bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2025–01–19
twenty-one papers selected by
Connor Rogerson, University of Cambridge
  1. NKX2.2 and KLF4 cooperate to regulate α-cell identity.
  2. TRIM28 is an essential regulator of three-dimensional chromatin state underpinning CD8+ T cell activation.
  3. Massively parallel characterization of transcriptional regulatory elements.
  4. ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A.
  5. LEDGF/p75 promotes transcriptional pausing through preventing SPT5 phosphorylation.
  6. Modular organization of enhancer network provides transcriptional robustness in mammalian development.
  7. Coordinated neuron-specific splicing events restrict nucleosome engagement of the LSD1 histone demethylase complex.
  8. Detecting copy-number alterations from single-cell chromatin sequencing data by AtaCNA.
  9. Uncovering the whole genome silencers of human cells via Ss-STARR-seq.
  10. A conserved switch to less catalytically active Polycomb repressive complexes in non-dividing cells.
  11. Single-molecule analysis of transcription activation: dynamics of SAGA coactivator recruitment.
  12. Mitotic chromatin marking governs the segregation of DNA damage.
  13. Dynamic PRC1-CBX8 stabilizes a porous structure of chromatin condensates.
  14. Deciphering transcription activity of mammalian early embryos unveils on/off of zygotic genome activation by protein translation/degradation.
  15. Single-cell RNA-seq identifies protracted mouse germline X chromosome reactivation dynamics directed by a PRC2-dependent mechanism.
  16. A multivalent engagement of ENL with MOZ.
  17. Evaluating the transcriptional regulators of arterial gene expression via a catalogue of characterized arterial enhancers.
  18. Stable centromere association of the yeast histone variant Cse4 requires its essential N-terminal domain.
  19. The chromatin remodeler ADNP regulates neurodevelopmental disorder risk genes and neocortical neurogenesis.
  20. Mutations to transcription factor MAX allosterically increase DNA selectivity by altering folding and binding pathways.
  21. A cardiac transcriptional enhancer is repurposed during regeneration to activate an anti-proliferative program.
  1. Genes Dev. 2024 Dec 20.
    NKX2.2 and KLF4 cooperate to regulate α-cell identity.
    Elliott P Brooks, McKenna R Casey, Kristen L Wells, Tsung-Yun Liu, Madeline Van Orman, Lori Sussel.
      Transcription factors (TFs) are indispensable for maintaining cell identity through regulating cell-specific gene expression. Distinct cell identities derived from a common progenitor are frequently perpetuated by shared TFs, yet the mechanisms that enable these TFs to regulate cell-specific targets are poorly characterized. We report that the TF NKX2.2 is critical for the identity of pancreatic islet α cells by directly activating α-cell genes and repressing alternate islet cell fate genes. When compared with the known role of NKX2.2 in islet β cells, we demonstrate that NKX2.2 regulates α-cell genes, facilitated in part by α-cell-specific DNA binding at gene promoters. Furthermore, we have identified the reprogramming factor KLF4 as having enriched expression in α cells, where it co-occupies NKX2.2-bound α-cell promoters, is necessary for NKX2.2 promoter occupancy in α cells, and coregulates many NKX2.2 α-cell transcriptional targets. Overexpression of Klf4 in β cells is sufficient to manipulate chromatin accessibility, increase binding of NKX2.2 at α-cell-specific promoter sites, and alter expression of NKX2.2-regulated cell-specific targets. This study identifies KLF4 as a novel α-cell factor that cooperates with NKX2.2 to regulate α-cell identity.
    Keywords:  KLF4; NKX2.2; pancreatic islets; transcriptional regulation; α cells
    DOI:  https://doi.org/10.1101/gad.352193.124
  2. Nat Commun. 2025 Jan 16. 16(1): 750
    TRIM28 is an essential regulator of three-dimensional chromatin state underpinning CD8+ T cell activation.
    Kun Wei, Ruifeng Li, Xiaohong Zhao, Bowen Xie, Tian Xie, Qinli Sun, Yongzhen Chen, Peng Wei, Wei Xu, Xinyi Guo, Zixuan Zhao, Han Feng, Ling Ni, Chen Dong.
      T cell activation is accompanied by extensive changes in epigenome. However, the high-ordered chromatin organization underpinning CD8+ T cell activation is not fully known. Here, we show extensive changes in the three-dimensional genome during CD8+ T cell activation, associated with changes in gene transcription. We show that CD8+ T-cell-specific deletion of Trim28 in mice disrupts autocrine IL-2 production and leads to impaired CD8+ T cell activation in vitro and in vivo. Mechanistically, TRIM28 binds to regulatory regions of genes associated with the formation of chromosomal loops during activation. At the loop anchor regions, TRIM28-occupancy overlaps with that of CTCF, a factor known for defining the boundaries of topologically associating domains and for forming of the loop anchors. In the absence of Trim28, RNA Pol II and cohesin binding to these regions diminishes, and the chromosomal structure required for the active state is disrupted. These results thus identify a critical role for TRIM28-dependent chromatin topology in gene transcription in activated CD8+ T cells.
    DOI:  https://doi.org/10.1038/s41467-025-56029-z
  3. Nature. 2025 Jan 15.
    Massively parallel characterization of transcriptional regulatory elements.
    Vikram Agarwal, Fumitaka Inoue, Max Schubach, Dmitry Penzar, Beth K Martin, Pyaree Mohan Dash, Pia Keukeleire, Zicong Zhang, Ajuni Sohota, Jingjing Zhao, Ilias Georgakopoulos-Soares, William S Noble, Galip Gürkan Yardımcı, Ivan V Kulakovskiy, Martin Kircher, Jay Shendure, Nadav Ahituv.
      The human genome contains millions of candidate cis-regulatory elements (cCREs) with cell-type-specific activities that shape both health and many disease states1. However, we lack a functional understanding of the sequence features that control the activity and cell-type-specific features of these cCREs. Here we used lentivirus-based massively parallel reporter assays (lentiMPRAs) to test the regulatory activity of more than 680,000 sequences, representing an extensive set of annotated cCREs among three cell types (HepG2, K562 and WTC11), and found that 41.7% of these sequences were active. By testing sequences in both orientations, we find promoters to have strand-orientation biases and their 200-nucleotide cores to function as non-cell-type-specific 'on switches' that provide similar expression levels to their associated gene. By contrast, enhancers have weaker orientation biases, but increased tissue-specific characteristics. Utilizing our lentiMPRA data, we develop sequence-based models to predict cCRE function and variant effects with high accuracy, delineate regulatory motifs and model their combinatorial effects. Testing a lentiMPRA library encompassing 60,000 cCREs in all three cell types further identified factors that determine cell-type specificity. Collectively, our work provides an extensive catalogue of functional CREs in three widely used cell lines and showcases how large-scale functional measurements can be used to dissect regulatory grammar.
    DOI:  https://doi.org/10.1038/s41586-024-08430-9
  4. iScience. 2025 Jan 17. 28(1): 111538
    ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A.
    Min Seong Kim, Raham Lee, Dong Hun Lee, Heesang Song, Taekyung Ha, Joo Kyung Kim, Bum-Yong Kang, Karl Agger, Kristian Helin, Donghyun Shin, Yunhee Kang, Changwon Park.
      ETV2/ER71, an ETS (E-twenty six) transcription factor, is critical for hematopoiesis and vascular development. However, research about the molecular mechanisms behind ETV2-mediated gene transcription is limited. Herein, we demonstrate that ETV2 and KDM4A, an H3K9 demethylase, regulate hematopoietic and endothelial genes. Etv2 -/- mouse embryonic stem cells (mESCs), which fail to generate hematopoietic and endothelial cells, exhibit enhanced H3K9me3 levels in hematopoietic and endothelial genes. ETV2 interacts with KDM4A, and the ETV2-mediated transcriptional activation of hematopoietic and endothelial genes depends on KDM4A histone demethylase activity. The ETV2 and KDM4A complex binds to the transcription regulatory regions of genes directly regulated by ETV2. Mice lacking Kdm4a and Etv2 in endothelial cells (Cdh5Cre:Kdm:Etv2 f/f mice) display a more severe perfusion recovery and neovascularization defect, compared with Cdh5Cre:Kdm4a f/f mice, Cdh5Cre:Etv2 f/f mice, and controls. Collectively, we demonstrate that ETV2 interacts with KDM4A, and that this interaction is critical for hematovascular lineage generation and vascular regeneration.
    Keywords:  Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2024.111538
  5. Sci Adv. 2025 Jan 17. 11(3): eadr2131
    LEDGF/p75 promotes transcriptional pausing through preventing SPT5 phosphorylation.
    Chenghao Guo, Shuhan Si, Haitong Fang, Shimin Shuai, Yadi Zhang, Xiaoyu Du, Bo Duan, Jiawei Wu, Honghong Yao, Zheng Ge, Chengqi Lin, Zhuojuan Luo.
      SPT5 exhibits versatile functions in RNA Pol II promoter proximal pausing, pause release, and elongation in metazoans. However, the mechanism underlying the functional switch of SPT5 during early elongation has not been fully understood. Here, we report that the phosphorylation site-rich domain (PRD)/CTR1 and the prion-like domain (PLD)/CTR2, which are situated adjacent to each other within the C-terminal repeat (CTR) in SPT5, play pivotal roles in Pol II pausing and elongation, respectively. Our study demonstrates that LEDGF/p75 is highly enriched at promoters, especially paused promoters, and prevents the phosphorylation of SPT5 PRD by the super elongation complex (SEC). Furthermore, deletion of LEDGF IBD leads to increased SEC occupancies and SPT5 PRD phosphorylation at promoters and also increased pause release. In sum, our study reveals that LEDGF and SEC function cooperatively on SPT5 distinct domains to ensure proper transcriptional transition from pausing to elongation.
    DOI:  https://doi.org/10.1126/sciadv.adr2131
  6. Nucleic Acids Res. 2025 Jan 11. pii: gkae1323. [Epub ahead of print]53(2):
    Modular organization of enhancer network provides transcriptional robustness in mammalian development.
    Hongli Lin, Xinyun Ye, Wenyan Chen, Danni Hong, Lifang Liu, Feng Chen, Ning Sun, Keying Ye, Jizhou Hong, Yalin Zhang, Falong Lu, Lei Li, Jialiang Huang.
      Enhancer clusters, pivotal in mammalian development and diseases, can organize as enhancer networks to control cell identity and disease genes; however, the underlying mechanism remains largely unexplored. Here, we introduce eNet 2.0, a comprehensive tool for enhancer networks analysis during development and diseases based on single-cell chromatin accessibility data. eNet 2.0 extends our previous work eNet 1.0 by adding network topology, comparison and dynamics analyses to its network construction function. We reveal modularly organized enhancer networks, where inter-module interactions synergistically affect gene expression. Moreover, network alterations correlate with abnormal and dynamic gene expression in disease and development. eNet 2.0 is robust across diverse datasets. To facilitate application, we introduce eNetDB (https://enetdb.huanglabxmu.com), an enhancer network database leveraging extensive scATAC-seq (single-cell assay for transposase-accessible chromatin sequencing) datasets from human and mouse tissues. Together, our work provides a powerful computational tool and reveals that modularly organized enhancer networks contribute to gene expression robustness in mammalian development and diseases.
    DOI:  https://doi.org/10.1093/nar/gkae1323
  7. Cell Rep. 2025 Jan 15. pii: S2211-1247(24)01564-X. [Epub ahead of print]44(1): 115213
    Coordinated neuron-specific splicing events restrict nucleosome engagement of the LSD1 histone demethylase complex.
    Robert S Porter, Sojin An, Maria C Gavilan, Masayoshi Nagai, Yumie Murata-Nakamura, Bo Zhou, Katherine M Bonefas, Olivier Dionne, Jeru Manoj Manuel, Joannie St-Germain, Suzanne Gascon, Jacqueline Kim, Liam Browning, Benoit Laurent, Uhn-Soo Cho, Shigeki Iwase.
      Chromatin regulatory proteins are expressed broadly and assumed to exert the same intrinsic function across cell types. Here, we report that 14 chromatin regulators undergo evolutionary-conserved neuron-specific splicing events involving microexons. Among them are two components of a histone demethylase complex: LSD1 H3K4 demethylase and the H3K4me0-reader PHF21A. We found that neuronal LSD1 splicing reduces the enzymes' affinity to the nucleosome. Meanwhile, neuronal PHF21A splicing significantly attenuates histone H3 binding and further ablates the DNA-binding function exerted by an AT-hook motif. Furthermore, in vitro reconstitution of the canonical and neuronal PHF21A-LSD1 complexes, combined with in vivo methylation mapping, identified the neuronal complex as a hypomorphic H3K4 demethylating machinery. The neuronal PHF21A, albeit with its weaker nucleosome binding, is necessary for normal gene expression and the H3K4 landscape in the developing brain. Thus, ubiquitously expressed chromatin regulatory complexes can exert neuron-specific functions via alternative splicing of their subunits.
    Keywords:  CP: Molecular biology; CP: Neuroscience; alternative splicing; cellular differentiation; chromatin regulation; genetics of autism spectrum disorder; methyl-histone regulation; neurodevelopment
    DOI:  https://doi.org/10.1016/j.celrep.2024.115213
  8. Cell Rep Methods. 2025 Jan 08. pii: S2667-2375(24)00329-1. [Epub ahead of print] 100939
    Detecting copy-number alterations from single-cell chromatin sequencing data by AtaCNA.
    Xiaochen Wang, Zijie Jin, Yang Shi, Ruibin Xi.
      Single-cell assay of transposase-accessible chromatin sequencing (scATAC-seq) unbiasedly profiles genome-wide chromatin accessibility in single cells. In single-cell tumor studies, identification of normal cells or tumor clonal structures often relies on copy-number alterations (CNAs). However, CNA detection from scATAC-seq is difficult due to the high noise, sparsity, and confounding factors. Here, we describe AtaCNA, a computational algorithm that accurately detects high-resolution CNAs from scATAC-seq data. We benchmark AtaCNA using simulation and real data and find AtaCNA's superior performance. Analyses of 10 scATAC-seq datasets show that AtaCNA could effectively distinguish malignant from non-malignant cells. In glioblastoma, endometrial, and ovarian cancer samples, AtaCNA identifies subclones at distinct cellular states, suggesting an important interplay between genetic and epigenetic plasticity. Some tumor subclones only differ in small-scale (10-20 Mb) CNAs, demonstrating the importance of high-resolution CNA detection. These data show that AtaCNA can aid in integrative analysis to understand the complex heterogeneity in cancer.
    Keywords:  Bayesian model; CP: Cancer biology; CP: Genetics; change points; copy-number alterations; high resolution; normalization; single-cell chromatin sequencing; tumor cell detection; tumor heterogeneity
    DOI:  https://doi.org/10.1016/j.crmeth.2024.100939
  9. Nat Commun. 2025 Jan 16. 16(1): 723
    Uncovering the whole genome silencers of human cells via Ss-STARR-seq.
    Xiusheng Zhu, Lei Huang, Chao Wang, Guoli Li, Biao Deng, Dashuai Kong, Xiaoxiao Wang, Rongrong Chang, Yi Gu, Qiuhan Wen, Siyuan Kong, Yuwen Liu, Yubo Zhang.
      Silencers, the yin to enhancers' yang, play a pivotal role in fine-tuning gene expression throughout the genome. However, despite their recognized importance, comprehensive identification of these regulatory elements in the genome is still in its early stages. We developed a method called Ss-STARR-seq to directly determine the activity of silencers in the whole genome. In this study, we applied Ss-STARR-seq to human cell lines K562, LNCaP, and 293 T, and identified 134,171, 137,753, and 125,307 silencers on a genome-wide scale, respectively, these silencers function in various cells in a cell-specific manner. Silencers exhibited a substantial enrichment of transcriptional-inhibitory motifs, including REST, and demonstrated overlap with the binding sites of repressor transcription factors within the endogenous environment. Interestingly, H3K27me3 did not reflect silencer activity but facilitated the silencer's inhibitory role on gene expression. Additionally, the silencer did not have any significant histone markers at the genome-wide level. Our findings unveil that aspect-silencers not only transition into enhancers throughout diverse cell lines but also achieve functional conversion with insulators. Regarding to biological effects, knockout experiments underscored the functional redundancy and specificity of silencers in regulating gene expression and cell proliferation. In summary, this study pioneers the elucidation of the genome-wide silencer landscape in human cells, delineates their global regulatory features, and identifies specific silencers influencing cancer cell proliferation.
    DOI:  https://doi.org/10.1038/s41467-025-55852-8
  10. Cell Rep. 2025 Jan 11. pii: S2211-1247(24)01543-2. [Epub ahead of print]44(1): 115192
    A conserved switch to less catalytically active Polycomb repressive complexes in non-dividing cells.
    Rachel McCole, James Nolan, David M Reck, Craig Monger, Samantha Rustichelli, Eric Conway, Gerard L Brien, Cheng Wang, Orla Deevy, Hannah K Neikes, Frances M Bashore, Aoibhinn Mooney, Richard Flavin, Elisabeth Vandenberghe, Sarena F Flanigan, Diego Pasini, Chen Davidovich, Michiel Vermeulen, Lindsey I James, Evan Healy, Adrian P Bracken.
      Polycomb repressive complex 2 (PRC2), composed of the core subunits EED, SUZ12, and either EZH1 or EZH2, is critical for maintaining cellular identity in multicellular organisms. PRC2 deposits H3K27me3, which is thought to recruit the canonical form of PRC1 (cPRC1) to promote gene repression. Here, we show that EZH1-PRC2 and cPRC1 are the primary Polycomb complexes on target genes in non-dividing, quiescent cells. Furthermore, these cells are resistant to PRC2 inhibitors. While PROTAC-mediated degradation of EZH1-PRC2 in quiescent cells does not reduce H3K27me3, it partially displaces cPRC1. Our results reveal an evolutionarily conserved switch to less catalytically active Polycomb complexes in non-dividing cells and raise concerns about using PRC2 inhibitors in cancers with significant populations of non-dividing cells.
    Keywords:  CBX7; CP: Molecular biology; EED; EZH1; EZH2; H3K27me3; PRC1; PRC2; PROTAC; Polycomb; cellular quiescence
    DOI:  https://doi.org/10.1016/j.celrep.2024.115192
  11. Nat Struct Mol Biol. 2025 Jan 14.
    Single-molecule analysis of transcription activation: dynamics of SAGA coactivator recruitment.
    Jongcheol Jeon, Larry J Friedman, Daniel H Zhou, Hogyu David Seo, Oluwatobi A Adeleke, Bria Graham, Emily F Patteson, Jeff Gelles, Stephen Buratowski.
      Transcription activators are said to stimulate gene expression by 'recruiting' coactivators, yet this vague term fits multiple kinetic models. To directly analyze the dynamics of activator-coactivator interactions, single-molecule microscopy was used to image promoter DNA, a transcription activator and the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex within yeast nuclear extract. SAGA readily but transiently binds nucleosome-free DNA without an activator, while chromatin association occurs primarily when an activator is present. On both templates, an activator increases SAGA association rates by an order of magnitude and dramatically extends occupancy time. These effects reflect sustained interactions with the transactivation domain, as VP16 or Rap1 activation domains produce different SAGA dynamics. SAGA preferentially associates with templates carrying more than one activator. Unexpectedly, SAGA binding is substantially improved by nucleoside triphosphates but not histone H3 or H4 tail tetra-acetylations. Thus, we observe two modes of SAGA-template interaction: short-lived activator-independent binding to non-nucleosomal DNA and tethering to promoter-bound transcription activators for up to several minutes.
    DOI:  https://doi.org/10.1038/s41594-024-01451-y
  12. Nat Commun. 2025 Jan 16. 16(1): 746
    Mitotic chromatin marking governs the segregation of DNA damage.
    Juliette Ferrand, Juliette Dabin, Odile Chevallier, Matteo Kane-Charvin, Ariana Kupai, Joel Hrit, Scott B Rothbart, Sophie E Polo.
      The faithful segregation of intact genetic material and the perpetuation of chromatin states through mitotic cell divisions are pivotal for maintaining cell function and identity across cell generations. However, most exogenous mutagens generate long-lasting DNA lesions that are segregated during mitosis. How this segregation is controlled is unknown. Here, we uncover a mitotic chromatin-marking pathway that governs the segregation of UV-induced damage in human cells. Our mechanistic analyses reveal two layers of control: histone ADP-ribosylation, and the incorporation of newly synthesized histones at UV damage sites, that both prevent local mitotic phosphorylations on histone H3 serine residues. Functionally, this chromatin-marking pathway controls the segregation of UV damage in the cell progeny with consequences on daughter cell fate. We propose that this mechanism may help preserve the integrity of stem cell compartments during asymmetric cell divisions.
    DOI:  https://doi.org/10.1038/s41467-025-56090-8
  13. Nat Struct Mol Biol. 2025 Jan 15.
    Dynamic PRC1-CBX8 stabilizes a porous structure of chromatin condensates.
    Michael Uckelmann, Vita Levina, Cyntia Taveneau, Xiao Han Ng, Varun Pandey, Jasmine Martinez, Shweta Mendiratta, Justin Houx, Marion Boudes, Hari Venugopal, Sylvain Trépout, Alex J Fulcher, Qi Zhang, Sarena Flanigan, Minrui Li, Emma Sierecki, Yann Gambin, Partha Pratim Das, Oliver Bell, Alex de Marco, Chen Davidovich.
      The compaction of chromatin is a prevalent paradigm in gene repression. Chromatin compaction is commonly thought to repress transcription by restricting chromatin accessibility. However, the spatial organization and dynamics of chromatin compacted by gene-repressing factors are unknown. Here, using cryo-electron tomography, we solved the three-dimensional structure of chromatin condensed by the polycomb repressive complex 1 (PRC1) in a complex with CBX8. PRC1-condensed chromatin is porous and stabilized through multivalent dynamic interactions of PRC1 with chromatin. Mechanistically, positively charged residues on the internally disordered regions of CBX8 mask negative charges on the DNA to stabilize the condensed state of chromatin. Within condensates, PRC1 remains dynamic while maintaining a static chromatin structure. In differentiated mouse embryonic stem cells, CBX8-bound chromatin remains accessible. These findings challenge the idea of rigidly compacted polycomb domains and instead provide a mechanistic framework for dynamic and accessible PRC1-chromatin condensates.
    DOI:  https://doi.org/10.1038/s41594-024-01457-6
  14. Cell Rep. 2025 Jan 16. pii: S2211-1247(24)01566-3. [Epub ahead of print]44(1): 115215
    Deciphering transcription activity of mammalian early embryos unveils on/off of zygotic genome activation by protein translation/degradation.
    Yi-Ran Zhang, Shi-Meng Guo, Xiao-Yan Shi, Yi-Wen Ding, Huai-Biao Li, Lei Li, Jia-Wei Xu, Ximiao He, Bing-Xin Ma, Ying Yin, Li-Quan Zhou.
      Quantification of transcription activities in mammalian preimplantation embryos is challenging due to a huge amount of maternally stored transcripts and paucity of research materials. Here, we investigate genome-wide transcription activities of mouse and human preimplantation embryos by quantifying elongating RNA polymerase II. Two transcriptional waves are identified in early mouse embryos, with summits at the 2-cell and 8-cell stages. Gene collections with different expression patterns are obtained, with genes mainly transcribed at the mouse early/late 2-cell stage designated as zygotic genome activation-early/late 2-cell (ZGA-E2C/L2C). ZGA-E2C genes are short and have low promoter CpG density. Protein translation/degradation not only regulates transcription activity through stepwise orchestration of histone modifications, transcriptional initiation, and elongation in early mouse embryos but also controls on/off switching of ZGA-E2C/L2C genes in maternal aged mouse embryos. Genes mainly transcribed at the mouse 2-cell stage can also be transcribed as early as the human 2-cell stage.
    Keywords:  CP: Developmental biology; CP: Molecular biology; embryo; maternal aging; preimplantation; transcription activity; zygotic genome activation
    DOI:  https://doi.org/10.1016/j.celrep.2024.115215
  15. Dev Cell. 2025 Jan 08. pii: S1534-5807(24)00767-6. [Epub ahead of print]
    Single-cell RNA-seq identifies protracted mouse germline X chromosome reactivation dynamics directed by a PRC2-dependent mechanism.
    Yaqiong Liu, Xianzhong Lau, Prabhakaran Munusamy, Carlos M Abascal Sherwell Sanchez, Daniel Snell, Mahesh Sangrithi.
      Female primordial germ cells (PGCs) undergo X chromosome reactivation (XCR) during genome-wide reprogramming. XCR kinetics and dynamics are poorly understood at a molecular level. Here, we apply single-cell RNA sequencing and chromatin profiling on germ cells from F1 mouse embryos, performing a precise appraisal of XCR spanning from migratory-stage PGCs to gonadal germ cells. Establishment of germ cell sexual dimorphism and X chromosome dosage compensation states in vivo are temporally linked to XCR. Allele-specific analysis evidence that the reactivating X chromosome is minimally active in embryonic day (E)9.5 female PGCs, reactivates gradually, and reaches parity to the active X chromosome in E16.5 oogonia. While Xist is repressed from E10.5 onward, epigenetic memory of X inactivation persists from self-sustained polycomb repressive complex 2 (PRC2) activity. The reactivating X is asymmetrically enriched for histone 3-lysine-27-trimethylation (H3K27me3) at E13.5, which is later reversed, permitting germline gene expression. Our findings relate XCR with PRC2 function in promoting female meiosis.
    Keywords:  PRC2; X chromosome reactivation; allele-specific expression; dosage compensation; genome-wide reprogramming; germ cells; single-cell RNA-seq
    DOI:  https://doi.org/10.1016/j.devcel.2024.12.028
  16. Nat Struct Mol Biol. 2025 Jan 10.
    A multivalent engagement of ENL with MOZ.
    Dustin C Becht, Karthik Selvam, Catherine Lachance, Valérie Côté, Kuai Li, Minh Chau Nguyen, Akshay Pareek, Xiaobing Shi, Hong Wen, M Andres Blanco, Jacques Côté, Tatiana G Kutateladze.
      The epigenetic cofactor ENL (eleven-nineteen-leukemia) and the acetyltransferase MOZ (monocytic leukemia zinc finger) have vital roles in transcriptional regulation and are implicated in aggressive forms of leukemia. Here, we describe the mechanistic basis for the intertwined association of ENL and MOZ. Genomic analysis shows that ENL and MOZ co-occupy active promoters and that MOZ recruits ENL to its gene targets. Structural studies reveal a multivalent assembly of ENL at the intrinsically disordered region (IDR) of MOZ. While the extraterminal (ET) domain of ENL recognizes the canonical ET-binding motif in IDR, the YEATS domains of ENL and homologous AF9 bind to a set of acetylation sites in the MOZ IDR that are generated by the acetyltransferase CBP (CREB-binding protein). Our findings suggest a multifaceted acetylation-dependent and independent coupling of ENL, MOZ and CBP/p300, which may contribute to leukemogenic activities of the ENL-MOZ assembly and chromosomal translocations of ENL, MOZ and CBP/p300.
    DOI:  https://doi.org/10.1038/s41594-024-01455-8
  17. Elife. 2025 Jan 17. pii: e102440. [Epub ahead of print]14
    Evaluating the transcriptional regulators of arterial gene expression via a catalogue of characterized arterial enhancers.
    Svanhild Nornes, Susann Bruche, Niharika Adak, Ian R McCracken, Sarah De Val.
      The establishment and growth of the arterial endothelium requires the coordinated expression of numerous genes. However, regulation of this process is not yet fully understood. Here, we combined in silico analysis with transgenic mice and zebrafish models to characterize arterial-specific enhancers associated with eight key arterial identity genes (Acvrl1/Alk1, Cxcr4, Cxcl12, Efnb2, Gja4/Cx37, Gja5/Cx40, Nrp1 and Unc5b). Next, to elucidate the regulatory pathways upstream of arterial gene transcription, we investigated the transcription factors binding each arterial enhancer compared to a similar assessment of non-arterial endothelial enhancers. These results found that binding of SOXF and ETS factors was a common occurrence at both arterial and pan-endothelial enhancers, suggesting neither are sufficient to direct arterial specificity. Conversely, FOX motifs independent of ETS motifs were over-represented at arterial enhancers. Further, MEF2 and RBPJ binding was enriched but not ubiquitous at arterial enhancers, potentially linked to specific patterns of behaviour within the arterial endothelium. Lastly, there was no shared or arterial-specific signature for WNT-associated TCF/LEF, TGFβ/BMP-associated SMAD1/5 and SMAD2/3, shear stress-associated KLF4 or venous-enriched NR2F2. This cohort of well characterized and in vivo-verified enhancers can now provide a platform for future studies into the interaction of different transcriptional and signalling pathways with arterial gene expression.
    Keywords:  developmental biology; genetics; genomics; mouse; zebrafish
    DOI:  https://doi.org/10.7554/eLife.102440
  18. EMBO J. 2025 Jan 14.
    Stable centromere association of the yeast histone variant Cse4 requires its essential N-terminal domain.
    Andrew R Popchock, Sabrine Hedouin, Yizi Mao, Charles L Asbury, Andrew B Stergachis, Sue Biggins.
      Chromosome segregation relies on kinetochores that assemble on specialized centromeric chromatin containing a histone H3 variant. In budding yeast, a single centromeric nucleosome containing Cse4 assembles at a sequence-defined 125 bp centromere. Yeast centromeric sequences are poor templates for nucleosome formation in vitro, suggesting the existence of mechanisms that specifically stabilize Cse4 nucleosomes in vivo. The extended Cse4 N-terminal tail binds to the chaperone Scm3, and a short essential region called END within the N-terminal tail binds the inner kinetochore complex Okp1/Ame1. To address the roles of these interactions, we utilized single-molecule fluorescence assays to monitor Cse4 during kinetochore assembly. We found that Okp1/Ame1 and Scm3 independently stabilize Cse4 at centromeres via their END interaction. Scm3 and Cse4 stability at the centromere are enhanced by Ipl1/Aurora B phosphorylation of the Cse4 END, identifying a previously unknown role for Ipl1 in ensuring Cse4 stability. Strikingly, a phosphomimetic mutation in the Cse4 END restores Cse4 recruitment in mutants defective in Okp1/Ame1 binding. Together, these data suggest that a key function of the essential Cse4 N-terminus is to ensure Cse4 localization at centromeres.
    Keywords:  Centromere; Centromeric Nucleosome; Chromosome Segregation; Kinetochore; TIRF Microscopy
    DOI:  https://doi.org/10.1038/s44318-024-00345-5
  19. Proc Natl Acad Sci U S A. 2025 Jan 21. 122(3): e2405981122
    The chromatin remodeler ADNP regulates neurodevelopmental disorder risk genes and neocortical neurogenesis.
    Samuel Clémot-Dupont, José Alex Lourenço Fernandes, Sarah Larrigan, Xiaoqi Sun, Suma Medisetti, Rory Stanley, Ziyad El Hankouri, Shrilaxmi V Joshi, David J Picketts, Karthik Shekhar, Pierre Mattar.
      Although chromatin remodelers are among the most important risk genes associated with neurodevelopmental disorders (NDDs), the roles of these complexes during brain development are in many cases unclear. Here, we focused on the recently discovered ChAHP chromatin remodeling complex. The zinc finger and homeodomain transcription factor ADNP is a core subunit of this complex, and de novo ADNP mutations lead to intellectual disability and autism spectrum disorder. However, germline Adnp knockout mice were previously shown to exhibit early embryonic lethality, obscuring subsequent roles for the ChAHP complex in neurogenesis. To circumvent this early developmental arrest, we generated a conditional Adnp mutant allele. Using single-cell transcriptomics, cut&run-seq, and histological approaches, we show that during neocortical development, Adnp orchestrates the production of late-born, upper-layer neurons through a two-step process. First, Adnp is required to sustain progenitor proliferation specifically during the developmental window for upper-layer cortical neurogenesis. Accordingly, we found that Adnp recruits the ChAHP subunit Chd4 to genes associated with progenitor proliferation. Second, in postmitotic differentiated neurons, we define a network of risk genes linked to NDDs that are regulated by Adnp and Chd4. Taken together, these data demonstrate that ChAHP is critical for driving the expansion of upper-layer cortical neurons and for regulating neuronal gene expression programs, suggesting that these processes may potentially contribute to NDD etiology.
    Keywords:  Adnp; ChAHP complex; chromatin remodeling; neocortex; neurodevelopmental disorders
    DOI:  https://doi.org/10.1073/pnas.2405981122
  20. Nat Commun. 2025 Jan 14. 16(1): 636
    Mutations to transcription factor MAX allosterically increase DNA selectivity by altering folding and binding pathways.
    Renee Hastings, Arjun K Aditham, Nicole DelRosso, Peter H Suzuki, Polly M Fordyce.
      Understanding how proteins discriminate between preferred and non-preferred ligands ('selectivity') is essential for predicting biological function and a central goal of protein engineering efforts, yet the biophysical mechanisms underpinning selectivity remain poorly understood. Towards this end, we study how variants of the promiscuous transcription factor (TF) MAX (H. sapiens) alter DNA specificity and selectivity, yielding >1700 Kds and >500 rate constants in complex with multiple DNA sequences. Twenty-two of the 240 assayed MAX point mutations enhance selectivity, yet none of these mutations occur at residues that contact nucleotides in published structures. By applying thermodynamic and kinetic models to these results and previous observations for the highly similar yet far more selective TF Pho4 (S. cerevisiae), we find that these mutations enhance selectivity by altering partitioning between or affinity within conformations with different intrinsic selectivity, providing a mechanistic basis for allosteric modulation of ligand selectivity. These results highlight the importance of conformational heterogeneity in determining sequence selectivity and can guide future efforts to engineer selective proteins.
    DOI:  https://doi.org/10.1038/s41467-024-55672-2
  21. Development. 2025 Jan 13. pii: dev.204458. [Epub ahead of print]
    A cardiac transcriptional enhancer is repurposed during regeneration to activate an anti-proliferative program.
    Anupama Rao, Andrew Russell, Jose Segura-Bermudez, Charles Franz, Rejenae Dockery, Anton Blatnik, Jacob Panten, Mateo Zevallos, Carson McNulty, Maciej Pietrzak, Joseph Aaron Goldman.
      Zebrafish have a high capacity to regenerate their hearts. Several studies have surveyed transcriptional enhancers to understand how gene expression is controlled during heart regeneration. We have identified REN or the runx1 enhancer that during regeneration regulates the expression of the nearby runx1 gene. We show that runx1 mRNA is reduced with deletion of REN (ΔREN) and cardiomyocyte proliferation is enhanced ΔREN mutants only during regeneration. Interestingly, in uninjured hearts, ΔREN mutants have reduced expression of adamts1, a nearby gene that encodes a Collagen protease. This results in excess Collagen within cardiac valves of uninjured hearts. The ΔREN Collagen phenotype is rescued by an allele with Δrunx1 mutations, suggesting that in uninjured hearts REN regulates adamts1 independently of runx1. Taken together, this suggests that REN is rewired from adamts1 in uninjured hearts to stimulate runx1 transcription during regeneration. Our data point to a previously unappreciated mechanism for gene regulation during zebrafish heart regeneration. We report that an enhancer is rewired from expression in a distal cardiac domain to activate a different gene in regenerating tissue.
    Keywords:  Gene regulation; Heart regeneration; Transcription enhancer; Zebrafish
    DOI:  https://doi.org/10.1242/dev.204458
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