriptional programs.
    Ting Xie, Adi Danieli-Mackay, Mariachiara Buccarelli, Mariano Barbieri, Ioanna Papadionysiou, Q Giorgio D'Alessandris, Claudia Robens, Nadine Übelmesser, Omkar Suhas Vinchure, Liverana Lauretti, Giorgio Fotia, Roland F Schwarz, Xiaotao Wang, Lucia Ricci-Vitiani, Jay Gopalakrishnan, Roberto Pallini, Argyris Papantonis.
      Glioblastoma multiforme (GBM) encompasses brain malignancies marked by phenotypic and transcriptional heterogeneity thought to render these tumors aggressive, resistant to therapy, and inevitably recurrent. However, little is known about how the spatial organization of GBM genomes underlies this heterogeneity and its effects. Here, we compile a cohort of 28 patient-derived glioblastoma stem cell-like lines (GSCs) known to reflect the properties of their tumor-of-origin; six of these were primary-relapse tumor pairs from the same patient. We generate and analyze 5 kbp-resolution chromosome conformation capture (Hi-C) data from all GSCs to systematically map thousands of standalone and complex structural variants (SVs) and the multitude of neoloops arising as a result. By combining Hi-C, histone modification, and gene expression data with chromatin folding simulations, we explain how the pervasive, uneven, and idiosyncratic occurrence of neoloops sustains tumor-specific transcriptional programs via the formation of new enhancer-promoter contacts. We also show how even moderately recurrent neoloops can relate to patient-specific vulnerabilities. Together, our data provide a resource for dissecting GBM biology and heterogeneity, as well as for informing therapeutic approaches.
    DOI:  https://doi.org/10.1038/s41467-024-48053-2
  12. Nat Methods. 2024 May 09.
    Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters.
    Jean-Benoît Lalanne, Samuel G Regalado, Silvia Domcke, Diego Calderon, Beth K Martin, Xiaoyi Li, Tony Li, Chase C Suiter, Choli Lee, Cole Trapnell, Jay Shendure.
      The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.
    DOI:  https://doi.org/10.1038/s41592-024-02260-3
  13. Nature. 2024 May 08.
    Mapping genotypes to chromatin accessibility profiles in single cells.
    Franco Izzo, Robert M Myers, Saravanan Ganesan, Levan Mekerishvili, Sanjay Kottapalli, Tamara Prieto, Elliot O Eton, Theo Botella, Andrew J Dunbar, Robert L Bowman, Jesus Sotelo, Catherine Potenski, Eleni P Mimitou, Maximilian Stahl, Sebastian El Ghaity-Beckley, JoAnn Arandela, Ramya Raviram, Daniel C Choi, Ronald Hoffman, Ronan Chaligné, Omar Abdel-Wahab, Peter Smibert, Irene M Ghobrial, Joseph M Scandura, Bridget Marcellino, Ross L Levine, Dan A Landau.
      In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.
    DOI:  https://doi.org/10.1038/s41586-024-07388-y
  14. Cell Stem Cell. 2024 May 06. pii: S1934-5909(24)00141-3. [Epub ahead of print]
    ARID1B controls transcriptional programs of axon projection in an organoid model of the human corpus callosum.
    Catarina Martins-Costa, Andrea Wiegers, Vincent A Pham, Jaydeep Sidhaye, Balint Doleschall, Maria Novatchkova, Thomas Lendl, Marielle Piber, Angela Peer, Paul Möseneder, Marlene Stuempflen, Siu Yu A Chow, Rainer Seidl, Daniela Prayer, Romana Höftberger, Gregor Kasprian, Yoshiho Ikeuchi, Nina S Corsini, Jürgen A Knoblich.
      Mutations in ARID1B, a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC), characterized by the absence of an interhemispheric white matter tract that connects distant cortical regions. Here, we find that neurons expressing SATB2, a determinant of callosal projection neuron (CPN) identity, show impaired maturation in ARID1B+/- neural organoids. Molecularly, a reduction in chromatin accessibility of genomic regions targeted by TCF-like, NFI-like, and ARID-like transcription factors drives the differential expression of genes required for corpus callosum (CC) development. Through an in vitro model of the CC tract, we demonstrate that this transcriptional dysregulation impairs the formation of long-range axonal projections, causing structural underconnectivity. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous axonogenesis defects in SATB2+ neurons as a cause of ACC in ARID1B patients.
    Keywords:  ARID1B; SATB2; axonogenesis; cerebral cortex; corpus callosum agenesis; mSWI/SNF; neural organoids
    DOI:  https://doi.org/10.1016/j.stem.2024.04.014
  15. Acta Neuropathol Commun. 2024 May 06. 12(1): 72
    Comprehensive analysis of the REST transcription factor regulatory networks in IDH mutant and IDH wild-type glioma cell lines and tumors.
    Malgorzata Perycz, Michal J Dabrowski, Marta Jardanowska-Kotuniak, Adria-Jaume Roura, Bartlomiej Gielniewski, Karolina Stepniak, Michał Dramiński, Iwona A Ciechomska, Bozena Kaminska, Bartosz Wojtas.
      The RE1-silencing transcription factor (REST) acts either as a repressor or activator of transcription depending on the genomic and cellular context. REST is a key player in brain cell differentiation by inducing chromatin modifications, including DNA methylation, in a proximity of its binding sites. Its dysfunction may contribute to oncogenesis. Mutations in IDH1/2 significantly change the epigenome contributing to blockade of cell differentiation and glioma development. We aimed at defining how REST modulates gene activation and repression in the context of the IDH mutation-related phenotype in gliomas. We studied the effects of REST knockdown, genome wide occurrence of REST binding sites, and DNA methylation of REST motifs in IDH wild type and IDH mutant gliomas. We found that REST target genes, REST binding patterns, and TF motif occurrence proximal to REST binding sites differed in IDH wild-type and mutant gliomas. Among differentially expressed REST targets were genes involved in glial cell differentiation and extracellular matrix organization, some of which were differentially methylated at promoters or gene bodies. REST knockdown differently impacted invasion of the parental or IDH1 mutant glioma cells. The canonical REST-repressed gene targets showed significant correlation with the GBM NPC-like cellular state. Interestingly, results of REST or KAISO silencing suggested the interplay between these TFs in regulation of REST-activated and repressed targets. The identified gene regulatory networks and putative REST cooperativity with other TFs, such as KAISO, show distinct REST target regulatory networks in IDH-WT and IDH-MUT gliomas, without concomitant DNA methylation changes. We conclude that REST could be an important therapeutic target in gliomas.
    Keywords:   IDH mutation; DNA methylation; Differentiation; Extracellular matrix; Glioblastoma; Invasion; KAISO; REST; Transcription factor; ZBTB33
    DOI:  https://doi.org/10.1186/s40478-024-01779-y
  16. NAR Cancer. 2024 Jun;6(2): zcae020
    Integrated enhancer regulatory network by enhancer-promoter looping in gastric cancer.
    Tianhui Zhu, Atsushi Okabe, Genki Usui, Ryoji Fujiki, Daichi Komiyama, Kie Kyon Huang, Motoaki Seki, Masaki Fukuyo, Hiroyuki Abe, Meng Ning, Tomoka Okada, Mizuki Minami, Makoto Matsumoto, Qin Fan, Bahityar Rahmutulla, Takayuki Hoshii, Patrick Tan, Teppei Morikawa, Tetsuo Ushiku, Atsushi Kaneda.
      Enhancer cis-regulatory elements play critical roles in gene regulation at many stages of cell growth. Enhancers in cancer cells also regulate the transcription of oncogenes. In this study, we performed a comprehensive analysis of long-range chromatin interactions, histone modifications, chromatin accessibility and expression in two gastric cancer (GC) cell lines compared to normal gastric epithelial cells. We found that GC-specific enhancers marked by histone modifications can activate a population of genes, including some oncogenes, by interacting with their proximal promoters. In addition, motif analysis of enhancer-promoter interacting enhancers showed that GC-specific transcription factors are enriched. Among them, we found that MYB is crucial for GC cell growth and activated by the enhancer with an enhancer-promoter loop and TCF7 upregulation. Clinical GC samples showed epigenetic activation of enhancers at the MYB locus and significant upregulation of TCF7 and MYB, regardless of molecular GC subtype and clinicopathological factors. Single-cell RNA sequencing of gastric mucosa with intestinal metaplasia showed high expression of TCF7 and MYB in intestinal stem cells. When we inactivated the loop-forming enhancer at the MYB locus using CRISPR interference (dCas9-KRAB), GC cell growth was significantly inhibited. In conclusion, we identified MYB as an oncogene activated by a loop-forming enhancer and contributing to GC cell growth.
    DOI:  https://doi.org/10.1093/narcan/zcae020
  17. NAR Genom Bioinform. 2024 Jun;6(2): lqae045
    Malat1 affects transcription and splicing through distinct pathways in mouse embryonic stem cells.
    Morteza Aslanzadeh, Laura Stanicek, Marcel Tarbier, Emilio Mármol-Sánchez, Inna Biryukova, Marc R Friedländer.
      Malat1 is a long-noncoding RNA with critical roles in gene regulation and cancer metastasis, however its functional role in stem cells is largely unexplored. We here perform a nuclear knockdown of Malat1 in mouse embryonic stem cells, causing the de-regulation of 320 genes and aberrant splicing of 90 transcripts, some of which potentially affecting the translated protein sequence. We find evidence that Malat1 directly interacts with gene bodies and aberrantly spliced transcripts, and that it locates upstream of down-regulated genes at their putative enhancer regions, in agreement with functional genomics data. Consistent with this, we find these genes affected at both exon and intron levels, suggesting that they are transcriptionally regulated by Malat1. Besides, the down-regulated genes are regulated by specific transcription factors and bear both activating and repressive chromatin marks, suggesting that some of them might be regulated by bivalent promoters. We propose a model in which Malat1 facilitates the transcription of genes involved in chromatid dynamics and mitosis in one pathway, and affects the splicing of transcripts that are themselves involved in RNA processing in a distinct pathway. Lastly, we compare our findings with Malat1 perturbation studies performed in other cell systems and in vivo.
    DOI:  https://doi.org/10.1093/nargab/lqae045
  18. Genome Biol. 2024 May 06. 25(1): 115
    Genome-wide distribution of 5-hydroxymethyluracil and chromatin accessibility in the Breviolum minutum genome.
    Georgi K Marinov, Xinyi Chen, Matthew P Swaffer, Tingting Xiang, Arthur R Grossman, William J Greenleaf.
       BACKGROUND: In dinoflagellates, a unique and extremely divergent genomic and nuclear organization has evolved. The highly unusual features of dinoflagellate nuclei and genomes include permanently condensed liquid crystalline chromosomes, primarily packaged by proteins other than histones, genes organized in very long unidirectional gene arrays, a general absence of transcriptional regulation, high abundance of the otherwise very rare DNA modification 5-hydroxymethyluracil (5-hmU), and many others. While most of these fascinating properties are originally identified in the 1970s and 1980s, they have not yet been investigated using modern genomic tools.
    RESULTS: In this work, we address some of the outstanding questions regarding dinoflagellate genome organization by mapping the genome-wide distribution of 5-hmU (using both immunoprecipitation-based and basepair-resolution chemical mapping approaches) and of chromatin accessibility in the genome of the Symbiodiniaceae dinoflagellate Breviolum minutum. We find that the 5-hmU modification is preferentially enriched over certain classes of repetitive elements, often coincides with the boundaries between gene arrays, and is generally correlated with decreased chromatin accessibility, the latter otherwise being largely uniform along the genome. We discuss the potential roles of 5-hmU in the functional organization of dinoflagellate genomes and its relationship to the transcriptional landscape of gene arrays.
    CONCLUSIONS: Our results provide the first window into the 5-hmU and chromatin accessibility landscapes in dinoflagellates.
    DOI:  https://doi.org/10.1186/s13059-024-03261-3
  19. Nucleic Acids Res. 2024 May 10. pii: gkae372. [Epub ahead of print]
    Reduction of ZFX levels decreases histone H4 acetylation and increases Pol2 pausing at target promoters.
    Emily Hsu, Katherine Hutchison, Yao Liu, Charles M Nicolet, Shannon Schreiner, Nathan R Zemke, Peggy J Farnham.
      The ZFX transcriptional activator binds to CpG island promoters, with a major peak at ∼200-250 bp downstream from transcription start sites. Because ZFX binds within the transcribed region, we investigated whether it regulates transcriptional elongation. We used GRO-seq to show that loss or reduction of ZFX increased Pol2 pausing at ZFX-regulated promoters. To further investigate the mechanisms by which ZFX regulates transcription, we determined regions of the protein needed for transactivation and for recruitment to the chromatin. Interestingly, although ZFX has 13 grouped zinc fingers, deletion of the first 11 fingers produces a protein that can still bind to chromatin and activate transcription. We next used TurboID-MS to detect ZFX-interacting proteins, identifying ZNF593, as well as proteins that interact with the N-terminal transactivation domain (which included histone modifying proteins), and proteins that interact with ZFX when it is bound to the chromatin (which included TAFs and other histone modifying proteins). Our studies support a model in which ZFX enhances elongation at target promoters by recruiting H4 acetylation complexes and reducing pausing.
    DOI:  https://doi.org/10.1093/nar/gkae372
  20. Sci Rep. 2024 05 04. 14(1): 10287
    Transcriptional repression of the oncofetal LIN28B gene by the transcription factor SOX6.
    Valentina Pastori, Gianluca Zambanini, Elisabetta Citterio, Tamina Weiss, Yukio Nakamura, Claudio Cantù, Antonella Ellena Ronchi.
      The identification of regulatory networks contributing to fetal/adult gene expression switches is a major challenge in developmental biology and key to understand the aberrant proliferation of cancer cells, which often reactivate fetal oncogenes. One key example is represented by the developmental gene LIN28B, whose aberrant reactivation in adult tissues promotes tumor initiation and progression. Despite the prominent role of LIN28B in development and cancer, the mechanisms of its transcriptional regulation are largely unknown. Here, by using quantitative RT-PCR and single cell RNA sequencing data, we show that in erythropoiesis the expression of the transcription factor SOX6 matched a sharp decline of LIN28B mRNA during human embryo/fetal to adult globin switching. SOX6 overexpression repressed LIN28B not only in a panel of fetal-like erythroid cells (K562, HEL and HUDEP1; ≈92% p < 0.0001, 54% p = 0.0009 and ≈60% p < 0.0001 reduction, respectively), but also in hepatoblastoma HepG2 and neuroblastoma SH-SY5H cells (≈99% p < 0.0001 and ≈59% p < 0.0001 reduction, respectively). SOX6-mediated repression caused downregulation of the LIN28B/Let-7 targets, including MYC and IGF2BP1, and rapidly blocks cell proliferation. Mechanistically, Lin28B repression is accompanied by SOX6 physical binding within its locus, suggesting a direct mechanism of LIN28B downregulation that might contribute to the fetal/adult erythropoietic transition and restrict cancer proliferation.
    DOI:  https://doi.org/10.1038/s41598-024-60438-3
  21. Genome Res. 2024 May 06.
    Suv39h-catalyzed H3K9me3 is critical for euchromatic genome organization and the maintenance of gene transcription.
    Christine R Keenan, Hannah D Coughlan, Nadia Iannarella, Andres Tapia Del Fierro, Andrew Keniry, Timothy M Johanson, Wing Fuk Chan, Alexandra L Garnham, Lachlan W Whitehead, Marnie E Blewitt, Gordon K Smyth, Rhys S Allan.
      H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes in differentiation and development. Here, we investigate the molecular consequences of heterochromatin loss in cells deficient in both SUV39H1 and SUV39H2 (Suv39DKO), the major mammalian histone methyltransferase enzymes that catalyze heterochromatic H3K9me3 deposition. We reveal a paradoxical repression of protein-coding genes in Suv39DKO cells, with these differentially expressed genes principally in euchromatic (Tn5-accessible, H3K4me3- and H3K27ac-marked) rather than heterochromatic (H3K9me3-marked) or polycomb (H3K27me3-marked) regions. Examination of the three-dimensional (3D) nucleome reveals that transcriptomic dysregulation occurs in euchromatic regions close to the nuclear periphery in 3D space. Moreover, this transcriptomic dysregulation is highly correlated with altered 3D genome organization in Suv39DKO cells. Together, our results suggest that the nuclear lamina-tethering of Suv39-dependent H3K9me3 domains provides an essential scaffold to support euchromatic genome organization and the maintenance of gene transcription for healthy cellular function.
    DOI:  https://doi.org/10.1101/gr.279119.124
  22. Nat Biotechnol. 2024 May 09.
    Tracking single-cell evolution using clock-like chromatin accessibility loci.
    Yu Xiao, Wan Jin, Lingao Ju, Jie Fu, Gang Wang, Mengxue Yu, Fangjin Chen, Kaiyu Qian, Xinghuan Wang, Yi Zhang.
      Single-cell chromatin accessibility sequencing (scATAC-seq) reconstructs developmental trajectory by phenotypic similarity. However, inferring the exact developmental trajectory is challenging. Previous studies showed age-associated DNA methylation (DNAm) changes in specific genomic regions, termed clock-like differential methylation loci (ClockDML). Age-associated DNAm could either result from or result in chromatin accessibility changes at ClockDML. As cells undergo mitosis, the heterogeneity of chromatin accessibility on clock-like loci is reduced, providing a measure of mitotic age. In this study, we developed a method, called EpiTrace, that counts the fraction of opened clock-like loci from scATAC-seq data to determine cell age and perform lineage tracing in various cell lineages and animal species. It shows concordance with known developmental hierarchies, correlates well with DNAm-based clocks and is complementary with mutation-based lineage tracing, RNA velocity and stemness predictions. Applying EpiTrace to scATAC-seq data reveals biological insights with clinically relevant implications, ranging from hematopoiesis, organ development, tumor biology and immunity to cortical gyrification.
    DOI:  https://doi.org/10.1038/s41587-024-02241-z
  23. Genes Dev. 2024 May 07.
    Oct4 redox sensitivity potentiates reprogramming and differentiation.
    Zuolian Shen, Yifan Wu, Asit Manna, Chongil Yi, Bradley R Cairns, Kimberley J Evason, Mahesh B Chandrasekharan, Dean Tantin.
      The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.
    Keywords:  Oct1 (Pou2f1); Oct4 (Pou5f1); induced pluripotent stem cells (iPSCs); oxidative stress; ubiquitylation
    DOI:  https://doi.org/10.1101/gad.351411.123
  24. Nat Genet. 2024 May 09.
    Direct transposition of native DNA for sensitive multimodal single-molecule sequencing.
    Arjun S Nanda, Ke Wu, Iryna Irkliyenko, Brian Woo, Megan S Ostrowski, Andrew S Clugston, Leanne C Sayles, Lingru Xu, Ansuman T Satpathy, Hao G Nguyen, E Alejandro Sweet-Cordero, Hani Goodarzi, Sivakanthan Kasinathan, Vijay Ramani.
      Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90-99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000-50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.
    DOI:  https://doi.org/10.1038/s41588-024-01748-0
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