bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024‒11‒17
thirty-one papers selected by
Connor Rogerson, University of Cambridge



  1. Nucleic Acids Res. 2024 Nov 11. pii: gkae958. [Epub ahead of print]
      PWWP-DOMAIN INTERACTOR OF POLYCOMBS (PWO) family proteins play a vital role in regulating plant development. However, the molecular mechanisms of how PWOs regulate chromatin structure is elusive. Our data show that the PWO1 binding sites are enriched with positive modifications but exclusive with H3K27me3. Moreover, PWO1 binds to the H3K27me3-enriched compartment domain (H3K27me3-CD) boundary regions, and functions to maintain the boundary strength. Meanwhile, we found that PWOs and Polycomb repressive complex 2 (PRC2) function parallelly in maintaining H3K27me3-CDs' structure. Loss of either PWOs or PRC2 leads to H3K27me3-CD strength reduction, B to A compartment switching as well as the H3K27me3-CD relocating away from the nuclear periphery. Additionally, PWOs and lamin-like proteins collaborate to regulate multiple chromatin structures to repress gene transcription within H3K27me3-CDs. We conclude that PWOs maintain H3K27me3-CDs' repressive state and regulate their spatial position in the nucleus.
    DOI:  https://doi.org/10.1093/nar/gkae958
  2. Genome Biol. 2024 Nov 14. 25(1): 293
      BACKGROUND: Inhomogeneous patterns of chromatin-chromatin contacts within 10-100-kb-sized regions of the genome are a generic feature of chromatin spatial organization. These features, termed topologically associating domains (TADs), have led to the loop extrusion factor (LEF) model. Currently, our ability to model TADs relies on the observation that in vertebrates TAD boundaries are correlated with DNA sequences that bind CTCF, which therefore is inferred to block loop extrusion. However, although TADs feature prominently in their Hi-C maps, non-vertebrate eukaryotes either do not express CTCF or show few TAD boundaries that correlate with CTCF sites. In all of these organisms, the counterparts of CTCF remain unknown, frustrating comparisons between Hi-C data and simulations.RESULTS: To extend the LEF model across the tree of life, here, we propose the conserved-current loop extrusion (CCLE) model that interprets loop-extruding cohesin as a nearly conserved probability current. From cohesin ChIP-seq data alone, we derive a position-dependent loop extrusion rate, allowing for a modified paradigm for loop extrusion, that goes beyond solely localized barriers to also include loop extrusion rates that vary continuously. We show that CCLE accurately predicts the TAD-scale Hi-C maps of interphase Schizosaccharomyces pombe, as well as those of meiotic and mitotic Saccharomyces cerevisiae, demonstrating its utility in organisms lacking CTCF.
    CONCLUSIONS: The success of CCLE in yeasts suggests that loop extrusion by cohesin is indeed the primary mechanism underlying TADs in these systems. CCLE allows us to obtain loop extrusion parameters such as the LEF density and processivity, which compare well to independent estimates.
    DOI:  https://doi.org/10.1186/s13059-024-03432-2
  3. Mol Cell. 2024 Nov 08. pii: S1097-2765(24)00860-8. [Epub ahead of print]
      The polymerase associated factor 1 (PAF1) complex (PAF1c) promotes RNA polymerase II (RNA Pol II) transcription at the elongation step; however, how PAF1c transcription activity is selectively regulated during cell fate transitions remains poorly understood. Here, we reveal that the alternative reading frame (ARF) tumor suppressor operates at two levels to restrain PAF1c-dependent oncogenic transcriptional programs upon p53 loss in mouse cells. First, ARF assembles into homo-oligomers to bind the PAF1 subunit to promote PAF1c disassembly, consequently dampening PAF1c interaction with RNA Pol II and PAF1c-dependent transcription. Second, ARF targets the RUNX family transcription factor 1 (RUNX1) to selectively tune gene transcription. Consistently, ARF loss triggers RUNX1- and PAF1c-dependent transcriptional activation of pro-growth ligands (growth differentiation factor/bone morphogenetic protein [GDF/BMP]), promoting a cell-intrinsic GDF/BMP-Smad1/5 axis that aberrantly induce cell growth. Notably, pharmacologic inactivation of GDF/BMP signaling and genetic perturbation of RUNX1 significantly attenuate cell proliferation mediated by dual p53 and ARF loss, offering therapeutic utility. Our data underscore the significance of selective ARF-mediated tumor-suppressive functions through a universal transcriptional regulator.
    Keywords:  ARF; CDKN2A; PAF1; RNA polymerase II; RUNX; cell proliferation; p53; transcription; tumor suppressor; tumorigenesis
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.020
  4. Elife. 2024 Nov 13. pii: RP95170. [Epub ahead of print]13
      Enhancers and promoters are classically considered to be bound by a small set of transcription factors (TFs) in a sequence-specific manner. This assumption has come under increasing skepticism as the datasets of ChIP-seq assays of TFs have expanded. In particular, high-occupancy target (HOT) loci attract hundreds of TFs with often no detectable correlation between ChIP-seq peaks and DNA-binding motif presence. Here, we used a set of 1003 TF ChIP-seq datasets (HepG2, K562, H1) to analyze the patterns of ChIP-seq peak co-occurrence in combination with functional genomics datasets. We identified 43,891 HOT loci forming at the promoter (53%) and enhancer (47%) regions. HOT promoters regulate housekeeping genes, whereas HOT enhancers are involved in tissue-specific process regulation. HOT loci form the foundation of human super-enhancers and evolve under strong negative selection, with some of these loci being located in ultraconserved regions. Sequence-based classification analysis of HOT loci suggested that their formation is driven by the sequence features, and the density of mapped ChIP-seq peaks across TF-bound loci correlates with sequence features and the expression level of flanking genes. Based on the affinities to bind to promoters and enhancers we detected five distinct clusters of TFs that form the core of the HOT loci. We report an abundance of HOT loci in the human genome and a commitment of 51% of all TF ChIP-seq binding events to HOT locus formation thus challenging the classical model of enhancer activity and propose a model of HOT locus formation based on the existence of large transcriptional condensates.
    Keywords:  HOT; computational biology; gene expression; genetics; genomics; high-occupancy target; human; systems biology; transcriptional condensates
    DOI:  https://doi.org/10.7554/eLife.95170
  5. Commun Biol. 2024 Nov 14. 7(1): 1505
      The genome is intricately folded into chromatin compartments, topologically associating domains (TADs) and loops unique to each cell type. How this higher-order genome organization regulates cell fate transition remains elusive. Here we show how a single non-neural progenitor transcription factor, PTF1A, reorchestrates the 3D genome during fibroblast transdifferentiation into neural stem cells (NSCs). Multiomics analyses integrating Hi-C data, PTF1A and CTCF DNA-binding profiles, H3K27ac modification, and gene expression, demonstrate that PTF1A binds to subTAD boundaries subsequently associated with elevated CTCF binding and enhanced boundary insulation, and reorganizes chromatin loops, leading to gene expression changes that drive transdifferentiation into NSCs. Moreover, PTF1A activates enhancers and super-enhancers near low-insulation boundaries and modulates H3K27ac deposition, promoting cell fate transitions. Together, our data implicate an involvement of 3D genome in transcriptional and cell fate alterations, and highlight an essential role for PTF1A in gene expression control and multiscale 3D genome remodeling during cell reprogramming.
    DOI:  https://doi.org/10.1038/s42003-024-07230-1
  6. Nat Commun. 2024 Nov 15. 15(1): 9932
      Multimodal single-cell assays profile multiple sets of features in the same cells and are widely used for identifying and mapping cell states between chromatin and mRNA and linking regulatory elements to target genes. However, the high dimensionality of input features and shallow sequencing depth compared to unimodal assays pose challenges in data analysis. Here we present scPair, a multimodal single-cell data framework that overcomes these challenges by employing an implicit feature selection approach. scPair uses dual encoder-decoder structures trained on paired data to align cell states across modalities and predict features from one modality to another. We demonstrate that scPair outperforms existing methods in accuracy and execution time, and facilitates downstream tasks such as trajectory inference. We further show scPair can augment smaller multimodal datasets with larger unimodal atlases to increase statistical power to identify groups of transcription factors active during different stages of neural differentiation.
    DOI:  https://doi.org/10.1038/s41467-024-53971-2
  7. Nat Commun. 2024 Nov 14. 15(1): 9864
      Transition of cytosine to thymine in CpG dinucleotides is the most frequent type of mutation in cancer. This increased mutability is commonly attributed to the spontaneous deamination of 5-methylcytosine (5mC), which is normally repaired by the base-excision repair (BER) pathway. However, the contribution of 5mC deamination in the increasing diversity of cancer mutational signatures remains poorly explored. We integrate mutational signatures analysis in a large series of tumor whole genomes with lineage-specific epigenomic data to draw a detailed view of 5mC deamination in cancer. We uncover tumor type-specific patterns of 5mC deamination signatures in CpG and non-CpG contexts. We demonstrate that the BER glycosylase MBD4 preferentially binds to active chromatin and early replicating DNA, which correlates with lower mutational burden in these domains. We validate our findings by modeling BER deficiencies in isogenic cell models. Here, we establish MBD4 as the main actor responsible for 5mC deamination repair in humans.
    DOI:  https://doi.org/10.1038/s41467-024-54223-z
  8. Dev Cell. 2024 Nov 05. pii: S1534-5807(24)00628-2. [Epub ahead of print]
      The ability of cancer cells to undergo identity changes (i.e., lineage plasticity) plays a key role in tumor progression and response to therapy. Loss of the pulmonary lineage specifier NKX2-1 in KRAS-driven lung adenocarcinoma (LUAD) enhances tumor progression and causes a FoxA1/2-dependent pulmonary-to-gastric lineage switch. However, the mechanisms by which FoxA1/2 activate a latent gastric identity in the lung remain largely unknown. Here, we show that FoxA1/2 reprogram the epigenetic landscape of gastric-specific genes after NKX2-1 loss in mouse models by facilitating ten-eleven translocation (TET)2/3 recruitment, DNA demethylation, histone 3 lysine 27 acetylation (H3K27ac) deposition, and three-dimensional (3D) chromatin interactions. FoxA1/2-mediated DNA methylation changes are highly conserved in human endodermal development and in progression of human lung and pancreatic neoplasia. Furthermore, oncogenic signaling is required for specific elements of FoxA1/2-dependent epigenetic reprogramming. This work demonstrates the role of FoxA1/2 in rewiring the DNA methylation and 3D chromatin landscape of NKX2-1-negative LUAD to drive cancer cell lineage switching.
    Keywords:  FOXA1/2; TET2/3; cancer lineage switching/plasticity; epigenetic reprogramming; lung adenocarcinoma
    DOI:  https://doi.org/10.1016/j.devcel.2024.10.009
  9. Nat Commun. 2024 Nov 07. 15(1): 9614
      Histone post-translational modifications play pivotal roles in eukaryotic gene expression. To date, most studies have focused on modifications in unstructured histone N-terminal tail domains and their binding proteins. However, transcriptional regulation by chromatin-effector proteins that directly recognize modifications in histone globular domains has yet to be clearly demonstrated, despite the richness of their multiple modifications. Here, we show that the ATP-dependent chromatin-remodeling BAF complex stimulates p53-dependent transcription through direct interaction with H3K56ac located on the lateral surface of the histone globular domain. Mechanistically, the BAF complex recognizes nucleosomal H3K56ac via the DPF domain in the DPF2 subunit and exhibits enhanced nucleosome-remodeling activity in the presence of H3K56ac. We further demonstrate that a defect in H3K56ac-BAF complex interaction leads to impaired p53-dependent gene expression and DNA damage responses. Our study provides direct evidence that histone globular domain modifications participate in the regulation of gene expression.
    DOI:  https://doi.org/10.1038/s41467-024-53981-0
  10. Brief Bioinform. 2024 Sep 23. pii: bbae592. [Epub ahead of print]25(6):
      Deciphering the underlying gene regulatory networks (GRNs) that govern early human embryogenesis is critical for understanding developmental mechanisms yet remains challenging due to limited sample availability and the inherent complexity of the biological processes involved. To address this, we developed InPheRNo-ChIP, a computational framework that integrates multimodal data, including RNA-seq, transcription factor (TF)-specific ChIP-seq, and phenotypic labels, to reconstruct phenotype-relevant GRNs associated with endoderm development. The core of this method is a probabilistic graphical model that models the simultaneous effect of TFs on their putative target genes to influence a particular phenotypic outcome. Unlike the majority of existing GRN inference methods that are agnostic to the phenotypic outcomes, InPheRNo-ChIP directly incorporates phenotypic information during GRN inference, enabling the distinction between lineage-specific and general regulatory interactions. We integrated data from three experimental studies and applied InPheRNo-ChIP to infer the GRN governing the differentiation of human embryonic stem cells into definitive endoderm. Benchmarking against a scRNA-seq CRISPRi study demonstrated InPheRNo-ChIP's ability to identify regulatory interactions involving endoderm markers FOXA2, SMAD2, and SOX17, outperforming other methods. This highlights the importance of incorporating the phenotypic context during network inference. Furthermore, an ablation study confirms the synergistic contribution of ChIP-seq, RNA-seq, and phenotypic data, highlighting the value of multimodal integration for accurate phenotype-relevant GRN reconstruction.
    Keywords:  ChIP-seq; RNA-seq; endoderm formation; phenotype-relevant gene regulatory network
    DOI:  https://doi.org/10.1093/bib/bbae592
  11. Mol Cell. 2024 Nov 13. pii: S1097-2765(24)00870-0. [Epub ahead of print]
      Transcription-coupled nucleotide excision repair (TC-NER) efficiently eliminates DNA damage that impedes gene transcription by RNA polymerase II (RNA Pol II). TC-NER is initiated by the recognition of lesion-stalled RNA Pol II by CSB, which recruits the CRL4CSA ubiquitin ligase and UVSSA. RNA Pol II ubiquitylation at RPB1-K1268 by CRL4CSA serves as a critical TC-NER checkpoint, governing RNA Pol II stability and initiating DNA damage excision by TFIIH recruitment. However, the precise regulatory mechanisms of CRL4CSA activity and TFIIH recruitment remain elusive. Here, we reveal human serine/threonine-protein kinase 19 (STK19) as a TC-NER factor, which is essential for correct DNA damage removal and subsequent transcription restart. Cryogenic electron microscopy (cryo-EM) studies demonstrate that STK19 is an integral part of the RNA Pol II-TC-NER complex, bridging CSA, UVSSA, RNA Pol II, and downstream DNA. STK19 stimulates TC-NER complex stability and CRL4CSA activity, resulting in efficient RNA Pol II ubiquitylation and correct UVSSA and TFIIH binding. These findings underscore the crucial role of STK19 as a core TC-NER component.
    Keywords:  CSA; CSB; DNA damage; DNA repair; RNA polymerase II; STK19; TC-NER; TFIIH; UVSSA; transcription-coupled nucleotide excision repair
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.030
  12. Proc Natl Acad Sci U S A. 2024 Nov 19. 121(47): e2410261121
      Early embryos undergo profound changes in their genomic architecture to establish the totipotent state, enabling pioneer factors to access chromatin and drive zygotic genome activation (ZGA). However, the mechanisms by which the totipotent state is established and properly interpreted by pioneer factors to allow orderly ZGA remain unknown. Here, we identify the H3.3-specific chaperone HIRA as a factor involving establishing totipotent-state chromatin in Drosophila early embryos. Through cophase separation with HIRA, the pioneer factor GAGA factor (GAF) efficiently binds to H3.3-marked nucleosomes to activate major-wave zygotic genes. Importantly, dPCIF1, a chromatin-associated protein, antagonized the GAF-HIRA interaction by competitively binding to HIRA, thereby restricting GAF on earlier chromatin and avoiding premature ZGA. Hence, the coordinated action of HIRA and dPCIF1 ensures sequential ZGA from the minor to major wave in early embryos. This study provides insights into understanding how a totipotent state is established and properly controlled during ZGA.
    Keywords:  Drosophila; early embryo; pioneer factor; zygotic genome activation
    DOI:  https://doi.org/10.1073/pnas.2410261121
  13. EMBO J. 2024 Nov 14.
      Transcription factors (TFs) orchestrating lineage-development often control genes required for cellular survival. However, it is not well understood how cells survive when such TFs are lost, for example in cancer. PU.1 is an essential TF for myeloid fate, and mice with downregulated PU.1 levels develop acute myeloid leukemia (AML). Combining a multi-omics approach with a functional genetic screen, we reveal that PU.1-downregulated cells fundamentally change their survival control from cytokine-driven pathways to overexpression of an autophagy-predominated stem cell gene program, for which we also find evidence in human AML. Control of this program involves redirected chromatin occupancy of the PU.1 partner TF Runx1 to a lineage-inappropriate binding site repertoire. Hence, genomic reallocation of TF binding upon loss of a partner TF can act as a pro-oncogenic failsafe mechanism by sustaining cell survival during leukemogenesis.
    Keywords:  Myeloid Development; Myeloid Leukemia; PU.1; RUNX1
    DOI:  https://doi.org/10.1038/s44318-024-00295-y
  14. Nat Commun. 2024 Nov 09. 15(1): 9699
      Nervous system cancers exhibit diverse transcriptional cell states influenced by normal development, injury response, and growth. However, the understanding of these states' regulation and pharmacological relevance remains limited. Here we present "single-cell regulatory-driven clustering" (scregclust), a method that reconstructs cellular regulatory programs from extensive collections of single-cell RNA sequencing (scRNA-seq) data from both tumors and developing tissues. The algorithm efficiently divides target genes into modules, predicting key transcription factors and kinases with minimal computational time. Applying this method to adult and childhood brain cancers, we identify critical regulators and suggest interventions that could improve temozolomide treatment in glioblastoma. Additionally, our integrative analysis reveals a meta-module regulated by SPI1 and IRF8 linked to an immune-mediated mesenchymal-like state. Finally, scregclust's flexibility is demonstrated across 15 tumor types, uncovering both pan-cancer and specific regulators. The algorithm is provided as an easy-to-use R package that facilitates the exploration of regulatory programs underlying cell plasticity.
    DOI:  https://doi.org/10.1038/s41467-024-53954-3
  15. Dev Cell. 2024 Nov 09. pii: S1534-5807(24)00636-1. [Epub ahead of print]
      Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes calls for mechanisms to limit inappropriate regulation. DNA elements called insulators serve this purpose by shielding promoters from an enhancer when interposed. Unlike promoters and enhancers, insulators have not been systematically characterized due to lacking high-throughput screening assays, and questions regarding how insulators are distributed and encoded in the genome remain. Here, we establish "insulator-seq" as a plasmid-based massively parallel reporter assay in Drosophila cultured cells to perform a systematic insulator screen of selected genomic loci. Screening developmental gene loci showed that not all insulator protein binding sites effectively block enhancer-promoter communication. Deep insulator mutagenesis identified sequences flexibly positioned around the CTCF insulator protein binding motif that are critical for functionality. The ability to screen millions of DNA sequences without positional effect has enabled functional mapping of insulators and provided further insights into the determinants of insulators.
    Keywords:  CTCF; Drosophila; TAD; enhancer-blocking; high-throughput screen; insulator; massively parallel reporter assay
    DOI:  https://doi.org/10.1016/j.devcel.2024.10.017
  16. J Genet Genomics. 2024 Nov 08. pii: S1673-8527(24)00286-8. [Epub ahead of print]
      In the mammalian genome, most CpGs are methylated. However, CpGs within the CpG islands (CGIs) are largely unmethylated, which are important for gene expression regulation. The mechanism underlying the low methylation levels at CGIs remains largely elusive. KDM2 proteins (KDM2A and KDM2B) are H3K36me2 demethylases known to bind specifically at CGIs. Here, we report that depletion of each or both KDM2 proteins, or mutation of all their JmjC domains that harbor the H3K36me2 demethylation activity, leads to an increase in DNA methylation at selective CGIs. The Kdm2a/2b double knockout shows a stronger increase in DNA methylation compared to the single mutant of Kdm2a or Kdm2b, indicating that KDM2A and KDM2B redundantly regulate DNA methylation at CGIs. In addition, the increase of CGI DNA methylation upon mutations of KDM2 proteins is associated with the chromatin environment. Our findings reveal that KDM2A and KDM2B function redundantly in regulating DNA methylation at a subset of CGIs in an H3K36me2 demethylation-dependent manner.
    Keywords:  CpG island; DNA methylation; Demethylation; Embryonic stem cell; H3K36me2; KDM2A; KDM2B
    DOI:  https://doi.org/10.1016/j.jgg.2024.10.012
  17. Cell Syst. 2024 Nov 06. pii: S2405-4712(24)00303-X. [Epub ahead of print]
      Chromatin states play important roles in the maintenance of cell identities, yet their spatial patterns remain poorly characterized at the organism scale. We developed a systematic approach to analyzing spatial epigenomic data and then applied it to a recently published spatial-CUT&Tag dataset that was obtained from a mouse embryo. We identified a set of spatial genes whose H3K4me3 patterns delineate tissue boundaries. These genes are enriched with tissue-specific transcription factors, and their corresponding genomic loci are marked by broad H3K4me3 domains. Integrative analysis with H3K27me3 profiles showed coordinated spatial transitions across tissue boundaries, which is marked by the continuous shortening of H3K4me3 domains and expansion of H3K27me3 domains. Motif-based analysis identified transcription factors whose activities change significantly during such transitions. Taken together, our systematic analyses reveal a strong connection between the genomic and spatial variations of chromatin states. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  CUT&Tag; chromatin state; computational biology; development; epigenomics; spatial biology
    DOI:  https://doi.org/10.1016/j.cels.2024.10.006
  18. Nat Commun. 2024 Nov 14. 15(1): 9861
      Functional divergence of transcription factors (TFs) has driven cellular and organismal complexity throughout evolution, but its mechanistic drivers remain poorly understood. Here we test for new mechanisms using CORONA (CNA) and PHABULOSA (PHB), two functionally diverged paralogs in the CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) family of TFs. We show that virtually all genes bound by PHB ( ~ 99%) are also bound by CNA, ruling out occupation of distinct sets of genes as a mechanism of functional divergence. Further, genes bound and regulated by both paralogs are almost always regulated in the same direction, ruling out opposite regulation of shared targets as a mechanistic driver. Functional divergence of CNA and PHB instead results from differential usage of shared binding sites, with hundreds of uniquely regulated genes emerging from a commonly bound genetic network. Regulation of a given gene by CNA or PHB is thus a function of whether a bound site is considered 'responsive' versus 'non-responsive' by each paralog. Discrimination between responsive and non-responsive sites is controlled, at least in part, by their lipid binding START domain. This suggests a model in which HD-ZIPIII TFs use information integrated by their START domain to generate paralog-specific transcriptional outcomes from a shared network architecture. Taken together, our study identifies a mechanism of HD-ZIPIII TF paralog divergence and proposes the ubiquitously distributed START evolutionary module as a driver of functional divergence.
    DOI:  https://doi.org/10.1038/s41467-024-54269-z
  19. Cell. 2024 Nov 12. pii: S0092-8674(24)01202-9. [Epub ahead of print]
      In transcription-coupled nucleotide excision repair (TC-NER), stalled RNA polymerase II (RNA Pol II) binds CSB and CRL4CSA, which cooperate with UVSSA and ELOF1 to recruit TFIIH. To explore the mechanism of TC-NER, we recapitulated this reaction in vitro. When a plasmid containing a site-specific lesion is transcribed in frog egg extract, error-free repair is observed that depends on CSB, CRL4CSA, UVSSA, and ELOF1. Repair also requires STK19, a factor previously implicated in transcription recovery after UV exposure. A 1.9-Å cryo-electron microscopy structure shows that STK19 binds the TC-NER complex through CSA and the RPB1 subunit of RNA Pol II. Furthermore, AlphaFold predicts that STK19 interacts with the XPD subunit of TFIIH, and disrupting this interface impairs cell-free repair. Molecular modeling suggests that STK19 positions TFIIH ahead of RNA Pol II for lesion verification. Our analysis of cell-free TC-NER suggests that STK19 couples RNA Pol II stalling to downstream repair events.
    Keywords:  AlphaFold; NER; STK19; TFIIH; cryo-EM; nucleotide excision repair; transcription; transcription-coupled DNA repair
    DOI:  https://doi.org/10.1016/j.cell.2024.10.020
  20. Nature. 2024 Nov 13.
      Epigenetic inheritance of silent chromatin domains is fundamental to cellular memory during embryogenesis, but it must overcome the dilution of repressive histone modifications during DNA replication1. One such modification, histone H2A lysine 119 monoubiquitination (H2AK119Ub), needs to be re-established by the Polycomb repressive complex 1 (PRC1) E3 ligase to restore the silent Polycomb domain2,3. However, the exact mechanism behind this restoration remains unknown. Here, combining cryo-electron microscopy (cryo-EM) and functional approaches, we characterize the read-write mechanism of the non-canonical PRC1-containing RYBP (ncPRC1RYBP). This mechanism, which functions as a positive-feedback loop in epigenetic regulation4,5, emphasizes the pivotal role of ncPRC1RYBP in restoring H2AK119Ub. We observe an asymmetrical binding of ncPRC1RYBP to H2AK119Ub nucleosomes, guided in part by the N-terminal zinc-finger domain of RYBP binding to residual H2AK119Ub on nascent chromatin. This recognition positions the RING domains of RING1B and BMI1 on the unmodified nucleosome side, enabling recruitment of the E2 enzyme to ubiquitinate H2AK119 within the same nucleosome (intra-nucleosome read-write) or across nucleosomes (inter-nucleosome read-write). Collectively, our findings provide key structural and mechanistic insights into the dynamic interplay of epigenetic regulation, highlighting the significance of ncPRC1RYBP in H2AK119Ub restoration to sustain repressive chromatin domains.
    DOI:  https://doi.org/10.1038/s41586-024-08183-5
  21. Nat Commun. 2024 Nov 15. 15(1): 9909
      Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to meet the high energy and biosynthetic demands required for their proliferation. High mobility group A1 (HMGA1) is a structural transcription factor and frequently overexpressed in human colorectal cancer (CRC). Here, we show that HMGA1 promotes CRC progression by driving lipid synthesis in a AOM/DSS-induced CRC mouse model. Using conditional knockout (Hmga1△IEC) and knock-in (Hmga1IEC-OE/+) mouse models, we demonstrate that HMGA1 enhances CRC cell proliferation and accelerates tumor development by upregulating fatty acid synthase (FASN). Mechanistically, HMGA1 increases the transcriptional activity of sterol regulatory element-binding protein 1 (SREBP1) on the FASN promoter, leading to increased lipid accumulation in intestinal epithelial cells. Moreover, a high-fat diet exacerbates CRC progression in Hmga1△IEC mice, while pharmacological inhibition of FASN by orlistat reduces tumor growth in Hmga1IEC-OE/+ mice. Our findings suggest that targeting lipid metabolism could offer a promising therapeutic strategy for CRC.
    DOI:  https://doi.org/10.1038/s41467-024-54400-0
  22. Commun Biol. 2024 Nov 10. 7(1): 1483
      LSD1 plays a crucial role in mammalian biology, regulated through interactions with coregulators and post-translational modifications. Here we show that the kinase NEK6 stimulates LSD1 activity in cells and observe a strong colocalization of NEK6 and LSD1 at distinct chromatin sub-compartments (CSCs). We demonstrate that LSD1 is a substrate for NEK6 phosphorylation at the N-terminal intrinsically disordered region (IDR) of LSD1, which shows phase separation behavior in vitro and in cells. The LSD1-IDR is important for LSD1 activity and functions to co-compartmentalize NEK6, histone peptides and DNA. The subsequent phosphorylation of LSD1 by NEK6 supports the concentration of LSD1 at these distinct CSCs, which is imperative for dynamic control of transcription. This suggest that phase separation is crucial for the regulatory function of LSD1 and our findings highlight the role of NEK6 in modulating LSD1 activity and phase separation, expanding our understanding of LSD1 regulation and its implications in cellular processes.
    DOI:  https://doi.org/10.1038/s42003-024-07199-x
  23. Cell Stem Cell. 2024 Nov 08. pii: S1934-5909(24)00374-6. [Epub ahead of print]
      Gene therapy using hematopoietic stem and progenitor cells is altering the therapeutic landscape for patients with hematologic, immunologic, and metabolic disorders but has not yet been successfully developed for individuals with the bone marrow failure syndrome Diamond-Blackfan anemia (DBA). More than 30 mutations cause DBA through impaired ribosome function and lead to inefficient translation of the erythroid master regulator GATA1, providing a potential avenue for therapeutic intervention applicable to all patients with DBA, irrespective of the underlying genotype. Here, we report the development of a clinical-grade lentiviral gene therapy that achieves erythroid lineage-restricted expression of GATA1. We show that this vector is capable of augmenting erythropoiesis in DBA models and diverse patient samples without impacting hematopoietic stem cell function or demonstrating any signs of premalignant clonal expansion. These preclinical safety and efficacy data provide strong support for the first-in-human universal gene therapy trial for DBA through regulated GATA1 expression.
    Keywords:  Diamond-Blackfan anemia; GATA1; bone marrow failure; enhancer; erythropoiesis; gene therapy; hematopoiesis; hematopoietic stem cell; hypoplastic anemia; lentivirus
    DOI:  https://doi.org/10.1016/j.stem.2024.10.012
  24. EMBO Rep. 2024 Nov 07.
      Transposable elements (TEs) are repressed in plants through transcriptional gene silencing (TGS), maintained epigenetic silencing marks such as DNA methylation. However, the mechanisms by which silencing is first installed remain poorly understood in plants. Small interfering (si)RNAs and post-transcriptional gene silencing (PTGS) are believed to mediate the initiation of TGS by guiding the first deposition of DNA methylation. To determine how this silencing installation works, we took advantage of ÉVADÉ (EVD), an endogenous retroelement in Arabidopsis, able to recapitulate true de novo silencing with a sequence of PTGS followed by a TGS. To test whether PTGS is required for TGS, we introduce active EVD into RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6) mutants, an essential PTGS component. EVD activity and silencing are monitored across several generations. In the absence of PTGS, silencing of EVD is still achieved through installation of RNA-directed DNA methylation (RdDM). Our study shows that PTGS is dispensable for de novo EVD silencing. Although we cannot rule out that PTGS might facilitate TGS, or control TE activity, initiation of epigenetic silencing can take place in its absence.
    Keywords:  Epigenetics; Plants; Silencing; Transposons; siRNAs
    DOI:  https://doi.org/10.1038/s44319-024-00304-5
  25. Nucleic Acids Res. 2024 Nov 14. pii: gkae1053. [Epub ahead of print]
      SETDB1 (SET domain bifurcated histone lysine methyltransferase 1) is a major protein lysine methyltransferase trimethylating lysine 9 on histone H3 (H3K9) which is involved in heterochromatin formation and silencing of repeat elements (REs). It contains a unique Triple Tudor Domain (3TD), which specifically binds the dual modification of H3K14ac in the presence of H3K9me1/2/3. Here, we explored the role of the 3TD H3-tail interaction for the H3K9 methylation activity of SETDB1. We generated a binding reduced 3TD mutant and demonstrate in biochemical methylation assays on peptides and recombinant nucleosomes containing H3K14ac and H3K14ac analogs, respectively, that H3K14 acetylation is crucial for the 3TD mediated recruitment of SETDB1. We also observe this effect in cells where SETDB1 binding and activity is globally correlated with H3K14ac, and knockout of the H3K14 acetyltransferase HBO1 causes a drastic reduction in H3K9me3 levels at SETDB1 dependent sites. Regions with DNA hypomethylation after SETDB1 knockout also show an enrichment in SETDB1-dependent H3K9me3 and H3K14ac. Further analyses revealed that 3TD is particularly important at specific target regions like L1M REs, where H3K9me3 cannot be efficiently reconstituted by the 3TD mutant of SETDB1. In summary, our data demonstrate that the H3K9me3 and H3K14ac are not antagonistic marks but rather the presence of H3K14ac is required for SETDB1 recruitment via 3TD binding to H3K9me1/2/3-K14ac regions and establishment of H3K9me3.
    DOI:  https://doi.org/10.1093/nar/gkae1053
  26. Cell Rep. 2024 Nov 06. pii: S2211-1247(24)01315-9. [Epub ahead of print]43(11): 114964
      Inactivating mutations in chromatin modifiers, like the α-thalassemia/mental retardation, X-linked (ATRX)-death domain-associated protein (DAXX) chromatin remodeling/histone H3.3 deposition complex, drive the cancer-specific alternative lengthening of telomeres (ALT) pathway. Prior studies revealed that HIRA, another histone H3.3 chaperone, compensates for ATRX-DAXX loss at telomeres to sustain ALT cancer cell survival. How HIRA rescues telomeres from the consequences of ATRX-DAXX deficiency remains unclear. Here, using an assay for transposase-accessible chromatin using sequencing (ATAC-seq) and cleavage under targets and release using nuclease (CUT&RUN), we establish that HIRA-mediated deposition of new H3.3 maintains telomeric chromatin accessibility to prevent the detrimental accumulation of nucleosome-free single-stranded DNA (ssDNA) in ATRX-DAXX-deficient ALT cells. We show that the HIRA-UBN1/UBN2 complex deposits new H3.3 to prevent TERRA R-loop buildup and transcription-replication conflicts (TRCs) at telomeres. Furthermore, HIRA-mediated H3.3 incorporation into telomeric chromatin links productive ALT to the phosphorylation of serine 31, an H3.3-specific amino acid, by Chk1. Therefore, we identify a critical role for HIRA-mediated H3.3 deposition that ensures the survival of ATRX-DAXX-deficient ALT cancer cells.
    Keywords:  ALT; CP: Cancer; CP: Molecular biology; HIRA; R-loop; cancer; histone; telomere
    DOI:  https://doi.org/10.1016/j.celrep.2024.114964
  27. PNAS Nexus. 2024 Nov;3(11): pgae484
      Nucleosome remodelers modify the local structure of chromatin to release the region from nucleosome-mediated transcriptional suppression. Overlapping dinucleosomes (OLDNs) are nucleoprotein complexes formed around transcription start sites as a result of remodeling, and they consist of two nucleosome moieties: a histone octamer wrapped by DNA (octasome) and a histone hexamer wrapped by DNA (hexasome). While OLDN formation alters chromatin accessibility to proteins, the structural mechanism behind this process is poorly understood. Thus, this study investigated the characteristics of structural fluctuations in OLDNs. First, multiple structures of the OLDN were visualized through cryoelectron microscopy (cryoEM), providing an overview of the tilting motion of the hexasome relative to the octasome at the near-atomistic resolution. Second, small-angle X-ray scattering (SAXS) revealed the presence of OLDN conformations with a larger radius of gyration than cryoEM structures. A more complete description of OLDN fluctuation was proposed by SAXS-based ensemble modeling, which included possible transient structures. The ensemble model supported the tilting motion of the OLDN outlined by the cryoEM models, further suggesting the presence of more diverse conformations. The amplitude of the relative tilting motion of the hexasome was larger, and the nanoscale fluctuation in distance between the octasome and hexasome was also proposed. The cryoEM models were found to be mapped in the energetically stable region of the conformational distribution of the ensemble. Exhaustive complex modeling using all conformations that appeared in the structural ensemble suggested that conformational and motional asymmetries of the OLDN result in asymmetries in the accessibility of OLDN-binding proteins.
    Keywords:  chromatin; chromatin remodeler; ensemble modeling; molecular dynamics simulation
    DOI:  https://doi.org/10.1093/pnasnexus/pgae484
  28. Mol Cell. 2024 Nov 05. pii: S1097-2765(24)00863-3. [Epub ahead of print]
      Long-term perturbation of de novo chromatin assembly during DNA replication has profound effects on epigenome maintenance and cell fate. The early mechanistic origin of these defects is unknown. Here, we combine acute degradation of chromatin assembly factor 1 (CAF-1), a key player in de novo chromatin assembly, with single-cell genomics, quantitative proteomics, and live microscopy to uncover these initiating mechanisms in human cells. CAF-1 loss immediately slows down DNA replication speed and renders nascent DNA hyper-accessible. A rapid cellular response, distinct from canonical DNA damage signaling, is triggered and lowers histone mRNAs. In turn, histone variants' usage and their modifications are altered, limiting transcriptional fidelity and delaying chromatin maturation within a single S-phase. This multi-level response induces a p53-dependent cell-cycle arrest after mitosis. Our work reveals the immediate consequences of defective de novo chromatin assembly during DNA replication, indicating how at later times the epigenome and cell fate can be altered.
    Keywords:  DNA replication; PCNA; SILAC proteomics; cell cycle; chromatin assembly; epigenome stability; histone chaperones; histone mRNA; single-cell sequencing
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.023
  29. Nat Cell Biol. 2024 Nov 08.
      As tissues develop, cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. Here, to determine the role of cell division in differentiation, we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single-cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues from early gastrulation to the end of segmentation. However, arresting cell division does slow down differentiation in some cell types, and it induces global stress responses. While differentiation is robust to blocking cell division, the proportions of cells across cell states are not, but show evidence of partial compensation. This work clarifies our understanding of the role of cell division in development and showcases the utility of combining embryo-wide perturbations with single-cell RNA sequencing to uncover the role of common biological processes across multiple tissues.
    DOI:  https://doi.org/10.1038/s41556-024-01546-0
  30. Cell. 2024 Nov 11. pii: S0092-8674(24)01200-5. [Epub ahead of print]
      Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and transcription factor IIH (TFIIH) around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryoelectron microscopy (cryo-EM) and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.
    Keywords:  CSA; CSB; DNA repair; ELOF1; RNA polymerase II; STK19; TFIIH; UVSSA; nucleotide excision repair; transcription
    DOI:  https://doi.org/10.1016/j.cell.2024.10.018
  31. Nucleic Acids Res. 2024 Nov 12. pii: gkae1081. [Epub ahead of print]
      microRNAs (miRNAs) are active in various biological processes by mediating gene expression, and the full investigation of miRNA transcription is crucial for understanding the mechanisms underlying miRNA deregulation in pathological conditions. Here an updated TransmiR v3.0 database is presented with more comprehensive miRNA transcription regulation information, which contains 5095 transcription factor (TF) -miRNA regulations curated from 2285 papers and >6 million TF-miRNA regulations derived from ChIP-seq data. Currently, TransmiR v3.0 covers 3260 TFs, 4253 miRNAs and 514 433 TF-miRNA regulation pairs across 29 organisms. Additionally, motif scanning of TF loci on promoter sequences of miRNAs from multiple species is employed to predict TF-miRNA regulations, generating 284 527 predicted TF-miRNA regulations. Besides the significant growth of data volume, we also improve the annotations for TFs and miRNAs by introducing the TF family, TFBS motif, and expression profiles for several species. Moreover, the functionality of the TransmiR v3.0 online database is enhanced, including allowing batch search for flexible queries and offering more extensive disease-specific, as well as newly sex-specific TF-miRNA regulation networks in the 'Network' module. TransmiR v3.0 provides a useful resource for studying miRNA biogenesis regulation and can be freely accessed at http://www.cuilab.cn/transmir.
    DOI:  https://doi.org/10.1093/nar/gkae1081