bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2023‒01‒01
nine papers selected by
Connor Rogerson
University of Cambridge

  1. Cell Rep. 2022 Dec 27. pii: S2211-1247(22)01761-2. [Epub ahead of print]41(13): 111865
      How transcription is regulated as development commences is fundamental to understand how the transcriptionally silent mature gametes are reprogrammed. The embryonic genome is activated for the first time during zygotic genome activation (ZGA). How RNA polymerase II (Pol II) and productive elongation are regulated during this process remains elusive. Here, we generate genome-wide maps of Serine 5 and Serine 2-phosphorylated Pol II during and after ZGA in mouse embryos. We find that both phosphorylated Pol II forms display similar distributions across genes during ZGA, with typical elongation enrichment of Pol II emerging after ZGA. Serine 2-phosphorylated Pol II occurs at genes prior to their activation, suggesting that Serine 2 phosphorylation may prime gene expression. Functional perturbations demonstrate that CDK9 and SPT5 are major ZGA regulators and that SPT5 prevents precocious activation of some genes. Overall, our work sheds molecular insights into transcriptional regulation at the beginning of mammalian development.
    Keywords:  CDK9; CP: Developmental biology; CP: Molecular biology; RNA polymerase; SPT5; ZGA; embryonic transcription; genome activation; mouse embryo; transcriptional elongation
  2. Nat Commun. 2022 12 24. 13(1): 7939
      The interplay between histone modifications and DNA methylation drives the establishment and maintenance of the cellular epigenomic landscape, but it remains challenging to investigate the complex relationship between these epigenetic marks across the genome. Here we describe a nanopore-sequencing-based-method, nanoHiMe-seq, for interrogating the genome-wide localization of histone modifications and DNA methylation from single DNA molecules. nanoHiMe-seq leverages a nonspecific methyltransferase to exogenously label adenine bases proximal to antibody-targeted modified nucleosomes in situ. The labelled adenines and the endogenous methylated CpG sites are simultaneously detected on individual nanopore reads using a hidden Markov model, which is implemented in the nanoHiMe software package. We demonstrate the utility, robustness and sensitivity of nanoHiMe-seq by jointly profiling DNA methylation and histone modifications at low coverage depths, concurrently determining phased patterns of DNA methylation and histone modifications, and probing the intrinsic connectivity between these epigenetic marks across the genome.
  3. Cell Rep. 2022 Dec 27. pii: S2211-1247(22)01754-5. [Epub ahead of print]41(13): 111858
      The histone chaperone FACT (facilitates chromatin transcription) enhances transcription in eukaryotic cells, targeting DNA-protein interactions. FACT, a heterodimer in humans, comprises SPT16 and SSRP1 subunits. We measure nucleosome stability and dynamics in the presence of FACT and critical component domains. Optical tweezers quantify FACT/subdomain binding to nucleosomes, displacing the outer wrap of DNA, disrupting direct DNA-histone (core site) interactions, altering the energy landscape of unwrapping, and increasing the kinetics of DNA-histone disruption. Atomic force microscopy reveals nucleosome remodeling, while single-molecule fluorescence quantifies kinetics of histone loss for disrupted nucleosomes, a process accelerated by FACT. Furthermore, two isolated domains exhibit contradictory functions; while the SSRP1 HMGB domain displaces DNA, SPT16 MD/CTD stabilizes DNA-H2A/H2B dimer interactions. However, only intact FACT tethers disrupted DNA to the histones and supports rapid nucleosome reformation over several cycles of force disruption/release. These results demonstrate that key FACT domains combine to catalyze both nucleosome disassembly and reassembly.
    Keywords:  AFM; CP: Molecular biology; DNA; FACT; chaperone; chromatin; confocal fluorescence; energy landscape; histones; nucleosomes; optical tweezers
  4. Genome Biol. 2022 Dec 27. 23(1): 270
      A major question in systems biology is how to identify the core gene regulatory circuit that governs the decision-making of a biological process. Here, we develop a computational platform, named NetAct, for constructing core transcription factor regulatory networks using both transcriptomics data and literature-based transcription factor-target databases. NetAct robustly infers regulators' activity using target expression, constructs networks based on transcriptional activity, and integrates mathematical modeling for validation. Our in silico benchmark test shows that NetAct outperforms existing algorithms in inferring transcriptional activity and gene networks. We illustrate the application of NetAct to model networks driving TGF-β-induced epithelial-mesenchymal transition and macrophage polarization.
    Keywords:  Cellular state transitions; Epithelial-mesenchymal transition; Gene regulatory circuits; Gene regulatory networks; Macrophage polarization; Mathematical modeling; Systems biology; Transcriptional activity
  5. Sci Rep. 2022 Dec 27. 12(1): 22425
      The chromodomain helicase DNA-binding protein CHD8 is the most frequently mutated gene in autism spectrum disorder. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is a chromatin regulator which binds to the promoters of actively transcribed genes through genomic targeting mechanisms which have yet to be fully defined. By generating a conditional loss-of-function and an endogenously tagged allele in human pluripotent stem cells, we investigated the molecular function and the interaction of CHD8 with chromatin in human neurons. Chromatin accessibility analysis and transcriptional profiling revealed that CHD8 functions as a transcriptional activator at its target genes in human neurons. Furthermore, we found that CHD8 chromatin targeting is cell context-dependent. In human neurons, CHD8 preferentially binds at ETS motif-enriched promoters. This enrichment is particularly prominent on the promoters of genes whose expression significantly changes upon the loss of CHD8. Indeed, among the ETS transcription factors, we identified ELK1 as being most highly correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our stem cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motif-containing sites. These findings imply that ELK1 and CHD8 functionally cooperate to regulate gene expression and chromatin states at MAPK/ERK target genes in human neurons. Our results suggest that the MAPK/ERK/ELK1 axis potentially contributes to the pathogenesis caused by CHD8 mutations in human neurodevelopmental disorders.
  6. Proc Natl Acad Sci U S A. 2023 Jan 03. 120(1): e2216611120
      Small molecules that bind in the minor groove of DNA are in clinical use as antibiotics and antitumor drugs. Two members of this class of molecules, netropsin and chromomycin, are shown here to displace DNA from the nucleosome and promote transfer of the histone octamer to an acceptor protein. The effects of these groove-binding molecules are exploited to address an outstanding problem in the mechanism of the RSC chromatin remodeling complex. RSC and other remodeling complexes are DNA translocases, acting near the center of the nucleosomal DNA, but translocation is apparently impossible because DNA cannot slide across the histone surface in the nucleosome. Netropsin and chromomycin promote the release of DNA from the histone surface, enhance the formation of a RSC-nucleosome complex, and synergize with RSC in chromatin remodeling. These findings are in keeping with an involvement of bulge translocation in chromatin remodeling.
    Keywords:  RSC; chromomycin; groove binders; netropsin; nucleosome
  7. BMC Genomics. 2022 Dec 27. 23(1): 854
      Phenotypic evolution is often caused by variation in gene expression resulting from altered gene regulatory mechanisms. Genetic variation affecting chromatin remodeling has been identified as a potential source of variable gene expression; however, the roles of specific chromatin remodeling factors remain unclear. Here, we address this knowledge gap by examining the relationship between variation in gene expression, variation in chromatin structure, and variation in binding of the pioneer factor Grainy head between imaginal wing discs of two divergent strains of Drosophila melanogaster and their F1 hybrid. We find that (1) variation in Grainy head binding is mostly due to sequence changes that act in cis but are located outside of the canonical Grainy head binding motif, (2) variation in Grainy head binding correlates with changes in chromatin accessibility, and (3) this variation in chromatin accessibility, coupled with variation in Grainy head binding, correlates with variation in gene expression in some cases but not others. Interactions among these three molecular layers is complex, but these results suggest that genetic variation affecting the binding of pioneer factors contributes to variation in chromatin remodeling and the evolution of gene expression.
    Keywords:  Chromatin accessibility; Evolution; Pioneer factor; Transcription factor
  8. Stem Cell Reports. 2022 Dec 26. pii: S2213-6711(22)00543-4. [Epub ahead of print]
      In direct lineage conversion, transcription factor (TF) overexpression reconfigures gene regulatory networks (GRNs) to reprogram cell identity. We previously developed CellOracle, a computational method to infer GRNs from single-cell transcriptome and epigenome data. Using inferred GRNs, CellOracle simulates gene expression changes in response to TF perturbation, enabling in silico interrogation of network reconfiguration. Here, we combine CellOracle analysis with lineage tracing of fibroblast to induced endoderm progenitor (iEP) conversion, a prototypical direct reprogramming paradigm. By linking early network state to reprogramming outcome, we reveal distinct network configurations underlying successful and failed fate conversion. Via in silico simulation of TF perturbation, we identify new factors to coax cells into successfully converting their identity, uncovering a central role for the AP-1 subunit Fos with the Hippo signaling effector, Yap1. Together, these results demonstrate the efficacy of CellOracle to infer and interpret cell-type-specific GRN configurations, providing new mechanistic insights into lineage reprogramming.
    Keywords:  cell fate prediction; direct lineage reprogramming; gene perturbation simulation; gene regulatory networks; machine learning; single-cell analysis
  9. Elife. 2022 Dec 28. pii: e81856. [Epub ahead of print]11
      CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal nonspecific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.
    Keywords:  genetics; genomics; human