bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2023‒04‒09
25 papers selected by
Connor Rogerson
University of Cambridge
  1. Acetylation of histone H2B marks active enhancers and predicts CBP/p300 target genes.
  2. Dynamic antagonism between key repressive pathways maintains the placental epigenome.
  3. Connectome and regulatory hubs of CAGE highly active enhancers.
  4. Structural and functional properties of mSWI/SNF chromatin remodeling complexes revealed through single-cell perturbation screens.
  5. Esearch3D: propagating gene expression in chromatin networks to illuminate active enhancers.
  6. Dual functions of TET1 in germ layer lineage bifurcation distinguished by genomic context and dependence on 5-methylcytosine oxidation.
  7. Enhancer-promoter contact formation requires RNAPII and antagonizes loop extrusion.
  8. Precise modulation of transcription factor levels identifies features underlying dosage sensitivity.
  9. Cellcano: supervised cell type identification for single cell ATAC-seq data.
  10. RBBP4 is an epigenetic barrier for the induced transition of pluripotent stem cells into totipotent 2C-like cells.
  11. Transfer learning identifies sequence determinants of cell-type specific regulatory element accessibility.
  12. Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns.
  13. Large-scale mapping and mutagenesis of human transcriptional effector domains.
  14. MLL3/MLL4 methyltransferase activities control early embryonic development and embryonic stem cell differentiation in a lineage-selective manner.
  15. mRNA recognition and packaging by the human transcription-export complex.
  16. Defining the fine structure of promoter activity on a genome-wide scale with CISSECTOR.
  17. GAGA-associated factor fosters loop formation in the Drosophila genome.
  18. Cohesin-independent STAG proteins interact with RNA and R-loops and promote complex loading.
  19. Yeast PIC-Mediator structure with RNA polymerase II C-terminal domain.
  20. eccDB: a comprehensive repository for eccDNA-mediated chromatin contacts in multi-species.
  21. Developmentally programmed histone H3 expression regulates cellular plasticity at the parental-to-early embryo transition.
  22. Condensed but liquid-like domain organization of active chromatin regions in living human cells.
  23. ETV4 mediates dosage-dependent prostate tumor initiation and cooperates with p53 loss to generate prostate cancer.
  24. Hypoxia switches TET1 from being tumor-suppressive to oncogenic.
  25. An epigenetic switch controls an alternative NR2F2 isoform that unleashes a metastatic program in melanoma.
  1. Nat Genet. 2023 Apr 06.
    Acetylation of histone H2B marks active enhancers and predicts CBP/p300 target genes.
    Takeo Narita, Yoshiki Higashijima, Sinan Kilic, Tim Liebner, Jonas Walter, Chunaram Choudhary.
      Chromatin features are widely used for genome-scale mapping of enhancers. However, discriminating active enhancers from other cis-regulatory elements, predicting enhancer strength and identifying their target genes is challenging. Here we establish histone H2B N-terminus multisite lysine acetylation (H2BNTac) as a signature of active enhancers. H2BNTac prominently marks candidate active enhancers and a subset of promoters and discriminates them from ubiquitously active promoters. Two mechanisms underlie the distinct H2BNTac specificity: (1) unlike H3K27ac, H2BNTac is specifically catalyzed by CBP/p300; (2) H2A-H2B, but not H3-H4, are rapidly exchanged through transcription-induced nucleosome remodeling. H2BNTac-positive candidate enhancers show a high validation rate in orthogonal enhancer activity assays and a vast majority of endogenously active enhancers are marked by H2BNTac and H3K27ac. Notably, H2BNTac intensity predicts enhancer strength and outperforms current state-of-the-art models in predicting CBP/p300 target genes. These findings have broad implications for generating fine-grained enhancer maps and modeling CBP/p300-dependent gene regulation.
    DOI:  https://doi.org/10.1038/s41588-023-01348-4
  2. Nat Cell Biol. 2023 Apr 06.
    Dynamic antagonism between key repressive pathways maintains the placental epigenome.
    Raha Weigert, Sara Hetzel, Nina Bailly, Chuck Haggerty, Ibrahim A Ilik, Philip Yuk Kwong Yung, Carmen Navarro, Adriano Bolondi, Abhishek Sampath Kumar, Chiara Anania, Björn Brändl, David Meierhofer, Darío G Lupiáñez, Franz-Josef Müller, Tugce Aktas, Simon J Elsässer, Helene Kretzmer, Zachary D Smith, Alexander Meissner.
      DNA and Histone 3 Lysine 27 methylation typically function as repressive modifications and operate within distinct genomic compartments. In mammals, the majority of the genome is kept in a DNA methylated state, whereas the Polycomb repressive complexes regulate the unmethylated CpG-rich promoters of developmental genes. In contrast to this general framework, the extra-embryonic lineages display non-canonical, globally intermediate DNA methylation levels, including disruption of local Polycomb domains. Here, to better understand this unusual landscape's molecular properties, we genetically and chemically perturbed major epigenetic pathways in mouse trophoblast stem cells. We find that the extra-embryonic epigenome reflects ongoing and dynamic de novo methyltransferase recruitment, which is continuously antagonized by Polycomb to maintain intermediate, locally disordered methylation. Despite its disorganized molecular appearance, our data point to a highly controlled equilibrium between counteracting repressors within extra-embryonic cells, one that can seemingly persist indefinitely without bistable features typically seen for embryonic forms of epigenetic regulation.
    DOI:  https://doi.org/10.1038/s41556-023-01114-y
  3. Sci Rep. 2023 Apr 05. 13(1): 5594
    Connectome and regulatory hubs of CAGE highly active enhancers.
    Mewen Briend, Anne Rufiange, Louis-Hippolyte Minvielle Moncla, Samuel Mathieu, Yohan Bossé, Patrick Mathieu.
      Evidence indicates that enhancers are transcriptionally active. Herein, we investigated transcriptionally active enhancers by using cap analysis of gene expression (CAGE) combined with epigenetic marks and chromatin interactions. We identified CAGE-tag highly active (CHA) enhancers as distant regulatory elements with CAGE-tag ≥ 90th percentile and overlapping with H3K27ac peaks (4.5% of enhancers). CHA enhancers were conserved between mouse and man and were independent from super-enhancers in predicting cell identity with lower P-values. CHA enhancers had increased open chromatin and a higher recruitment of cell-specific transcription factors as well as molecules involved in 3D genome interactions. HiChIP analysis of enhancer-promoter looping indicated that CHA enhancers had a higher density of anchor loops when compared to regular enhancers. A subset of CHA enhancers and promoters characterized by a high density of chromatin loops and forming hub regulatory units were connected to the promoter of immediate early response genes, genes involved in cancer and encoding for transcription factors. Promoter of genes within hub CHA regulatory units were less likely to be paused. CHA enhancers were enriched in gene variants associated with autoimmune disorders and had looping with causal candidate genes as revealed by Mendelian randomization. Hence, CHA enhancers form a dense hierarchical network of chromatin interactions between regulatory elements and genes involved in cell identity and disorders.
    DOI:  https://doi.org/10.1038/s41598-023-32669-3
  4. Mol Cell. 2023 Apr 01. pii: S1097-2765(23)00203-4. [Epub ahead of print]
    Structural and functional properties of mSWI/SNF chromatin remodeling complexes revealed through single-cell perturbation screens.
    Jordan E Otto, Oana Ursu, Alexander P Wu, Evan B Winter, Michael S Cuoco, Sai Ma, Kristin Qian, Brittany C Michel, Jason D Buenrostro, Bonnie Berger, Aviv Regev, Cigall Kadoch.
      The mammalian SWI/SNF (mSWI/SNF or BAF) family of chromatin remodeling complexes play critical roles in regulating DNA accessibility and gene expression. The three final-form subcomplexes-cBAF, PBAF, and ncBAF-are distinct in biochemical componentry, chromatin targeting, and roles in disease; however, the contributions of their constituent subunits to gene expression remain incompletely defined. Here, we performed Perturb-seq-based CRISPR-Cas9 knockout screens targeting mSWI/SNF subunits individually and in select combinations, followed by single-cell RNA-seq and SHARE-seq. We uncovered complex-, module-, and subunit-specific contributions to distinct regulatory networks and defined paralog subunit relationships and shifted subcomplex functions upon perturbations. Synergistic, intra-complex genetic interactions between subunits reveal functional redundancy and modularity. Importantly, single-cell subunit perturbation signatures mapped across bulk primary human tumor expression profiles both mirror and predict cBAF loss-of-function status in cancer. Our findings highlight the utility of Perturb-seq to dissect disease-relevant gene regulatory impacts of heterogeneous, multi-component master regulatory complexes.
    Keywords:  ATAC-seq; ATP-dependent chromatin remodeling; BAF complex; Perturb-seq; SHARE-Seq; cancer; chromatin accessibility; gene expression; mammalian SWI/SNF complexes; pediatric cancer; rare diseases
    DOI:  https://doi.org/10.1016/j.molcel.2023.03.013
  5. Nucleic Acids Res. 2023 Apr 06. pii: gkad229. [Epub ahead of print]
    Esearch3D: propagating gene expression in chromatin networks to illuminate active enhancers.
    Maninder Heer, Luca Giudice, Claudia Mengoni, Rosalba Giugno, Daniel Rico.
      Most cell type-specific genes are regulated by the interaction of enhancers with their promoters. The identification of enhancers is not trivial as enhancers are diverse in their characteristics and dynamic in their interaction partners. We present Esearch3D, a new method that exploits network theory approaches to identify active enhancers. Our work is based on the fact that enhancers act as a source of regulatory information to increase the rate of transcription of their target genes and that the flow of this information is mediated by the folding of chromatin in the three-dimensional (3D) nuclear space between the enhancer and the target gene promoter. Esearch3D reverse engineers this flow of information to calculate the likelihood of enhancer activity in intergenic regions by propagating the transcription levels of genes across 3D genome networks. Regions predicted to have high enhancer activity are shown to be enriched in annotations indicative of enhancer activity. These include: enhancer-associated histone marks, bidirectional CAGE-seq, STARR-seq, P300, RNA polymerase II and expression quantitative trait loci (eQTLs). Esearch3D leverages the relationship between chromatin architecture and transcription, allowing the prediction of active enhancers and an understanding of the complex underpinnings of regulatory networks. The method is available at: https://github.com/InfOmics/Esearch3D and https://doi.org/10.5281/zenodo.7737123.
    DOI:  https://doi.org/10.1093/nar/gkad229
  6. Nucleic Acids Res. 2023 Apr 06. pii: gkad231. [Epub ahead of print]
    Dual functions of TET1 in germ layer lineage bifurcation distinguished by genomic context and dependence on 5-methylcytosine oxidation.
    Bernard K van der Veer, Lehua Chen, Colin Custers, Paraskevi Athanasouli, Mariana Schroiff, Riet Cornelis, Jonathan Sai-Hong Chui, Richard H Finnell, Frederic Lluis, Kian Peng Koh.
      Gastrulation begins when the epiblast forms the primitive streak or becomes definitive ectoderm. During this lineage bifurcation, the DNA dioxygenase TET1 has bipartite functions in transcriptional activation and repression, but the mechanisms remain unclear. By converting mouse embryonic stem cells (ESCs) into neuroprogenitors, we defined how Tet1-/- cells switch from neuroectoderm fate to form mesoderm and endoderm. We identified the Wnt repressor Tcf7l1 as a TET1 target that suppresses Wnt/β-catenin and Nodal signalling. ESCs expressing catalytic dead TET1 retain neural potential but activate Nodal and subsequently Wnt/β-catenin pathways to generate also mesoderm and endoderm. At CpG-poor distal enhancers, TET1 maintains accessible chromatin at neuroectodermal loci independently of DNA demethylation. At CpG-rich promoters, DNA demethylation by TET1 affects the expression of bivalent genes. In ESCs, a non-catalytic TET1 cooperation with Polycomb represses primitive streak genes; post-lineage priming, the interaction becomes antagonistic at neuronal genes, when TET1's catalytic activity is further involved by repressing Wnt signalling. The convergence of repressive DNA and histone methylation does not inhibit neural induction in Tet1-deficient cells, but some DNA hypermethylated loci persist at genes with brain-specific functions. Our results reveal versatile switching of non-catalytic and catalytic TET1 activities based on genomic context, lineage and developmental stage.
    DOI:  https://doi.org/10.1093/nar/gkad231
  7. Nat Genet. 2023 Apr 03.
    Enhancer-promoter contact formation requires RNAPII and antagonizes loop extrusion.
    Shu Zhang, Nadine Übelmesser, Mariano Barbieri, Argyris Papantonis.
      Homotypic chromatin interactions and loop extrusion are thought to be the two main drivers of mammalian chromosome folding. Here we tested the role of RNA polymerase II (RNAPII) across different scales of interphase chromatin organization in a cellular system allowing for its rapid, auxin-mediated degradation. We combined Micro-C and computational modeling to characterize subsets of loops differentially gained or lost upon RNAPII depletion. Gained loops, extrusion of which was antagonized by RNAPII, almost invariably formed by engaging new or rewired CTCF anchors. Lost loops selectively affected contacts between enhancers and promoters anchored by RNAPII, explaining the repression of most genes. Surprisingly, promoter-promoter interactions remained essentially unaffected by polymerase depletion, and cohesin occupancy was sustained. Together, our findings reconcile the role of RNAPII in transcription with its direct involvement in setting-up regulatory three-dimensional chromatin contacts genome wide, while also revealing an impact on cohesin loop extrusion.
    DOI:  https://doi.org/10.1038/s41588-023-01364-4
  8. Nat Genet. 2023 Apr 06.
    Precise modulation of transcription factor levels identifies features underlying dosage sensitivity.
    Sahin Naqvi, Seungsoo Kim, Hanne Hoskens, Harold S Matthews, Richard A Spritz, Ophir D Klein, Benedikt Hallgrímsson, Tomek Swigut, Peter Claes, Jonathan K Pritchard, Joanna Wysocka.
      Transcriptional regulation exhibits extensive robustness, but human genetics indicates sensitivity to transcription factor (TF) dosage. Reconciling such observations requires quantitative studies of TF dosage effects at trait-relevant ranges, largely lacking so far. TFs play central roles in both normal-range and disease-associated variation in craniofacial morphology; we therefore developed an approach to precisely modulate TF levels in human facial progenitor cells and applied it to SOX9, a TF associated with craniofacial variation and disease (Pierre Robin sequence (PRS)). Most SOX9-dependent regulatory elements (REs) are buffered against small decreases in SOX9 dosage, but REs directly and primarily regulated by SOX9 show heightened sensitivity to SOX9 dosage; these RE responses partially predict gene expression responses. Sensitive REs and genes preferentially affect functional chondrogenesis and PRS-like craniofacial shape variation. We propose that such REs and genes underlie the sensitivity of specific phenotypes to TF dosage, while buffering of other genes leads to robust, nonlinear dosage-to-phenotype relationships.
    DOI:  https://doi.org/10.1038/s41588-023-01366-2
  9. Nat Commun. 2023 Apr 03. 14(1): 1864
    Cellcano: supervised cell type identification for single cell ATAC-seq data.
    Wenjing Ma, Jiaying Lu, Hao Wu.
      Computational cell type identification is a fundamental step in single-cell omics data analysis. Supervised celltyping methods have gained increasing popularity in single-cell RNA-seq data because of the superior performance and the availability of high-quality reference datasets. Recent technological advances in profiling chromatin accessibility at single-cell resolution (scATAC-seq) have brought new insights to the understanding of epigenetic heterogeneity. With continuous accumulation of scATAC-seq datasets, supervised celltyping method specifically designed for scATAC-seq is in urgent need. Here we develop Cellcano, a computational method based on a two-round supervised learning algorithm to identify cell types from scATAC-seq data. The method alleviates the distributional shift between reference and target data and improves the prediction performance. After systematically benchmarking Cellcano on 50 well-designed celltyping tasks from various datasets, we show that Cellcano is accurate, robust, and computationally efficient. Cellcano is well-documented and freely available at https://marvinquiet.github.io/Cellcano/ .
    DOI:  https://doi.org/10.1038/s41467-023-37439-3
  10. Nucleic Acids Res. 2023 Apr 06. pii: gkad219. [Epub ahead of print]
    RBBP4 is an epigenetic barrier for the induced transition of pluripotent stem cells into totipotent 2C-like cells.
    Wangfang Ping, Yingliang Sheng, Gongcheng Hu, Hongxin Zhong, Yaoyi Li, YanJiang Liu, Wei Luo, Chenghong Yan, Yulin Wen, Xinxiu Wang, Qing Li, Rong Guo, Jie Zhang, Ake Liu, Guangjin Pan, Hongjie Yao.
      Cellular totipotency is critical for whole-organism generation, yet how totipotency is established remains poorly illustrated. Abundant transposable elements (TEs) are activated in totipotent cells, which is critical for embryonic totipotency. Here, we show that the histone chaperone RBBP4, but not its homolog RBBP7, is indispensable for maintaining the identity of mouse embryonic stem cells (mESCs). Auxin-induced degradation of RBBP4, but not RBBP7, reprograms mESCs to the totipotent 2C-like cells. Also, loss of RBBP4 enhances transition from mESCs to trophoblast cells. Mechanistically, RBBP4 binds to the endogenous retroviruses (ERVs) and functions as an upstream regulator by recruiting G9a to deposit H3K9me2 on ERVL elements, and recruiting KAP1 to deposit H3K9me3 on ERV1/ERVK elements, respectively. Moreover, RBBP4 facilitates the maintenance of nucleosome occupancy at the ERVK and ERVL sites within heterochromatin regions through the chromatin remodeler CHD4. RBBP4 depletion leads to the loss of the heterochromatin marks and activation of TEs and 2C genes. Together, our findings illustrate that RBBP4 is required for heterochromatin assembly and is a critical barrier for inducing cell fate transition from pluripotency to totipotency.
    DOI:  https://doi.org/10.1093/nar/gkad219
  11. NAR Genom Bioinform. 2023 Jun;5(2): lqad026
    Transfer learning identifies sequence determinants of cell-type specific regulatory element accessibility.
    Marco Salvatore, Marc Horlacher, Annalisa Marsico, Ole Winther, Robin Andersson.
      Dysfunction of regulatory elements through genetic variants is a central mechanism in the pathogenesis of disease. To better understand disease etiology, there is consequently a need to understand how DNA encodes regulatory activity. Deep learning methods show great promise for modeling of biomolecular data from DNA sequence but are limited to large input data for training. Here, we develop ChromTransfer, a transfer learning method that uses a pre-trained, cell-type agnostic model of open chromatin regions as a basis for fine-tuning on regulatory sequences. We demonstrate superior performances with ChromTransfer for learning cell-type specific chromatin accessibility from sequence compared to models not informed by a pre-trained model. Importantly, ChromTransfer enables fine-tuning on small input data with minimal decrease in accuracy. We show that ChromTransfer uses sequence features matching binding site sequences of key transcription factors for prediction. Together, these results demonstrate ChromTransfer as a promising tool for learning the regulatory code.
    DOI:  https://doi.org/10.1093/nargab/lqad026
  12. EMBO J. 2023 Apr 04. e113519
    Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns.
    Leonid Serebreni, Lisa-Marie Pleyer, Vanja Haberle, Oliver Hendy, Anna Vlasova, Vincent Loubiere, Filip Nemčko, Katharina Bergauer, Elisabeth Roitinger, Karl Mechtler, Alexander Stark.
      Recruitment of RNA polymerase II (Pol II) to promoters is essential for transcription. Despite conflicting evidence, the Pol II preinitiation complex (PIC) is often thought to have a uniform composition and to assemble at all promoters via an identical mechanism. Here, using Drosophila melanogaster S2 cells as a model, we demonstrate that different promoter classes function via distinct PICs. Promoter DNA of developmentally regulated genes readily associates with the canonical Pol II PIC, whereas housekeeping promoters do not, and instead recruit other factors such as DREF. Consistently, TBP and DREF are differentially required by distinct promoter types. TBP and its paralog TRF2 also function at different promoter types in a partially redundant manner. In contrast, TFIIA is required at all promoters, and we identify factors that can recruit and/or stabilize TFIIA at housekeeping promoters and activate transcription. Promoter activation by tethering these factors is sufficient to induce the dispersed transcription initiation patterns characteristic of housekeeping promoters. Thus, different promoter classes utilize distinct mechanisms of transcription initiation, which translate into different focused versus dispersed initiation patterns.
    Keywords:  RNA polymerase II preinitiation complex; promoters; transcription initiation
    DOI:  https://doi.org/10.15252/embj.2023113519
  13. Nature. 2023 Apr 05.
    Large-scale mapping and mutagenesis of human transcriptional effector domains.
    Nicole DelRosso, Josh Tycko, Peter Suzuki, Cecelia Andrews, Aradhana, Adi Mukund, Ivan Liongson, Connor Ludwig, Kaitlyn Spees, Polly Fordyce, Michael C Bassik, Lacramioara Bintu.
      Human gene expression is regulated by more than 2,000 transcription factors and chromatin regulators1,2. Effector domains within these proteins can activate or repress transcription. However, for many of these regulators we do not know what type of effector domains they contain, their location in the protein, their activation and repression strengths, and the sequences that are necessary for their functions. Here, we systematically measure the effector activity of more than 100,000 protein fragments tiling across most chromatin regulators and transcription factors in human cells (2,047 proteins). By testing the effect they have when recruited at reporter genes, we annotate 374 activation domains and 715 repression domains, roughly 80% of which are new and have not been previously annotated3-5. Rational mutagenesis and deletion scans across all the effector domains reveal aromatic and/or leucine residues interspersed with acidic, proline, serine and/or glutamine residues are necessary for activation domain activity. Furthermore, most repression domain sequences contain sites for small ubiquitin-like modifier (SUMO)ylation, short interaction motifs for recruiting corepressors or are structured binding domains for recruiting other repressive proteins. We discover bifunctional domains that can both activate and repress, some of which dynamically split a cell population into high- and low-expression subpopulations. Our systematic annotation and characterization of effector domains provide a rich resource for understanding the function of human transcription factors and chromatin regulators, engineering compact tools for controlling gene expression and refining predictive models of effector domain function.
    DOI:  https://doi.org/10.1038/s41586-023-05906-y
  14. Nat Genet. 2023 Apr 03.
    MLL3/MLL4 methyltransferase activities control early embryonic development and embryonic stem cell differentiation in a lineage-selective manner.
    Guojia Xie, Ji-Eun Lee, Anna D Senft, Young-Kwon Park, Younghoon Jang, Shreeta Chakraborty, Joyce J Thompson, Kaitlin McKernan, Chengyu Liu, Todd S Macfarlan, Pedro P Rocha, Weiqun Peng, Kai Ge.
      H3K4me1 methyltransferases MLL3 (KMT2C) and MLL4 (KMT2D) are critical for enhancer activation, cell differentiation and development. However, roles of MLL3/4 enzymatic activities and MLL3/4-mediated enhancer H3K4me1 in these processes remain unclear. Here we report that constitutive elimination of both MLL3 and MLL4 enzymatic activities prevents initiation of gastrulation and leads to early embryonic lethality in mice. However, selective elimination of MLL3/4 enzymatic activities in embryonic, but not extraembryonic, lineages leaves gastrulation largely intact. Consistent with this, embryonic stem cells (ESCs) lacking MLL3/4 enzymatic activities can differentiate toward the three embryonic germ layers but show aberrant differentiation to extraembryonic endoderm (ExEn) and trophectoderm. The failure in ExEn differentiation can be attributed to markedly reduced enhancer-binding of the lineage-determining transcription factor GATA6. Furthermore, we show that MLL3/4-catalyzed H3K4me1 is largely dispensable for enhancer activation during ESC differentiation. Together, our findings suggest a lineage-selective, but enhancer activation-independent, role of MLL3/4 methyltransferase activities in early embryonic development and ESC differentiation.
    DOI:  https://doi.org/10.1038/s41588-023-01356-4
  15. Nature. 2023 Apr 05.
    mRNA recognition and packaging by the human transcription-export complex.
    Belén Pacheco-Fiallos, Matthias K Vorländer, Daria Riabov-Bassat, Laura Fin, Francis J O'Reilly, Farja I Ayala, Ulla Schellhaas, Juri Rappsilber, Clemens Plaschka.
      Newly made mRNAs are processed and packaged into mature ribonucleoprotein complexes (mRNPs) and are recognized by the essential transcription-export complex (TREX) for nuclear export1,2. However, the mechanisms of mRNP recognition and three-dimensional mRNP organization are poorly understood3. Here we report cryo-electron microscopy and tomography structures of reconstituted and endogenous human mRNPs bound to the 2-MDa TREX complex. We show that mRNPs are recognized through multivalent interactions between the TREX subunit ALYREF and mRNP-bound exon junction complexes. Exon junction complexes can multimerize through ALYREF, which suggests a mechanism for mRNP organization. Endogenous mRNPs form compact globules that are coated by multiple TREX complexes. These results reveal how TREX may simultaneously recognize, compact and protect mRNAs to promote their packaging for nuclear export. The organization of mRNP globules provides a framework to understand how mRNP architecture facilitates mRNA biogenesis and export.
    DOI:  https://doi.org/10.1038/s41586-023-05904-0
  16. Nucleic Acids Res. 2023 Apr 04. pii: gkad232. [Epub ahead of print]
    Defining the fine structure of promoter activity on a genome-wide scale with CISSECTOR.
    Vincent D FitzPatrick, Christ Leemans, Joris van Arensbergen, Bas van Steensel, Harmen J Bussemaker.
      Classic promoter mutagenesis strategies can be used to study how proximal promoter regions regulate the expression of particular genes of interest. This is a laborious process, in which the smallest sub-region of the promoter still capable of recapitulating expression in an ectopic setting is first identified, followed by targeted mutation of putative transcription factor binding sites. Massively parallel reporter assays such as survey of regulatory elements (SuRE) provide an alternative way to study millions of promoter fragments in parallel. Here we show how a generalized linear model (GLM) can be used to transform genome-scale SuRE data into a high-resolution genomic track that quantifies the contribution of local sequence to promoter activity. This coefficient track helps identify regulatory elements and can be used to predict promoter activity of any sub-region in the genome. It thus allows in silico dissection of any promoter in the human genome to be performed. We developed a web application, available at cissector.nki.nl, that lets researchers easily perform this analysis as a starting point for their research into any promoter of interest.
    DOI:  https://doi.org/10.1093/nar/gkad232
  17. Mol Cell. 2023 Mar 28. pii: S1097-2765(23)00168-5. [Epub ahead of print]
    GAGA-associated factor fosters loop formation in the Drosophila genome.
    Xiao Li, Xiaona Tang, Xinyang Bing, Christopher Catalano, Taibo Li, Gabriel Dolsten, Carl Wu, Michael Levine.
      The impact of genome organization on the control of gene expression persists as a major challenge in regulatory biology. Most efforts have focused on the role of CTCF-enriched boundary elements and TADs, which enable long-range DNA-DNA associations via loop extrusion processes. However, there is increasing evidence for long-range chromatin loops between promoters and distal enhancers formed through specific DNA sequences, including tethering elements, which bind the GAGA-associated factor (GAF). Previous studies showed that GAF possesses amyloid properties in vitro, bridging separate DNA molecules. In this study, we investigated whether GAF functions as a looping factor in Drosophila development. We employed Micro-C assays to examine the impact of defined GAF mutants on genome topology. These studies suggest that the N-terminal POZ/BTB oligomerization domain is important for long-range associations of distant GAGA-rich tethering elements, particularly those responsible for promoter-promoter interactions that coordinate the activities of distant paralogous genes.
    Keywords:  GAGA-associated factor; POZ/BTB domain; Q-rich moiety; chromatin loop; long-range enhancer-promoter interactions; promoter-promoter associations connecting distant paralogous genes; tethering elements; toperons; topological operons
    DOI:  https://doi.org/10.1016/j.molcel.2023.03.011
  18. Elife. 2023 Apr 03. pii: e79386. [Epub ahead of print]12
    Cohesin-independent STAG proteins interact with RNA and R-loops and promote complex loading.
    Hayley Porter, Yang Li, Maria Victoria Neguembor, Manuel Beltran, Wazeer Varsally, Laura Martin, Manuel Tavares Cornejo, Dubravka Pezic, Amandeep Bhamra, Silvia Surinova, Richard G Jenner, Maria Pia Cosma, Suzana Hadjur.
      Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA, RNA binding proteins and R-loops, even in the absence of cohesin. Our results place SA1 on chromatin upstream of the cohesin ring and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease.
    Keywords:  cell biology; chromosomes; gene expression; human
    DOI:  https://doi.org/10.7554/eLife.79386
  19. Proc Natl Acad Sci U S A. 2023 Apr 11. 120(15): e2220542120
    Yeast PIC-Mediator structure with RNA polymerase II C-terminal domain.
    Sandra Schilbach, Haibo Wang, Christian Dienemann, Patrick Cramer.
      For transcription initiation, RNA polymerase II (Pol II) forms a preinitiation complex (PIC) that associates with the general coactivator Mediator. Whereas atomic models of the human PIC-Mediator structure have been reported, structures for its yeast counterpart remain incomplete. Here, we present an atomic model for the yeast PIC with core Mediator, including the Mediator middle module that was previously poorly resolved and including subunit Med1 that was previously lacking. We observe three peptide regions containing eleven of the 26 heptapeptide repeats of the flexible C-terminal repeat domain (CTD) of Pol II. Two of these CTD regions bind between the Mediator head and middle modules and form defined CTD-Mediator interactions. CTD peptide 1 binds between the Med6 shoulder and Med31 knob domains, whereas CTD peptide 2 forms additional contacts with Med4. The third CTD region (peptide 3) binds in the Mediator cradle and associates with the Mediator hook. Comparisons with the human PIC-Mediator structure show that the central region in peptide 1 is similar and forms conserved contacts with Mediator, whereas peptides 2 and 3 exhibit distinct structures and Mediator interactions.
    Keywords:  Mediator, RNA polymerase II C-terminal domain; RNA polymerase II initiation; cryo-electron microscopy; gene transcription
    DOI:  https://doi.org/10.1073/pnas.2220542120
  20. Bioinformatics. 2023 Apr 05. pii: btad173. [Epub ahead of print]
    eccDB: a comprehensive repository for eccDNA-mediated chromatin contacts in multi-species.
    Min Yang, Bo Qiu, Guo-You He, Jian-Yuan Zhou, Hao-Jie Yu, Yu-Ying Zhang, Yan-Shang Li, Tai-Song Li, Jin-Cheng Guo, Xue-Cang Li, Jian-Jun Xie.
      SUMMARY: We developed the eccDB database to integrate available resources for eccDNA data. eccDB is a comprehensive repository for storing, browsing, searching, and analyzing extrachromosomal circular DNAs (eccDNAs) from multi-species. The database provides regulatory and epigenetic information on eccDNAs, with a focus on analyzing intrachromosomal and interchromosomal interactions to predict their transcriptional regulatory functions. Moreover, eccDB identifies eccDNAs from unknown DNA sequences and analyzes the functional and evolutionary relationships of eccDNAs among different species. Overall, eccDB offers web-based analytical tools and a comprehensive resource for biologists and clinicians to decipher the molecular regulatory mechanisms of eccDNAs.AVAILABILITY: eccDB is freely available at http://www.xiejjlab.bio/eccDB.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btad173
  21. Sci Adv. 2023 Apr 07. 9(14): eadh0411
    Developmentally programmed histone H3 expression regulates cellular plasticity at the parental-to-early embryo transition.
    Ryan J Gleason, Yanrui Guo, Christopher S Semancik, Cindy Ow, Gitanjali Lakshminarayanan, Xin Chen.
      During metazoan development, the marked change in developmental potential from the parental germline to the embryo raises an important question regarding how the next life cycle is reset. As the basic unit of chromatin, histones are essential for regulating chromatin structure and function and, accordingly, transcription. However, the genome-wide dynamics of the canonical, replication-coupled (RC) histones during gametogenesis and embryogenesis remain unknown. In this study, we use CRISPR-Cas9-mediated gene editing in Caenorhabditis elegans to investigate the expression pattern and role of individual RC histone H3 genes and compare them to the histone variant, H3.3. We report a tightly regulated epigenome landscape change from the germline to embryos that are regulated through differential expression of distinct histone gene clusters. Together, this study reveals that a change from a H3.3- to H3-enriched epigenome during embryogenesis restricts developmental plasticity and uncovers distinct roles for individual H3 genes in regulating germline chromatin.
    DOI:  https://doi.org/10.1126/sciadv.adh0411
  22. Sci Adv. 2023 04 05. 9(14): eadf1488
    Condensed but liquid-like domain organization of active chromatin regions in living human cells.
    Tadasu Nozaki, Soya Shinkai, Satoru Ide, Koichi Higashi, Sachiko Tamura, Masa A Shimazoe, Masaki Nakagawa, Yutaka Suzuki, Yasushi Okada, Masaki Sasai, Shuichi Onami, Ken Kurokawa, Shiori Iida, Kazuhiro Maeshima.
      In eukaryotes, higher-order chromatin organization is spatiotemporally regulated as domains, for various cellular functions. However, their physical nature in living cells remains unclear (e.g., condensed domains or extended fiber loops; liquid-like or solid-like). Using novel approaches combining genomics, single-nucleosome imaging, and computational modeling, we investigated the physical organization and behavior of early DNA replicated regions in human cells, which correspond to Hi-C contact domains with active chromatin marks. Motion correlation analysis of two neighbor nucleosomes shows that nucleosomes form physically condensed domains with ~150-nm diameters, even in active chromatin regions. The mean-square displacement analysis between two neighbor nucleosomes demonstrates that nucleosomes behave like a liquid in the condensed domain on the ~150 nm/~0.5 s spatiotemporal scale, which facilitates chromatin accessibility. Beyond the micrometers/minutes scale, chromatin seems solid-like, which may contribute to maintaining genome integrity. Our study reveals the viscoelastic principle of the chromatin polymer; chromatin is locally dynamic and reactive but globally stable.
    DOI:  https://doi.org/10.1126/sciadv.adf1488
  23. Sci Adv. 2023 04 05. 9(14): eadc9446
    ETV4 mediates dosage-dependent prostate tumor initiation and cooperates with p53 loss to generate prostate cancer.
    Dan Li, Yu Zhan, Naitao Wang, Fanying Tang, Cindy J Lee, Gabriella Bayshtok, Amanda R Moore, Elissa W P Wong, Mohini R Pachai, Yuanyuan Xie, Jessica Sher, Jimmy L Zhao, Makhzuna Khudoynazarova, Anuradha Gopalan, Joseph Chan, Ekta Khurana, Peter Shepherd, Nora M Navone, Ping Chi, Yu Chen.
      The mechanisms underlying ETS-driven prostate cancer initiation and progression remain poorly understood due to a lack of model systems that recapitulate this phenotype. We generated a genetically engineered mouse with prostate-specific expression of the ETS factor, ETV4, at lower and higher protein dosage through mutation of its degron. Lower-level expression of ETV4 caused mild luminal cell expansion without histologic abnormalities, and higher-level expression of stabilized ETV4 caused prostatic intraepithelial neoplasia (mPIN) with 100% penetrance within 1 week. Tumor progression was limited by p53-mediated senescence and Trp53 deletion cooperated with stabilized ETV4. The neoplastic cells expressed differentiation markers such as Nkx3.1 recapitulating luminal gene expression features of untreated human prostate cancer. Single-cell and bulk RNA sequencing showed that stabilized ETV4 induced a previously unidentified luminal-derived expression cluster with signatures of cell cycle, senescence, and epithelial-to-mesenchymal transition. These data suggest that ETS overexpression alone, at sufficient dosage, can initiate prostate neoplasia.
    DOI:  https://doi.org/10.1126/sciadv.adc9446
  24. Oncogene. 2023 Apr 05.
    Hypoxia switches TET1 from being tumor-suppressive to oncogenic.
    Qi Yang, Hui Dang, Jiaxin Liu, Xingye Wang, Jingyuan Wang, Xinhui Lan, Meiju Ji, Mingzhao Xing, Peng Hou.
      The classical oxidizing enzymatic activity of Ten Eleven Translocation 1 (TET1) and its tumor suppressor role are well known. Here, we find that high TET1 expression is associated with poor patient survival in solid cancers often having hypoxia, which is inconsistent with its tumor suppressor role. Through a series of in vitro and in vivo studies, using thyroid cancer as a model, we demonstrate that TET1 plays a tumor suppressor function in normoxia and, surprisingly, an oncogenic function in hypoxia. Mechanistically, TET1 mediates HIF1α-p300 interaction by acting as a co-activator of HIF1α to promote CK2B transcription under hypoxia, which is independent of its enzymatic activity; CK2 activates the AKT/GSK3β signaling pathway to promote oncogenesis. Activated AKT/GSK3β signaling in turn maintains HIF1α at elevated levels by preventing its K48-linked ubiquitination and degradation, creating a feedback loop to enhance the oncogenicity of TET1 in hypoxia. Thus, this study uncovers a novel oncogenic mechanism in which TET1 promotes oncogenesis and cancer progression through a non-enzymatic interaction between TET1 and HIF1α in hypoxia, providing novel therapeutic targeting implications for cancer.
    DOI:  https://doi.org/10.1038/s41388-023-02659-w
  25. Nat Commun. 2023 Apr 04. 14(1): 1867
    An epigenetic switch controls an alternative NR2F2 isoform that unleashes a metastatic program in melanoma.
    Veronica Davalos, Claudia D Lovell, Richard Von Itter, Igor Dolgalev, Praveen Agrawal, Gillian Baptiste, David J Kahler, Elena Sokolova, Sebastian Moran, Laia Piqué, Eleazar Vega-Saenz de Miera, Barbara Fontanals-Cirera, Alcida Karz, Aristotelis Tsirigos, Chi Yun, Farbod Darvishian, Heather C Etchevers, Iman Osman, Manel Esteller, Markus Schober, Eva Hernando.
      Metastatic melanoma develops once transformed melanocytic cells begin to de-differentiate into migratory and invasive melanoma cells with neural crest cell (NCC)-like and epithelial-to-mesenchymal transition (EMT)-like features. However, it is still unclear how transformed melanocytes assume a metastatic melanoma cell state. Here, we define DNA methylation changes that accompany metastatic progression in melanoma patients and discover Nuclear Receptor Subfamily 2 Group F, Member 2 - isoform 2 (NR2F2-Iso2) as an epigenetically regulated metastasis driver. NR2F2-Iso2 is transcribed from an alternative transcriptional start site (TSS) and it is truncated at the N-terminal end which encodes the NR2F2 DNA-binding domain. We find that NR2F2-Iso2 expression is turned off by DNA methylation when NCCs differentiate into melanocytes. Conversely, this process is reversed during metastatic melanoma progression, when NR2F2-Iso2 becomes increasingly hypomethylated and re-expressed. Our functional and molecular studies suggest that NR2F2-Iso2 drives metastatic melanoma progression by modulating the activity of full-length NR2F2 (Isoform 1) over EMT- and NCC-associated target genes. Our findings indicate that DNA methylation changes play a crucial role during metastatic melanoma progression, and their control of NR2F2 activity allows transformed melanocytes to acquire NCC-like and EMT-like features. This epigenetically regulated transcriptional plasticity facilitates cell state transitions and metastatic spread.
    DOI:  https://doi.org/10.1038/s41467-023-36967-2
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