bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024–09–22
23 papers selected by
Connor Rogerson, University of Cambridge
  1. Prediction of Enhancer-Gene Interactions Using Chromatin-Conformation Capture and Epigenome Data Using STARE.
  2. Enhancer-promoter hubs organize transcriptional networks promoting oncogenesis and drug resistance.
  3. RNA fine-tunes estrogen receptor-alpha binding on low-affinity DNA motifs for transcriptional regulation.
  4. Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements.
  5. Identifying genetic variants associated with chromatin looping and genome function.
  6. Multimodal Hox5 activity generates motor neuron diversity.
  7. MITF regulates IDH1, NNT, and a transcriptional program protecting melanoma from reactive oxygen species.
  8. Nucleosomal DNA unwinding pathway through canonical and non-canonical histone disassembly.
  9. EnhancerNet: A predictive model of cell identity dynamics through enhancer selection.
  10. OVO positively regulates essential maternal pathways by binding near the transcriptional start sites in the Drosophila female germline.
  11. Pharmacological restriction of genomic binding sites redirects PU.1 pioneer transcription factor activity.
  12. Ion-mediated condensation controls the mechanics of mitotic chromosomes.
  13. Transcription start site scanning requires the fungi-specific hydrophobic loop of Tfb3.
  14. PRDM3/16 regulate chromatin accessibility required for NKX2-1 mediated alveolar epithelial differentiation and function.
  15. Integrative identification of non-coding regulatory regions driving metastatic prostate cancer.
  16. How to scan naked DNA using promiscuous recognition and no clamping: a model for pioneer transcription factors.
  17. Fasting shapes chromatin architecture through an mTOR/RNA Pol I axis.
  18. Antagonism between H3K27me3 and genome-lamina association drives atypical spatial genome organization in the totipotent embryo.
  19. Dynamic phosphorylation of FOXA1 by Aurora B guides post-mitotic gene reactivation.
  20. A simple, robust, cost-effective, and low-input ChIP-seq method for profiling histone modifications and Pol II in plants.
  21. Hippo effector, Yorkie, is a tumor suppressor in select Drosophila squamous epithelia.
  22. Nanoscale imaging of DNA-RNA identifies transcriptional plasticity at heterochromatin.
  23. PPARγ and C/EBPα enable adipocyte differentiation upon inhibition of histone methyltransferase PRC2 in malignant tumors.
  1. Methods Mol Biol. 2025 ;2856 327-339
    Prediction of Enhancer-Gene Interactions Using Chromatin-Conformation Capture and Epigenome Data Using STARE.
    Dennis Hecker, Marcel H Schulz.
      Disentangling the relationship of enhancers and genes is an ongoing challenge in epigenomics. We present STARE, our software to quantify the strength of enhancer-gene interactions based on enhancer activity and chromatin contact data. It implements the generalized Activity-by-Contact (gABC) score, which allows predicting putative target genes of candidate enhancers over any desired genomic distance. The only requirement for its application is a measurement of enhancer activity. In addition to regulatory interactions, STARE calculates transcription factor (TF) affinities on gene level. We illustrate its usage on a public single-cell data set of the human heart by predicting regulatory interactions on cell type level, by giving examples on how to integrate them with other data modalities, and by constructing TF affinity matrices.
    Keywords:  Chromatin contact data; Enhancer–gene links; Gene regulation; Regulatory regions; Transcription factors
    DOI:  https://doi.org/10.1007/978-1-0716-4136-1_20
  2. Nat Commun. 2024 Sep 14. 15(1): 8070
    Enhancer-promoter hubs organize transcriptional networks promoting oncogenesis and drug resistance.
    Brent S Perlman, Noah Burget, Yeqiao Zhou, Gregory W Schwartz, Jelena Petrovic, Zora Modrusan, Robert B Faryabi.
      Recent advances in high-resolution mapping of spatial interactions among regulatory elements support the existence of complex topological assemblies of enhancers and promoters known as enhancer-promoter hubs or cliques. Yet, organization principles of these multi-interacting enhancer-promoter hubs and their potential role in regulating gene expression in cancer remain unclear. Here, we systematically identify enhancer-promoter hubs in breast cancer, lymphoma, and leukemia. We find that highly interacting enhancer-promoter hubs form at key oncogenes and lineage-associated transcription factors potentially promoting oncogenesis of these diverse cancer types. Genomic and optical mapping of interactions among enhancer and promoter elements further show that topological alterations in hubs coincide with transcriptional changes underlying acquired resistance to targeted therapy in T cell leukemia and B cell lymphoma. Together, our findings suggest that enhancer-promoter hubs are dynamic and heterogeneous topological assemblies with the potential to control gene expression circuits promoting oncogenesis and drug resistance.
    DOI:  https://doi.org/10.1038/s41467-024-52375-6
  3. EMBO J. 2024 Sep 16.
    RNA fine-tunes estrogen receptor-alpha binding on low-affinity DNA motifs for transcriptional regulation.
    Deepanshu Soota, Bharath Saravanan, Rajat Mann, Tripti Kharbanda, Dimple Notani.
      Transcription factors (TFs) regulate gene expression by binding with varying strengths to DNA via their DNA-binding domain. Additionally, some TFs also interact with RNA, which modulates transcription factor binding to chromatin. However, whether RNA-mediated TF binding results in differential transcriptional outcomes remains unknown. In this study, we demonstrate that estrogen receptor α (ERα), a ligand-activated TF, interacts with RNA in a ligand-dependent manner. Defects in RNA binding lead to genome-wide loss of ERα recruitment, particularly at weaker ERα-motifs. Furthermore, ERα mobility in the nucleus increases in the absence of its RNA-binding capacity. Unexpectedly, this increased mobility coincides with robust polymerase loading and transcription of ERα-regulated genes that harbor low-strength motifs. However, highly stable binding of ERα on chromatin negatively impacts ligand-dependent transcription. Collectively, our results suggest that RNA interactions spatially confine ERα on low-affinity sites to fine-tune gene transcription.
    Keywords:  Chromatin; DNA-motifs; Estrogen Receptor-Alpha; Non-Coding RNA; Transcription Factors
    DOI:  https://doi.org/10.1038/s44318-024-00225-y
  4. Nat Commun. 2024 Sep 18. 15(1): 8209
    Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements.
    Florence M Chardon, Troy A McDiarmid, Nicholas F Page, Riza M Daza, Beth K Martin, Silvia Domcke, Samuel G Regalado, Jean-Benoît Lalanne, Diego Calderon, Xiaoyi Li, Lea M Starita, Stephan J Sanders, Nadav Ahituv, Jay Shendure.
      CRISPR-based gene activation (CRISPRa) is a strategy for upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1 Mb, including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate genes in a cell type-specific manner.
    DOI:  https://doi.org/10.1038/s41467-024-52490-4
  5. Nat Commun. 2024 Sep 18. 15(1): 8174
    Identifying genetic variants associated with chromatin looping and genome function.
    Sourya Bhattacharyya, Ferhat Ay.
      Here we present a comprehensive HiChIP dataset on naïve CD4 T cells (nCD4) from 30 donors and identify QTLs that associate with genotype-dependent and/or allele-specific variation of HiChIP contacts defining loops between active regulatory regions (iQTLs). We observe a substantial overlap between iQTLs and previously defined eQTLs and histone QTLs, and an enrichment for fine-mapped QTLs and GWAS variants. Furthermore, we describe a distinct subset of nCD4 iQTLs, for which the significant variation of chromatin contacts in nCD4 are translated into significant eQTL trends in CD4 T cell memory subsets. Finally, we define connectivity-QTLs as iQTLs that are significantly associated with concordant genotype-dependent changes in chromatin contacts over a broad genomic region (e.g., GWAS SNP in the RNASET2 locus). Our results demonstrate the importance of chromatin contacts as a complementary modality for QTL mapping and their power in identifying previously uncharacterized QTLs linked to cell-specific gene expression and connectivity.
    DOI:  https://doi.org/10.1038/s41467-024-52296-4
  6. Commun Biol. 2024 Sep 17. 7(1): 1166
    Multimodal Hox5 activity generates motor neuron diversity.
    Ritesh Kc, Raquel López de Boer, Minshan Lin, Alicia N Vagnozzi, Lucie Jeannotte, Polyxeni Philippidou.
      Motor neurons (MNs) are the final output of circuits driving fundamental behaviors, such as respiration and locomotion. Hox proteins are essential in generating the MN diversity required for accomplishing these functions, but the transcriptional mechanisms that enable Hox paralogs to assign distinct MN subtype identities despite their promiscuous DNA binding motif are not well understood. Here we show that Hoxa5 modifies chromatin accessibility in all mouse spinal cervical MN subtypes and engages TALE co-factors to directly bind and regulate subtype-specific genes. We identify a paralog-specific interaction of Hoxa5 with the phrenic MN-specific transcription factor Scip and show that heterologous expression of Hoxa5 and Scip is sufficient to suppress limb-innervating MN identity. We also demonstrate that phrenic MN identity is stable after Hoxa5 downregulation and identify Klf proteins as potential regulators of phrenic MN maintenance. Our data identify multiple modes of Hoxa5 action that converge to induce and maintain MN identity.
    DOI:  https://doi.org/10.1038/s42003-024-06835-w
  7. Sci Rep. 2024 09 14. 14(1): 21527
    MITF regulates IDH1, NNT, and a transcriptional program protecting melanoma from reactive oxygen species.
    Elisabeth Roider, Alexandra I T Lakatos, Alicia M McConnell, Poguang Wang, Alina Mueller, Akinori Kawakami, Jennifer Tsoi, Botond L Szabolcs, Anna A Ascsillán, Yusuke Suita, Vivien Igras, Jennifer A Lo, Jennifer J Hsiao, Rebecca Lapides, Dorottya M P Pál, Anna S Lengyel, Alexander Navarini, Arimichi Okazaki, Othon Iliopoulos, István Németh, Thomas G Graeber, Leonard Zon, Roger W Giese, Lajos V Kemeny, David E Fisher.
      Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte function, development and plays a significant role in melanoma pathogenesis. MITF genomic amplification promotes melanoma development, and it can facilitate resistance to multiple therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo. Some of the MITF target genes involved, such as IDH1 and NNT, are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state.
    DOI:  https://doi.org/10.1038/s41598-024-72031-9
  8. Commun Biol. 2024 Sep 14. 7(1): 1144
    Nucleosomal DNA unwinding pathway through canonical and non-canonical histone disassembly.
    Hikaru Nozawa, Fritz Nagae, Satoshi Ogihara, Rina Hirano, Hirohito Yamazaki, Ryo Iizuka, Munetaka Akatsu, Tomoya Kujirai, Shoji Takada, Hitoshi Kurumizaka, Sotaro Uemura.
      The nucleosome including H2A.B, a mammalian-specific H2A variant, plays pivotal roles in spermatogenesis, embryogenesis, and oncogenesis, indicating unique involvement in transcriptional regulation distinct from canonical H2A nucleosomes. Despite its significance, the exact regulatory mechanism remains elusive. This study utilized solid-state nanopores to investigate DNA unwinding dynamics, applying local force between DNA and histones. Comparative analysis of canonical H2A and H2A.B nucleosomes demonstrated that the H2A.B variant required a lower voltage for complete DNA unwinding. Furthermore, synchronization analysis and molecular dynamics simulations indicate that the H2A.B variant rapidly unwinds DNA, causing the H2A-H2B dimer to dissociate from DNA immediately upon disassembly of the histone octamer. In contrast, canonical H2A nucleosomes unwind DNA at a slower rate, suggesting that the H2A-H2B dimer undergoes a state of stacking at the pore. These findings suggest that nucleosomal DNA in the H2A.B nucleosomes undergoes a DNA unwinding process involving histone octamer disassembly distinct from that of canonical H2A nucleosomes, enabling smoother unwinding. The integrated approach of MD simulations and nanopore measurements is expected to evolve into a versatile tool for studying molecular interactions, not only within nucleosomes but also through the forced dissociation of DNA-protein complexes.
    DOI:  https://doi.org/10.1038/s42003-024-06856-5
  9. Development. 2024 Sep 17. pii: dev.202997. [Epub ahead of print]
    EnhancerNet: A predictive model of cell identity dynamics through enhancer selection.
    Omer Karin.
      Understanding how cell identity is encoded by the genome and acquired during differentiation is a central challenge in cell biology. We have developed a theoretical framework called EnhancerNet, which models the regulation of cell identity through the lens of transcription factor (TF)-enhancer interactions. We demonstrate that autoregulation in these interactions imposes a constraint on the model, resulting in simplified dynamics that can be parameterized from observed cell identities. Despite its simplicity, EnhancerNet recapitulates a broad range of experimental observations on cell identity dynamics, including enhancer selection, cell fate induction, hierarchical differentiation through multipotent progenitor states, and direct reprogramming by TF overexpression. The model makes specific quantitative predictions, reproducing known reprogramming recipes and the complex hematopoietic differentiation hierarchy without fitting unobserved parameters. EnhancerNet provides insights into how new cell types could evolve and highlights the functional importance of distal regulatory elements with dynamic chromatin in multicellular evolution.
    Keywords:  Cell Fate; Cell Identity; Dynamical Systems; Enhancer Selection; Statistical Physics; Systems Biology
    DOI:  https://doi.org/10.1242/dev.202997
  10. Elife. 2024 Sep 18. pii: RP94631. [Epub ahead of print]13
    OVO positively regulates essential maternal pathways by binding near the transcriptional start sites in the Drosophila female germline.
    Leif Benner, Savannah Muron, Jillian G Gomez, Brian Oliver.
      Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO's role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5'-TAACNGT-3' OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.
    Keywords:  D. melanogaster; chromosomes; developmental biology; embryonic development; gene expression; gene regulation; germ cells; transcription factor
    DOI:  https://doi.org/10.7554/eLife.94631
  11. Nat Genet. 2024 Sep 18.
    Pharmacological restriction of genomic binding sites redirects PU.1 pioneer transcription factor activity.
    Samuel J Taylor, Jacob Stauber, Oliver Bohorquez, Goichi Tatsumi, Rajni Kumari, Joyeeta Chakraborty, Boris A Bartholdy, Emily Schwenger, Sriram Sundaravel, Abdelbasset A Farahat, Justin C Wheat, Mendel Goldfinger, Amit Verma, Arvind Kumar, David W Boykin, Kristy R Stengel, Gregory M K Poon, Ulrich Steidl.
      Transcription factor (TF) DNA-binding dynamics govern cell fate and identity. However, our ability to pharmacologically control TF localization is limited. Here we leverage chemically driven binding site restriction leading to robust and DNA-sequence-specific redistribution of PU.1, a pioneer TF pertinent to many hematopoietic malignancies. Through an innovative technique, 'CLICK-on-CUT&Tag', we characterize the hierarchy of de novo PU.1 motifs, predicting occupancy in the PU.1 cistrome under binding site restriction. Temporal and single-molecule studies of binding site restriction uncover the pioneering dynamics of native PU.1 and identify the paradoxical activation of an alternate target gene set driven by PU.1 localization to second-tier binding sites. These transcriptional changes were corroborated by genetic blockade and site-specific reporter assays. Binding site restriction and subsequent PU.1 network rewiring causes primary human leukemia cells to differentiate. In summary, pharmacologically induced TF redistribution can be harnessed to govern TF localization, actuate alternate gene networks and direct cell fate.
    DOI:  https://doi.org/10.1038/s41588-024-01911-7
  12. Nat Mater. 2024 Sep 16.
    Ion-mediated condensation controls the mechanics of mitotic chromosomes.
    Hannes Witt, Janni Harju, Emma M J Chameau, Charlotte M A Bruinsma, Tinka V M Clement, Christian F Nielsen, Ian D Hickson, Erwin J G Peterman, Chase P Broedersz, Gijs J L Wuite.
      During mitosis in eukaryotic cells, mechanical forces generated by the mitotic spindle pull the sister chromatids into the nascent daughter cells. How do mitotic chromosomes achieve the necessary mechanical stiffness and stability to maintain their integrity under these forces? Here we use optical tweezers to show that ions involved in physiological chromosome condensation are crucial for chromosomal stability, stiffness and viscous dissipation. We combine these experiments with high-salt histone depletion and theory to show that chromosomal elasticity originates from the chromatin fibre behaving as a flexible polymer, whereas energy dissipation can be explained by modelling chromatin loops as an entangled polymer solution. Taken together, we show how collective properties of mitotic chromosomes, a biomaterial of incredible complexity, emerge from molecular properties, and how they are controlled by the physico-chemical environment.
    DOI:  https://doi.org/10.1038/s41563-024-01975-0
  13. Nucleic Acids Res. 2024 Sep 17. pii: gkae805. [Epub ahead of print]
    Transcription start site scanning requires the fungi-specific hydrophobic loop of Tfb3.
    Chun Yang, Pratik Basnet, Samah Sharmin, Hui Shen, Craig D Kaplan, Kenji Murakami.
      RNA polymerase II (pol II) initiates transcription from transcription start sites (TSSs) located ∼30-35 bp downstream of the TATA box in metazoans, whereas in the yeast Saccharomyces cerevisiae, pol II scans further downstream TSSs located ∼40-120 bp downstream of the TATA box. Previously, we found that removal of the kinase module TFIIK (Kin28-Ccl1-Tfb3) from TFIIH shifts the TSS in a yeast in vitro system upstream to the location observed in metazoans and that addition of recombinant Tfb3 back to TFIIH-ΔTFIIK restores the downstream TSS usage. Here, we report that this biochemical activity of yeast TFIIK in TSS scanning is attributable to the Tfb3 RING domain at the interface with pol II in the pre-initiation complex (PIC): especially, swapping Tfb3 Pro51-a residue conserved among all fungi-with Ala or Ser as in MAT1, the metazoan homolog of Tfb3, confers an upstream TSS shift in vitro in a similar manner to the removal of TFIIK. Yeast genetic analysis suggests that both Pro51 and Arg64 of Tfb3 are required to maintain the stability of the Tfb3-pol II interface in the PIC. Cryo-electron microscopy analysis of a yeast PIC lacking TFIIK reveals considerable variability in the orientation of TFIIH, which impairs TSS scanning after promoter opening.
    DOI:  https://doi.org/10.1093/nar/gkae805
  14. Nat Commun. 2024 Sep 16. 15(1): 8112
    PRDM3/16 regulate chromatin accessibility required for NKX2-1 mediated alveolar epithelial differentiation and function.
    Hua He, Sheila M Bell, Ashley Kuenzi Davis, Shuyang Zhao, Anusha Sridharan, Cheng-Lun Na, Minzhe Guo, Yan Xu, John Snowball, Daniel T Swarr, William J Zacharias, Jeffrey A Whitsett.
      While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Combined deletion of the histone methyl transferases Prdm3 and Prdm16 in early lung endoderm causes perinatal lethality due to respiratory failure from loss of AT2 cells and the accumulation of partially differentiated AT1 cells. Combination of single-cell RNA-seq, bulk ATAC-seq, and CUT&RUN data demonstrate that PRDM3 and PRDM16 regulate chromatin accessibility at NKX2-1 transcriptional targets critical for perinatal AT2 cell differentiation and surfactant homeostasis. Lineage specific deletion of PRDM3/16 in AT2 cells leads to lineage infidelity, with PRDM3/16 null cells acquiring partial AT1 fate. Together, these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiation is mediated by epigenomic modulation via PRDM3/16.
    DOI:  https://doi.org/10.1038/s41467-024-52154-3
  15. Cell Rep. 2024 Sep 13. pii: S2211-1247(24)01115-X. [Epub ahead of print]43(9): 114764
    Integrative identification of non-coding regulatory regions driving metastatic prostate cancer.
    Brian J Woo, Ruhollah Moussavi-Baygi, Heather Karner, Mehran Karimzadeh, Hassan Yousefi, Sean Lee, Kristle Garcia, Tanvi Joshi, Keyi Yin, Albertas Navickas, Luke A Gilbert, Bo Wang, Hosseinali Asgharian, Felix Y Feng, Hani Goodarzi.
      Large-scale sequencing efforts have been undertaken to understand the mutational landscape of the coding genome. However, the vast majority of variants occur within non-coding genomic regions. We designed an integrative computational and experimental framework to identify recurrently mutated non-coding regulatory regions that drive tumor progression. Applying this framework to sequencing data from a large prostate cancer patient cohort revealed a large set of candidate drivers. We used (1) in silico analyses, (2) massively parallel reporter assays, and (3) in vivo CRISPR interference screens to systematically validate metastatic castration-resistant prostate cancer (mCRPC) drivers. One identified enhancer region, GH22I030351, acts on a bidirectional promoter to simultaneously modulate expression of the U2-associated splicing factor SF3A1 and chromosomal protein CCDC157. SF3A1 and CCDC157 promote tumor growth in vivo. We nominated a number of transcription factors, notably SOX6, to regulate expression of SF3A1 and CCDC157. Our integrative approach enables the systematic detection of non-coding regulatory regions that drive human cancers.
    Keywords:  CP: Cancer; CP: Molecular biology; bidirectional; genomics; modeling; noncoding
    DOI:  https://doi.org/10.1016/j.celrep.2024.114764
  16. Nucleic Acids Res. 2024 Sep 17. pii: gkae790. [Epub ahead of print]
    How to scan naked DNA using promiscuous recognition and no clamping: a model for pioneer transcription factors.
    Rama Reddy Goluguri, Catherine Ghosh, Joshua Quintong, Mourad Sadqi, Victor Muñoz.
      Most DNA scanning proteins uniquely recognize their cognate sequence motif and slide on DNA assisted by some sort of clamping interface. The pioneer transcription factors that control cell fate in eukaryotes must forgo both elements to gain access to DNA in naked and chromatin forms; thus, whether or how these factors scan naked DNA is unknown. Here, we use single-molecule techniques to investigate naked DNA scanning by the Engrailed homeodomain (enHD) as paradigm of highly promiscuous recognition and open DNA binding interface. We find that enHD scans naked DNA quite effectively, and about 200000-fold faster than expected for a continuous promiscuous slide. To do so, enHD scans about 675 bp of DNA in 100 ms and then redeploys stochastically to another location 530 bp afar in just 10 ms. During the scanning phase enHD alternates between slow- and medium-paced modes every 3 and 40 ms, respectively. We also find that enHD binds nucleosomes and does so with enhanced affinity relative to naked DNA. Our results demonstrate that pioneer-like transcription factors can in principle do both, target nucleosomes and scan active DNA efficiently. The hybrid scanning mechanism used by enHD appears particularly well suited for the highly complex genomic signals of eukaryotic cells.
    DOI:  https://doi.org/10.1093/nar/gkae790
  17. Nat Cell Biol. 2024 Sep 19.
    Fasting shapes chromatin architecture through an mTOR/RNA Pol I axis.
    Nada Al-Refaie, Francesco Padovani, Johanna Hornung, Lorenz Pudelko, Francesca Binando, Andrea Del Carmen Fabregat, Qiuxia Zhao, Benjamin D Towbin, Elif Sarinay Cenik, Nicholas Stroustrup, Jan Padeken, Kurt M Schmoller, Daphne S Cabianca.
      Chromatin architecture is a fundamental mediator of genome function. Fasting is a major environmental cue across the animal kingdom, yet how it impacts three-dimensional (3D) genome organization is unknown. Here we show that fasting induces an intestine-specific, reversible and large-scale spatial reorganization of chromatin in Caenorhabditis elegans. This fasting-induced 3D genome reorganization requires inhibition of the nutrient-sensing mTOR pathway, acting through the regulation of RNA Pol I, but not Pol II nor Pol III, and is accompanied by remodelling of the nucleolus. By uncoupling the 3D genome configuration from the animal's nutritional status, we find that the expression of metabolic and stress-related genes increases when the spatial reorganization of chromatin occurs, showing that the 3D genome might support the transcriptional response in fasted animals. Our work documents a large-scale chromatin reorganization triggered by fasting and reveals that mTOR and RNA Pol I shape genome architecture in response to nutrients.
    DOI:  https://doi.org/10.1038/s41556-024-01512-w
  18. Nat Genet. 2024 Sep 16.
    Antagonism between H3K27me3 and genome-lamina association drives atypical spatial genome organization in the totipotent embryo.
    Isabel Guerreiro, Franka J Rang, Yumiko K Kawamura, Carla Kroon-Veenboer, Jeroen Korving, Femke C Groenveld, Ramada E van Beek, Silke J A Lochs, Ellen Boele, Antoine H M F Peters, Jop Kind.
      In mammals, early embryonic development exhibits highly unusual spatial positioning of genomic regions at the nuclear lamina, but the mechanisms underpinning this atypical genome organization remain elusive. Here, we generated single-cell profiles of lamina-associated domains (LADs) coupled with transcriptomics, which revealed a striking overlap between preimplantation-specific LAD dissociation and noncanonical broad domains of H3K27me3. Loss of H3K27me3 resulted in a restoration of canonical LAD profiles, suggesting an antagonistic relationship between lamina association and H3K27me3. Tethering of H3K27me3 to the nuclear periphery showed that the resultant relocalization is partially dependent on the underlying DNA sequence. Collectively, our results suggest that the atypical organization of LADs in early developmental stages is the result of a tug-of-war between intrinsic affinity for the nuclear lamina and H3K27me3, constrained by the available space at the nuclear periphery. This study provides detailed insight into the molecular mechanisms regulating nuclear organization during early mammalian development.
    DOI:  https://doi.org/10.1038/s41588-024-01902-8
  19. Cell Rep. 2024 Sep 13. pii: S2211-1247(24)01090-8. [Epub ahead of print]43(9): 114739
    Dynamic phosphorylation of FOXA1 by Aurora B guides post-mitotic gene reactivation.
    Ting Zhang, Shuaiyu Liu, Olanrewaju Durojaye, Fangyuan Xiong, Zhiyou Fang, Tahir Ullah, Chuanhai Fu, Bo Sun, Hao Jiang, Peng Xia, Zhikai Wang, Xuebiao Yao, Xing Liu.
      FOXA1 serves as a crucial pioneer transcription factor during developmental processes and plays a pivotal role as a mitotic bookmarking factor to perpetuate gene expression profiles and maintain cellular identity. During mitosis, the majority of FOXA1 dissociates from specific DNA binding sites and redistributes to non-specific binding sites; however, the regulatory mechanisms governing molecular dynamics and activity of FOXA1 remain elusive. Here, we show that mitotic kinase Aurora B specifies the different DNA binding modes of FOXA1 and guides FOXA1 biomolecular condensation in mitosis. Mechanistically, Aurora B kinase phosphorylates FOXA1 at Serine 221 (S221) to liberate the specific, but not the non-specific, DNA binding. Interestingly, the phosphorylation of S221 attenuates the FOXA1 condensation that requires specific DNA binding. Importantly, perturbation of the dynamic phosphorylation impairs accurate gene reactivation and cell proliferation, suggesting that reversible mitotic protein phosphorylation emerges as a fundamental mechanism for the spatiotemporal control of mitotic bookmarking.
    Keywords:  Aurora B; CP: Cell biology; FOXA1; biomolecular condensation; bookmarking factor; gene reactivation; mitosis; phosphorylation
    DOI:  https://doi.org/10.1016/j.celrep.2024.114739
  20. New Phytol. 2024 Sep 15.
    A simple, robust, cost-effective, and low-input ChIP-seq method for profiling histone modifications and Pol II in plants.
    Danling Zhu, Yi Wen, Yifang Tan, Xi Chen, Zhe Wu.
      Chromatin immunoprecipitation and sequencing (vs ChIP-seq) is an essential tool for epigenetic and molecular genetic studies. Although being routinely used, ChIP-seq is expensive, requires grams of plant materials, and is challenging for samples that enrich fatty acids such as seeds. Here, we developed an Ultrasensitive Plant ChIP-seq (UP-ChIP) method based on native ChIP-seq combined with Tn5 tagmentation-based library construction strategy. UP-ChIP is generally applicable for profiling both histone modification and Pol II in a wide range of plant samples, such as a single Arabidopsis seedling, a few Arabidopsis seeds, and sorted nuclei. Compared with conventional ChIP-seq, UP-ChIP is much less labor intensive and only consumes 1 μg of antibody and 10 μl of Protein-A/G conjugated beads for each IP and can work effectively with the amount of starting material down to a few milligrams. By performing UP-ChIP in various conditions and genotypes, we showed that UP-ChIP is highly reliable, sensitive, and quantitative for studying histone modifications. Detailed UP-ChIP protocol is provided. We recommend UP-ChIP as an alternative to traditional ChIP-seq for profiling histone modifications and Pol II, offering the advantages of reduced labor intensity, decreased costs, and low-sample input.
    Keywords:  ChIP‐seq; Pol II; epigenetics; histone modifications; seeds
    DOI:  https://doi.org/10.1111/nph.20125
  21. Proc Natl Acad Sci U S A. 2024 Sep 24. 121(39): e2319666121
    Hippo effector, Yorkie, is a tumor suppressor in select Drosophila squamous epithelia.
    Rachita Bhattacharya, Jaya Kumari, Shweta Banerjee, Jyoti Tripathi, Saurabh Singh Parihar, Nitin Mohan, Pradip Sinha.
      Mammalian Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) and Drosophila Yorkie (Yki) are transcription cofactors of the highly conserved Hippo signaling pathway. It has been long assumed that the YAP/TAZ/Yki signaling drives cell proliferation during organ growth. However, its instructive role in regulating developmentally programmed organ growth, if any, remains elusive. Out-of-context gain of YAP/TAZ/Yki signaling often turns oncogenic. Paradoxically, mechanically strained, and differentiated squamous epithelia display developmentally programmed constitutive nuclear YAP/TAZ/Yki signaling. The unknown, therefore, is how a growth-promoting YAP/TAZ/Yki signaling restricts proliferation in differentiated squamous epithelia. Here, we show that reminiscent of a tumor suppressor, Yki negatively regulates the cell growth-promoting PI3K/Akt/TOR signaling in the squamous epithelia of Drosophila tubular organs. Thus, downregulation of Yki signaling in the squamous epithelium of the adult male accessory gland (MAG) up-regulates PI3K/Akt/TOR signaling, inducing cell hypertrophy, exit from their cell cycle arrest, and, finally, culminating in squamous cell carcinoma (SCC). Thus, blocking PI3K/Akt/TOR signaling arrests Yki loss-induced MAG-SCC. Further, MAG-SCCs, like other lethal carcinomas, secrete a cachectin, Impl2-the Drosophila homolog of mammalian IGFBP7-inducing cachexia and shortening the lifespan of adult males. Moreover, in the squamous epithelium of other tubular organs, like the dorsal trunk of larval tracheal airways or adult Malpighian tubules, downregulation of Yki signaling triggers PI3K/Akt/TOR-induced cell hypertrophy. Our results reveal that Yki signaling plays an instructive, antiproliferative role in the squamous epithelia of tubular organs.
    Keywords:  Drosophila; TOR; Yki; squamous cell carcinoma; tubular organs
    DOI:  https://doi.org/10.1073/pnas.2319666121
  22. Life Sci Alliance. 2024 Dec;pii: e202402849. [Epub ahead of print]7(12):
    Nanoscale imaging of DNA-RNA identifies transcriptional plasticity at heterochromatin.
    Christelle Guillermier, Naveen Vg Kumar, Ronan C Bracken, Diana Alvarez, John O'Keefe, Aditi Gurkar, Jonathan D Brown, Matthew L Steinhauser.
      The three-dimensional structure of DNA is a biophysical determinant of transcription. The density of chromatin condensation is one determinant of transcriptional output. Chromatin condensation is generally viewed as enforcing transcriptional suppression, and therefore, transcriptional output should be inversely proportional to DNA compaction. We coupled stable isotope tracers with multi-isotope imaging mass spectrometry to quantify and image nanovolumetric relationships between DNA density and newly made RNA within individual nuclei. Proliferative cell lines and cycling cells in the murine small intestine unexpectedly demonstrated no consistent relationship between DNA density and newly made RNA, even though localized examples of this phenomenon were detected at nuclear-cytoplasmic transitions. In contrast, non-dividing hepatocytes demonstrated global reduction in newly made RNA and an inverse relationship between DNA density and transcription, driven by DNA condensates at the nuclear periphery devoid of newly made RNA. Collectively, these data support an evolving model of transcriptional plasticity that extends at least to a subset of chromatin at the extreme of condensation as expected of heterochromatin.
    DOI:  https://doi.org/10.26508/lsa.202402849
  23. J Biol Chem. 2024 Sep 12. pii: S0021-9258(24)02266-X. [Epub ahead of print] 107765
    PPARγ and C/EBPα enable adipocyte differentiation upon inhibition of histone methyltransferase PRC2 in malignant tumors.
    Jiaqi Zhao, Hui Qian, Yang An, Liping Chu, Dongxia Tan, Chenyang Qin, Qianying Sun, Yunpeng Wang, Wei Qi.
      Loss of terminal differentiation is a hallmark of cancer and offers a potential mechanism for differentiation therapy. Polycomb Repressive Complex 2 (PRC2) serves as the methyltransferase for K27 of histone H3 that is crucial in development. While PRC2 inhibitors show promise in treating various cancers, the underlying mechanisms remain incompletely understood. Here, we demonstrated that the inhibition or depletion of PRC2 enhanced adipocyte differentiation in malignant rhabdoid tumors (MRTs) and mesenchymal stem cells (MSCs), through upregulation of PPARG and CEBPA. Mechanistically, PRC2 directly represses their transcription through H3K27 methylation, as both genes exhibit a bivalent state in MSCs. Knockout of PPARG compromised C/EBPα expression and impeded the PRC2 inhibitor-induced differentiation into adipocytes. Furthermore, the combination of the PPARγ agonist rosiglitazone and the PRC2 inhibitor MAK683 exhibited a higher inhibition on Ki67 positivity in tumor xenograft compared to MAK683 alone. High CEBPA, PLIN1 and FABP4 levels positively correlated with favorable prognosis in sarcoma patients in TCGA cohort. Together, these findings unveil an epigenetic regulatory mechanism for PPARG and highlight the essential role of PPARγ and C/EBPα in the adipocyte differentiation of MRTs and sarcomas with a potential clinical implication.
    Keywords:  C/EBPα sarcoma; PPARγ; PRC2 inhibitors; adipocyte differentiation
    DOI:  https://doi.org/10.1016/j.jbc.2024.107765
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