bims-crepig 2024-10-20 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2024–10–20
twenty-six papers selected by
Connor Rogerson, University of Cambridge

Activity-assembled nBAF complex mediates rapid immediate early gene transcription by regulating RNA polymerase II productive elongation.
Redirecting the pioneering function of FOXA1 with covalent small molecules.
Massively parallel binding assay (MPBA) reveals limited transcription factor binding cooperativity, challenging models of specificity.
Double-strand breaks in facultative heterochromatin require specific movements and chromatin changes for efficient repair.
Massively parallel reporter assays identify enhancer elements in oesophageal Adenocarcinoma.
Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness.
Notch induces transcription by stimulating release of paused RNA polymerase II.
Chromatin remodeller Chd7 is developmentally regulated in the neural crest by tissue-specific transcription factors.
Analysis of single-cell CRISPR perturbations indicates that enhancers predominantly act multiplicatively.
Droplet Hi-C enables scalable, single-cell profiling of chromatin architecture in heterogeneous tissues.
Mechanism of polyadenylation-independent RNA polymerase II termination.
Heat Shock Factor 1 forms nuclear condensates and restructures the yeast genome before activating target genes.
Phosphorylation regulates the chromatin remodeler SMARCAD1 in nucleosome binding, ATP hydrolysis, and histone exchange.
Identifying transcription factors with cell-type specific DNA binding signatures.
A vector system encoding histone H3 mutants facilitates manipulations of the neuronal epigenome.
HDAC7 promotes cardiomyocyte proliferation by suppressing Myocyte Enhancer Factor 2.
LINE1 and PRC2 control nucleolar organization and repression of the 8C state in human ESCs.
A distinct metabolic and epigenetic state drives trained immunity in HSC-derived macrophages from autoimmune mice.
Chromatin interaction maps identify oncogenic targets of enhancer duplications in cancer.
Heterogeneous force response of chromatin in isolated nuclei.
MethyLasso: a segmentation approach to analyze DNA methylation patterns and identify differentially methylated regions from whole-genome datasets.
A Plasmodium falciparum MORC protein complex modulates epigenetic control of gene expression through interaction with heterochromatin.
INO80 regulates chromatin accessibility to facilitate suppression of sex-linked gene expression during mouse spermatogenesis.
Gene regulatory network inference from CRISPR perturbations in primary CD4+ T cells elucidates the genomic basis of immune disease.
MoNETA: MultiOmics Network Embedding for SubType Analysis.
Scalable identification of lineage-specific gene regulatory networks from metacells with NetID.

Cell Rep. 2024 Oct 15. pii: S2211-1247(24)01228-2. [Epub ahead of print]43(11): 114877

Activity-assembled nBAF complex mediates rapid immediate early gene transcription by regulating RNA polymerase II productive elongation.

Karen G Cornejo, Andie Venegas, Morgan H Sono, Madeline Door, Brenda Gutierrez-Ruiz, Lucy B Karabedian, Supratik G Nandi, Marco Hadisurya, W Andy Tao, Emily C Dykhuizen, Ramendra N Saha.

  Signal-dependent RNA polymerase II (RNA Pol II) productive elongation is an integral component of gene transcription, including that of immediate early genes (IEGs) induced by neuronal activity. However, it remains unclear how productively elongating RNA Pol II overcomes nucleosomal barriers. Using RNAi, three degraders, and several small-molecule inhibitors, we show that the mammalian switch/sucrose non-fermentable (SWI/SNF) complex of neurons (neuronal BRG1/BRM-associated factor or nBAF) is required for activity-induced transcription of neuronal IEGs, including Arc. The nBAF complex facilitates promoter-proximal RNA Pol II pausing and signal-dependent RNA Pol II recruitment (loading) and, importantly, mediates productive elongation in the gene body via interaction with the elongation complex and elongation-competent RNA Pol II. Mechanistically, RNA Pol II elongation is mediated by activity-induced nBAF assembly (especially ARID1A recruitment) and its ATPase activity. Together, our data demonstrate that the nBAF complex regulates several aspects of RNA Pol II transcription and reveal mechanisms underlying activity-induced RNA Pol II elongation. These findings may offer insights into human maladies etiologically associated with mutational interdiction of BAF functions.

Keywords:  Arc; BD98; CP: Molecular biology; CP: Neuroscience; RNA Pol II; RNA polymerase II; SWI/SNF; immediate early gene; nBAF; neuron; productive elongation; transcription

DOI:  https://doi.org/10.1016/j.celrep.2024.114877
Mol Cell. 2024 Oct 09. pii: S1097-2765(24)00780-9. [Epub ahead of print]

Redirecting the pioneering function of FOXA1 with covalent small molecules.

Sang Joon Won, Yuxiang Zhang, Christopher J Reinhardt, Lauren M Hargis, Nicole S MacRae, Kristen E DeMeester, Evert Njomen, Jarrett R Remsberg, Bruno Melillo, Benjamin F Cravatt, Michael A Erb.

  Pioneer transcription factors (TFs) bind to and open closed chromatin, facilitating engagement by other regulatory factors involved in gene activation or repression. Chemical probes are lacking for pioneer TFs, which has hindered their mechanistic investigation in cells. Here, we report the chemical proteomic discovery of electrophilic compounds that stereoselectively and site-specifically bind the pioneer TF forkhead box protein A1 (FOXA1) at a cysteine (C258) within the forkhead DNA-binding domain. We show that these covalent ligands react with FOXA1 in a DNA-dependent manner and rapidly remodel its pioneer activity in prostate cancer cells reflected in redistribution of FOXA1 binding across the genome and directionally correlated changes in chromatin accessibility. Motif analysis supports a mechanism where the ligands relax the canonical DNA-binding preference of FOXA1 by strengthening interactions with suboptimal sequences in predicted proximity to C258. Our findings reveal a striking plasticity underpinning the pioneering function of FOXA1 that can be controlled by small molecules.

Keywords:  ATAC-seq; ChIP-seq; FOXA1; activity-based protein profiling; chromatin; covalent; cysteine; pioneer transcription factor; proteomics

DOI:  https://doi.org/10.1016/j.molcel.2024.09.024
Nucleic Acids Res. 2024 Oct 16. pii: gkae846. [Epub ahead of print]

Massively parallel binding assay (MPBA) reveals limited transcription factor binding cooperativity, challenging models of specificity.

Tamar Jana Lang, Sagie Brodsky, Wajd Manadre, Matan Vidavski, Gili Valinsky, Vladimir Mindel, Guy Ilan, Miri Carmi, Felix Jonas, Naama Barkai.

DNA-binding domains (DBDs) within transcription factors (TFs) recognize short sequence motifs that are highly abundant in genomes. In vivo, TFs bind only a small subset of motif occurrences, which is often attributed to the cooperative binding of interacting TFs at proximal motifs. However, large-scale testing of this model is still lacking. Here, we describe a novel method allowing parallel measurement of TF binding to thousands of designed sequences within yeast cells and apply it to quantify the binding of dozens of TFs to libraries of regulatory regions containing clusters of binding motifs, systematically mutating all motif combinations. With few exceptions, TF occupancies were well explained by independent binding to individual motifs, with motif cooperation being of only limited effects. Our results challenge the general role of motif combinatorics in directing TF genomic binding and open new avenues for exploring the basis of protein-DNA interactions within cells.

DOI: https://doi.org/10.1093/nar/gkae846
Nat Commun. 2024 Oct 17. 15(1): 8984

Double-strand breaks in facultative heterochromatin require specific movements and chromatin changes for efficient repair.

Marieke R Wensveen, Aditya A Dixit, Robin van Schendel, Apfrida Kendek, Jan-Paul Lambooij, Marcel Tijsterman, Serafin U Colmenares, Aniek Janssen.

DNA double-strand breaks (DSBs) must be properly repaired within diverse chromatin domains to maintain genome stability. Whereas euchromatin has an open structure and is associated with transcription, facultative heterochromatin is essential to silence developmental genes and forms compact nuclear condensates, called polycomb bodies. Whether the specific chromatin properties of facultative heterochromatin require distinct DSB repair mechanisms remains unknown. Here, we integrate single DSB systems in euchromatin and facultative heterochromatin in Drosophila melanogaster and find that heterochromatic DSBs rapidly move outside polycomb bodies. These DSB movements coincide with a break-proximal reduction in the canonical heterochromatin mark histone H3 Lysine 27 trimethylation (H3K27me3). We demonstrate that DSB movement and loss of H3K27me3 at heterochromatic DSBs depend on the histone demethylase dUtx. Moreover, loss of dUtx specifically disrupts completion of homologous recombination at heterochromatic DSBs. We conclude that DSBs in facultative heterochromatin require dUtx-mediated loss of H3K27me3 to promote DSB movement and repair.

DOI: https://doi.org/10.1038/s41467-024-53313-2
NAR Cancer. 2024 Dec;6(4): zcae041

Massively parallel reporter assays identify enhancer elements in oesophageal Adenocarcinoma.

Shen-Hsi Yang, Ibrahim Ahmed, Yaoyong Li, Christopher W Bleaney, Andrew D Sharrocks.

Cancer is a disease underpinned by aberrant gene expression. Enhancers are regulatory elements that play a major role in transcriptional control and changes in active enhancer function are likely critical in the pathogenesis of oesophageal adenocarcinoma (OAC). Here, we utilise STARR-seq to profile the genome-wide enhancer landscape in OAC and identify hundreds of high-confidence enhancer elements. These regions are enriched in enhancer-associated chromatin marks, are actively transcribed and exhibit high levels of associated gene activity in OAC cells. These characteristics are maintained in human patient samples, demonstrating their disease relevance. This relevance is further underlined by their responsiveness to oncogenic ERBB2 inhibition and increased activity compared to the pre-cancerous Barrett's state. Mechanistically, these enhancers are linked to the core OAC transcriptional network and in particular KLF5 binding is associated with high level activity, providing further support for a role of this transcription factor in defining the OAC transcriptome. Our results therefore uncover a set of enhancer elements with physiological significance, that widen our understanding of the molecular alterations in OAC and point to mechanisms through which response to targeted therapy may occur.

DOI: https://doi.org/10.1093/narcan/zcae041
Nat Commun. 2024 Oct 17. 15(1): 8966

Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness.

Bess P Rosen, Qing V Li, Hyein S Cho, Dingyu Liu, Dapeng Yang, Sarah Graff, Jielin Yan, Renhe Luo, Nipun Verma, Jeyaram R Damodaran, Hanuman T Kale, Samuel J Kaplan, Michael A Beer, Simone Sidoli, Danwei Huangfu.

Pluripotent stem cells have remarkable self-renewal capacity: the ability to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into almost any cell type in the body. To investigate the interplay between these two aspects of self-renewal, we perform four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSCs and the dissolution of primed pluripotent identity during early differentiation. These screens distinguish genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further identify a core set of genes controlling both stem cell fitness and pluripotent identity, including a network of chromatin factors. Here, unbiased screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide a valuable resource for exploring pluripotent stem cell identity versus cell fitness, and offer a framework for categorizing gene function.

DOI: https://doi.org/10.1038/s41467-024-53284-4
Genes Dev. 2024 Oct 16.

Notch induces transcription by stimulating release of paused RNA polymerase II.

Julia M Rogers, Claudia A Mimoso, Benjamin J E Martin, Alexandre P Martin, Jon C Aster, Karen Adelman, Stephen C Blacklow.

  Notch proteins undergo ligand-induced proteolysis to release a nuclear effector that influences a wide range of cellular processes by regulating transcription. Despite years of study, however, how Notch induces the transcription of its target genes remains unclear. Here, we comprehensively examine the response to human Notch1 across a time course of activation using high-resolution genomic assays of chromatin accessibility and nascent RNA production. Our data reveal that Notch induces target gene transcription primarily by releasing paused RNA polymerase II (RNAPII). Moreover, in contrast to prevailing models suggesting that Notch acts by promoting chromatin accessibility, we found that open chromatin was established at Notch-responsive regulatory elements prior to Notch signal induction through SWI/SNF-mediated remodeling. Together, these studies show that the nuclear response to Notch signaling is dictated by the pre-existing chromatin state and RNAPII distribution at the time of signal activation.

Keywords:  ATAC-seq; NOTCH1; Notch signaling; PRO-seq; SWI/SNF; TT-seq

DOI:  https://doi.org/10.1101/gad.352108.124
PLoS Biol. 2024 Oct 17. 22(10): e3002786

Chromatin remodeller Chd7 is developmentally regulated in the neural crest by tissue-specific transcription factors.

Ruth M Williams, Guneş Taylor, Irving T C Ling, Ivan Candido-Ferreira, Daniel M Fountain, Sarah Mayes, Perihan Seda Ateş-Kalkan, Julianna O Haug, Andrew J Price, Sean A McKinney, Yavor K Bozhilovh, Richard C V Tyser, Shankar Srinivas, Jim R Hughes, Tatjana Sauka-Spengler.

Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed to pathogenic variants in the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD7 in neural crest development is well documented, how this factor is specifically up-regulated in neural crest cells is not understood. Here, we use epigenomic profiling of chick and human neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7, showing tissue-specific regulation of a globally acting chromatin remodeller. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation.

DOI: https://doi.org/10.1371/journal.pbio.3002786
Cell Genom. 2024 Oct 10. pii: S2666-979X(24)00291-X. [Epub ahead of print] 100672

Analysis of single-cell CRISPR perturbations indicates that enhancers predominantly act multiplicatively.

Jessica L Zhou, Karthik Guruvayurappan, Shushan Toneyan, Hsiuyi V Chen, Aaron R Chen, Peter Koo, Graham McVicker.

  A single gene may have multiple enhancers, but how they work in concert to regulate transcription is poorly understood. To analyze enhancer interactions throughout the genome, we developed a generalized linear modeling framework, GLiMMIRS, for interrogating enhancer effects from single-cell CRISPR experiments. We applied GLiMMIRS to a published dataset and tested for interactions between 46,166 enhancer pairs and corresponding genes, including 264 "high-confidence" enhancer pairs. We found that enhancer effects combine multiplicatively but with limited evidence for further interactions. Only 31 enhancer pairs exhibited significant interactions (false discovery rate <0.1), none of which came from the high-confidence set, and 20 were driven by outlier expression values. Additional analyses of a second CRISPR dataset and in silico enhancer perturbations with Enformer both support a multiplicative model of enhancer effects without interactions. Altogether, our results indicate that enhancer interactions are uncommon or have small effects that are difficult to detect.

Keywords:  CRISPR; data simulation; enhancers; gene expression; generalized linear models; genome-wide CRISPR screen; regulatory screen; single-cell sequencing; statistical modeling; transcriptional regulation

DOI:  https://doi.org/10.1016/j.xgen.2024.100672
Nat Biotechnol. 2024 Oct 18.

Droplet Hi-C enables scalable, single-cell profiling of chromatin architecture in heterogeneous tissues.

Lei Chang, Yang Xie, Brett Taylor, Zhaoning Wang, Jiachen Sun, Ethan J Armand, Shreya Mishra, Jie Xu, Melodi Tastemel, Audrey Lie, Zane A Gibbs, Hannah S Indralingam, Tuyet M Tan, Rafael Bejar, Clark C Chen, Frank B Furnari, Ming Hu, Bing Ren.

Current methods for analyzing chromatin architecture are not readily scalable to heterogeneous tissues. Here we introduce Droplet Hi-C, which uses a commercial microfluidic device for high-throughput, single-cell chromatin conformation profiling in droplets. Using Droplet Hi-C, we mapped the chromatin architecture of the mouse cortex and analyzed gene regulatory programs in major cortical cell types. In addition, we used this technique to detect copy number variations, structural variations and extrachromosomal DNA in human glioblastoma, colorectal and blood cancer cells, revealing clonal dynamics and other oncogenic events during treatment. We refined the technique to allow joint profiling of chromatin architecture and transcriptome in single cells, facilitating exploration of the links between chromatin architecture and gene expression in both normal tissues and tumors. Thus, Droplet Hi-C both addresses critical gaps in chromatin analysis of heterogeneous tissues and enhances understanding of gene regulation.

DOI: https://doi.org/10.1038/s41587-024-02447-1
Nat Struct Mol Biol. 2024 Oct 18.

Mechanism of polyadenylation-independent RNA polymerase II termination.

Srinivasan Rengachari, Thomas Hainthaler, Christiane Oberthuer, Michael Lidschreiber, Patrick Cramer.

The mechanisms underlying the initiation and elongation of RNA polymerase II (Pol II) transcription are well-studied, whereas termination remains poorly understood. Here we analyze the mechanism of polyadenylation-independent Pol II termination mediated by the yeast Sen1 helicase. Cryo-electron microscopy structures of two pretermination intermediates show that Sen1 binds to Pol II and uses its adenosine triphosphatase activity to pull on exiting RNA in the 5' direction. This is predicted to push Pol II forward, induce an unstable hypertranslocated state and destabilize the transcription bubble, thereby facilitating termination. This mechanism of transcription termination may be widely used because it is conceptually conserved in the bacterial transcription system.

DOI: https://doi.org/10.1038/s41594-024-01409-0
Elife. 2024 Oct 15. pii: RP92464. [Epub ahead of print]12

Heat Shock Factor 1 forms nuclear condensates and restructures the yeast genome before activating target genes.

Linda S Rubio, Suman Mohajan, David S Gross.

  In insects and mammals, 3D genome topology has been linked to transcriptional states yet whether this link holds for other eukaryotes is unclear. Using both ligation proximity and fluorescence microscopy assays, we show that in Saccharomyces cerevisiae, Heat Shock Response (HSR) genes dispersed across multiple chromosomes and under the control of Heat Shock Factor (Hsf1) rapidly reposition in cells exposed to acute ethanol stress and engage in concerted, Hsf1-dependent intergenic interactions. Accompanying 3D genome reconfiguration is equally rapid formation of Hsf1-containing condensates. However, in contrast to the transience of Hsf1-driven intergenic interactions that peak within 10-20 min and dissipate within 1 hr in the presence of 8.5% (v/v) ethanol, transcriptional condensates are stably maintained for hours. Moreover, under the same conditions, Pol II occupancy of HSR genes, chromatin remodeling, and RNA expression are detectable only later in the response and peak much later (>1 hr). This contrasts with the coordinate response of HSR genes to thermal stress (39°C) where Pol II occupancy, transcription, histone eviction, intergenic interactions, and formation of Hsf1 condensates are all rapid yet transient (peak within 2.5-10 min and dissipate within 1 hr). Therefore, Hsf1 forms condensates, restructures the genome and transcriptionally activates HSR genes in response to both forms of proteotoxic stress but does so with strikingly different kinetics. In cells subjected to ethanol stress, Hsf1 forms condensates and repositions target genes before transcriptionally activating them.

Keywords:  3D genome; RNA Pol II; S. cerevisiae; chromatin; chromosomes; ethanol stress; gene expression; heat shock response; transcriptional condensates

DOI:  https://doi.org/10.7554/eLife.92464
J Biol Chem. 2024 Oct 16. pii: S0021-9258(24)02395-0. [Epub ahead of print] 107893

Phosphorylation regulates the chromatin remodeler SMARCAD1 in nucleosome binding, ATP hydrolysis, and histone exchange.

Briana L Aboulache, Nicole M Hoitsma, Karolin Luger.

  Maintaining the dynamic structure of chromatin is critical for regulating the cellular processes that require access to the DNA template, such as DNA damage repair, transcription, and replication. Histone chaperones and ATP-dependent chromatin remodeling factors facilitate transitions in chromatin structure by assembling and positioning nucleosomes through a variety of enzymatic activities. SMARCAD1 is a unique chromatin remodeler that combines the ATP-dependent ability to exchange histones, with the chaperone-like activity of nucleosome deposition. We have shown previously that phosphorylated SMARCAD1 exhibits reduced binding to nucleosomes. However, it is unknown how phosphorylation affects SMARCAD1's ability to perform its various enzymatic activities. Here we use mutational analysis, activity assays, and mass spectrometry, to probe SMARCAD1 regulation and to investigate the role of its flexible N-terminal region. We show that phosphorylation affects SMARCAD1 binding to nucleosomes, DNA, and histones H2A-H2B as well as ATP hydrolysis and histone exchange. Conversely, we report only a marginal effect of phosphorylation for histone H3-H4 binding and nucleosome assembly. In addition, the SMARCAD1 N-terminal region is revealed to be critical for nucleosome assembly and histone exchange. Together, this work examines the intricacies of how phosphorylation governs SMARCAD1 activity and provides insight into its complex regulation and diverse activities.

Keywords:  ATP hydrolysis; SMARCAD1; chromatin remodeling; histone chaperone; histone exchange; nucleosome; nucleosome assembly; phosphorylation; post-translational modification (PTM)

DOI:  https://doi.org/10.1016/j.jbc.2024.107893
BMC Genomics. 2024 Oct 14. 25(1): 957

Identifying transcription factors with cell-type specific DNA binding signatures.

Aseel Awdeh, Marcel Turcotte, Theodore J Perkins.

   BACKGROUND: Transcription factors (TFs) bind to different parts of the genome in different types of cells, but it is usually assumed that the inherent DNA-binding preferences of a TF are invariant to cell type. Yet, there are several known examples of TFs that switch their DNA-binding preferences in different cell types, and yet more examples of other mechanisms, such as steric hindrance or cooperative binding, that may result in a "DNA signature" of differential binding.
RESULTS: To survey this phenomenon systematically, we developed a deep learning method we call SigTFB (Signatures of TF Binding) to detect and quantify cell-type specificity in a TF's known genomic binding sites. We used ENCODE ChIP-seq data to conduct a wide scale investigation of 169 distinct TFs in up to 14 distinct cell types. SigTFB detected statistically significant DNA binding signatures in approximately two-thirds of TFs, far more than might have been expected from the relatively sparse evidence in prior literature. We found that the presence or absence of a cell-type specific DNA binding signature is distinct from, and indeed largely uncorrelated to, the degree of overlap between ChIP-seq peaks in different cell types, and tended to arise by two mechanisms: using established motifs in different frequencies, and by selective inclusion of motifs for distint TFs.
CONCLUSIONS: While recent results have highlighted cell state features such as chromatin accessibility and gene expression in predicting TF binding, our results emphasize that, for some TFs, the DNA sequences of the binding sites contain substantial cell-type specific motifs.

Keywords:  Cell-type specificity; Deep learning; Differential binding; Transcription factor binding

DOI:  https://doi.org/10.1186/s12864-024-10859-1
Sci Rep. 2024 Oct 18. 14(1): 24415

A vector system encoding histone H3 mutants facilitates manipulations of the neuronal epigenome.

Sophie Warren, Sen Xiong, Daisy Robles-Magallanes, José-Manuel Baizabal.

The differentiation of developmental cell lineages is associated with genome-wide modifications in histone H3 methylation. However, the causal role of histone H3 methylation in transcriptional regulation and cell differentiation has been difficult to test in mammals. The experimental overexpression of histone H3 mutants carrying lysine-to-methionine (K-to-M) substitutions has emerged as an alternative tool for inhibiting the endogenous levels of histone H3 methylation at specific lysine residues. Here, we leverage the use of histone K-to-M mutants by creating Enhanced Episomal Vectors that enable the simultaneous depletion of multiple levels of histone H3 lysine 4 (H3K4) or lysine 9 (H3K9) methylation in projection neurons of the mouse cerebral cortex. Our approach also facilitates the simultaneous depletion of H3K9 and H3K27 trimethylation (H3K9me3 and H3K27me3, respectively) in cortical neurons. In addition, we report a tamoxifen-inducible Cre-FLEX system that allows the activation of mutant histones at specific developmental time points or in the adult cortex, leading to the depletion of specific histone marks. The tools presented here can be implemented in other experimental systems, such as human in vitro models, to test the combinatorial role of histone methylations in developmental fate decisions and the maintenance of cell identity.

DOI: https://doi.org/10.1038/s41598-024-74270-2
J Mol Cell Biol. 2024 Oct 11. pii: mjae044. [Epub ahead of print]

HDAC7 promotes cardiomyocyte proliferation by suppressing Myocyte Enhancer Factor 2.

Jihyun Jang, Mette Bentsen, Jin Bu, Ling Chen, Alexandre Rosa Campos, Mario Looso, Deqiang Li.

  Postnatal mammalian cardiomyocytes (CMs) rapidly lose proliferative capacity and exit the cell cycle and undergo further differentiation and maturation. Cell cycle activation has been a major strategy to stimulate postnatal CM proliferation, albeit achieving modest effects. One impediment is that postnatal CMs may need to undergo dedifferentiation before proliferation, if not simultaneously. Here, we report that overexpression of Hdac7 in neonatal mouse CMs results in significant CM dedifferentiation and proliferation. Mechanistically, we show that HDAC7-mediated CM proliferation is contingent on dedifferentiation, which is accomplished through suppressing MEF2. Hdac7 overexpression in CM shifts the chromatin state from binding MEF2, which favors the differentiation transcriptional program to AP-1, which favors the proliferative transcriptional program. Further, we found that HDAC7 interacts with minichromosome maintenance complex (MCM) components to initiate cell cycle progression. Our findings reveal that HDAC7 promotes CM proliferation by its dual action on CM dedifferentiation and proliferation, uncovering a potential new strategy for heart regeneration/repair.

Keywords:  HDAC7; cardiomyocyte; dedifferentiation; proliferation

DOI:  https://doi.org/10.1093/jmcb/mjae044
Dev Cell. 2024 Oct 14. pii: S1534-5807(24)00574-4. [Epub ahead of print]

LINE1 and PRC2 control nucleolar organization and repression of the 8C state in human ESCs.

Juan Zhang, Lamisa Ataei, Kirti Mittal, Liang Wu, Lauren Caldwell, Linh Huynh, Shahil Sarajideen, Kevin Tse, Marie-Michelle Simon, Md Abdul Mazid, David P Cook, Daniel Trcka, Tony Kwan, Michael M Hoffman, Jeffrey L Wrana, Miguel A Esteban, Miguel Ramalho-Santos.

  The mechanisms that ensure developmental progression in the early human embryo remain largely unknown. Here, we show that the family of long interspersed nuclear element 1 (LINE1) transposons prevents the reversion of naive human embryonic stem cells (hESCs) to 8-cell-like cells (8CLCs). LINE1 RNA contributes to maintenance of H3K27me3 levels, particularly at chromosome 19 (Chr19). Chr19 is enriched for key 8C regulators, H3K27me3, and genes derepressed upon LINE1 knockdown or PRC2 inhibition. Moreover, Chr19 is strongly associated with the nucleolus in hESCs but less in 8CLCs. Direct inhibition of PRC2 activity induces the 8C program and leads to a relocalization of Chr19 away from the nucleolus. LINE1 KD or PRC2 inhibition induces nucleolar stress, and disruption of nucleolar architecture is sufficient to de-repress the 8C program. These results indicate that LINE1 RNA and PRC2 maintain H3K27me3-mediated gene repression and 3D nuclear organization to prevent developmental reversion of hESCs.

Keywords:  8-cell-like cells; H3K27me3; LINE1; chromosome 19; human embryonic stem cells; nuclear compartmentalization; nucleolus; polycomb repressive complex 2

DOI:  https://doi.org/10.1016/j.devcel.2024.09.024
Cell Stem Cell. 2024 Oct 14. pii: S1934-5909(24)00324-2. [Epub ahead of print]

A distinct metabolic and epigenetic state drives trained immunity in HSC-derived macrophages from autoimmune mice.

Taylor S Mills, Bailee Kain, Matt A Burchill, Etienne Danis, Erin D Lucas, Rachel Culp-Hill, Courtney M Cowan, Wolfgang E Schleicher, Sweta B Patel, Brandon T Tran, Ruoqiong Cao, Andrew Goodspeed, Sarah Ferrara, Shaun Bevers, Beth A Jirón Tamburini, James R Roede, Angelo D'Alessandro, Katherine Y King, Eric M Pietras.

  Here, we investigate the contribution of long-term hematopoietic stem cells (HSCsLT) to trained immunity (TI) in the setting of chronic autoimmune disease. Using a mouse model of systemic lupus erythematosus (SLE), we show that bone marrow-derived macrophages (BMDMs) from autoimmune mice exhibit hallmark features of TI, including increased Mycobacterium avium killing and inflammatory cytokine production, which are mechanistically linked to increased glycolytic metabolism. We show that HSCs from autoimmune mice constitute a transplantable, long-term reservoir for macrophages that exhibit the functional properties of TI. However, these BMDMs exhibit reduced glycolytic activity and chromatin accessibility at metabolic genes while retaining elevated expression of TI-associated transcriptional regulators. Hence, HSC exposed to autoimmune inflammation can give rise to macrophages in which the functional and metabolic properties of TI are decoupled. Our data support a model in which TI is characterized by a spectrum of molecular and metabolic states driving augmented immune function.

Keywords:  autoimmune disease; bone marrow-derived macrophage; hematopoietic stem cell; inflammation; metabolism; trained immunity

DOI:  https://doi.org/10.1016/j.stem.2024.09.010
Genome Res. 2024 Oct 18.

Chromatin interaction maps identify oncogenic targets of enhancer duplications in cancer.

Yueqiang Song, Fuyuan Li, Shangzi Wang, Yuntong Wang, Cong Lai, Lian Chen, Ning Jiang, Jin Li, Xingdong Chen, Swneke D Bailey, Xiaoyang Zhang.

As a major type of structural variants, tandem duplication plays a critical role in tumorigenesis by increasing oncogene dosage. Recent work has revealed that noncoding enhancers are also affected by duplications leading to the activation of oncogenes that are inside or outside of the duplicated regions. However, the prevalence of enhancer duplication and the identity of their target genes remains largely unknown in the cancer genome. Here, by analyzing whole-genome sequencing data in a non-gene-centric manner, we identify 881 duplication hotspots in 13 major cancer types, most of which do not contain protein-coding genes. We show that the hotspots are enriched with distal enhancer elements and are highly lineage-specific. We develop a HiChIP-based methodology that navigates enhancer-promoter contact maps to prioritize the target genes for the duplication hotspots harboring enhancer elements. The methodology identifies many novel enhancer duplication events activating oncogenes such as ESR1, FOXA1, GATA3, GATA6, TP63, and VEGFA, as well as potentially novel oncogenes such as GRHL2, IRF2BP2, and CREB3L1 In particular, we identify a duplication hotspot on Chromosome 10p15 harboring a cluster of enhancers, which skips over two genes, through a long-range chromatin interaction, to activate an oncogenic isoform of the NET1 gene to promote migration of gastric cancer cells. Focusing on tandem duplications, our study substantially extends the catalog of noncoding driver alterations in multiple cancer types, revealing attractive targets for functional characterization and therapeutic intervention.

DOI: https://doi.org/10.1101/gr.278418.123
Cell Rep. 2024 Oct 15. pii: S2211-1247(24)01203-8. [Epub ahead of print]43(10): 114852

Heterogeneous force response of chromatin in isolated nuclei.

Giulia Bergamaschi, Andreas S Biebricher, Hannes Witt, Fitzroy J Byfield, Xamanie M R Seymonson, Cornelis Storm, Paul A Janmey, Gijs J L Wuite.

  A quantitative description of nuclear mechanics is crucial for understanding its role in force sensing within eukaryotic cells. Recent studies indicate that the chromatin within the nucleus cannot be treated as a homogeneous material. To elucidate its material properties, we combine optical tweezers manipulation of isolated nuclei with multi-color fluorescence imaging of lamin and chromatin to map the response of nuclei to local deformations. Force spectroscopy reveals nuclear strain stiffening and an exponential force dependence, well described by a hierarchical chain model. Simultaneously, fluorescence data show a higher compliance of chromatin compared to the nuclear envelope at strains <30%. Micrococcal nuclease (MNase) digestion of chromatin results in nuclear softening and can be captured by our model. Additionally, we observe stretching responses showing a lipid tether signature, suggesting that these tethers originate from the nuclear membrane. Our combined approach allows us to elucidate the nuclear force response while mapping the deformation of lamin, (eu)chromatin, and membrane.

Keywords:  CP: Cell biology; CP: Molecular biology; Mechanobiology; force spectroscopy; nuclear mechanics; optical tweezers

DOI:  https://doi.org/10.1016/j.celrep.2024.114852
Nucleic Acids Res. 2024 Oct 18. pii: gkae880. [Epub ahead of print]

MethyLasso: a segmentation approach to analyze DNA methylation patterns and identify differentially methylated regions from whole-genome datasets.

Delphine Balaramane, Yannick G Spill, Michaël Weber, Anaïs Flore Bardet.

DNA methylation is an epigenetic mark involved in the regulation of gene expression, and patterns of DNA methylation anticorrelate with chromatin accessibility and transcription factor binding. DNA methylation can be profiled at the single cytosine resolution in the whole genome and has been performed in many cell types and conditions. Computational approaches are then essential to study DNA methylation patterns in a single condition or capture dynamic changes of DNA methylation levels across conditions. Toward this goal, we developed MethyLasso, a new approach to segment DNA methylation data. We use it as an all-in-one tool to perform the identification of low-methylated regions, unmethylated regions, DNA methylation valleys and partially methylated domains in a single condition as well as differentially methylated regions between two conditions. We performed a rigorous benchmarking comparing existing approaches by evaluating the agreement of the regions across tools, their number, size, level of DNA methylation, boundaries, cytosine-guanine content and coverage using several real datasets as well as the sensitivity and precision of the approaches using simulated data and show that MethyLasso performs best overall. MethyLasso is freely available at https://github.com/bardetlab/methylasso.

DOI: https://doi.org/10.1093/nar/gkae880
Elife. 2024 Oct 16. pii: RP92201. [Epub ahead of print]12

A Plasmodium falciparum MORC protein complex modulates epigenetic control of gene expression through interaction with heterochromatin.

Maneesh Kumar Singh, Victoria Ann Bonnell, Israel Tojal Da Silva, Verônica Feijoli Santiago, Miriam Santos Moraes, Jack Adderley, Christian Doerig, Giuseppe Palmisano, Manuel Llinas, Celia R S Garcia.

  Dynamic control of gene expression is critical for blood stage development of malaria parasites. Here, we used multi-omic analyses to investigate transcriptional regulation by the chromatin-associated microrchidia protein, MORC, during asexual blood stage development of the human malaria parasite Plasmodium falciparum. We show that PfMORC (PF3D7_1468100) interacts with a suite of nuclear proteins, including APETALA2 (ApiAP2) transcription factors (PfAP2-G5, PfAP2-O5, PfAP2-I, PF3D7_0420300, PF3D7_0613800, PF3D7_1107800, and PF3D7_1239200), a DNA helicase DS60 (PF3D7_1227100), and other chromatin remodelers (PfCHD1 and PfEELM2). Transcriptomic analysis of PfMORCHA-glmS knockdown parasites revealed 163 differentially expressed genes belonging to hypervariable multigene families, along with upregulation of genes mostly involved in host cell invasion. In vivo genome-wide chromatin occupancy analysis during both trophozoite and schizont stages of development demonstrates that PfMORC is recruited to repressed, multigene families, including the var genes in subtelomeric chromosomal regions. Collectively, we find that PfMORC is found in chromatin complexes that play a role in the epigenetic control of asexual blood stage transcriptional regulation and chromatin organization.

Keywords:  P. falciparum; PfMORC; Plasmodium falciparum; infectious disease; malaria; microbiology

DOI:  https://doi.org/10.7554/eLife.92201
PLoS Genet. 2024 Oct 15. 20(10): e1011431

INO80 regulates chromatin accessibility to facilitate suppression of sex-linked gene expression during mouse spermatogenesis.

Prabuddha Chakraborty, Terry Magnuson.

The INO80 protein is the main catalytic subunit of the INO80-chromatin remodeling complex, which is critical for DNA repair and transcription regulation in murine spermatocytes. In this study, we explored the role of INO80 in silencing genes on meiotic sex chromosomes in male mice. INO80 immunolocalization at the XY body in pachytene spermatocytes suggested a role for INO80 in the meiotic sex body. Subsequent deletion of Ino80 resulted in high expression of sex-linked genes. Furthermore, the active form of RNA polymerase II at the sex chromosomes of Ino80-null pachytene spermatocytes indicates incomplete inactivation of sex-linked genes. A reduction in the recruitment of initiators of meiotic sex chromosome inhibition (MSCI) argues for INO80-facilitated recruitment of DNA repair factors required for silencing sex-linked genes. This role of INO80 is independent of a common INO80 target, H2A.Z. Instead, in the absence of INO80, a reduction in chromatin accessibility at DNA repair sites occurs on the sex chromosomes. These data suggest a role for INO80 in DNA repair factor localization, thereby facilitating the silencing of sex-linked genes during the onset of pachynema.

DOI: https://doi.org/10.1371/journal.pgen.1011431
Cell Genom. 2024 Oct 10. pii: S2666-979X(24)00290-8. [Epub ahead of print] 100671

Gene regulatory network inference from CRISPR perturbations in primary CD4+ T cells elucidates the genomic basis of immune disease.

Joshua S Weinstock, Maya M Arce, Jacob W Freimer, Mineto Ota, Alexander Marson, Alexis Battle, Jonathan K Pritchard.

  The effects of genetic variation on complex traits act mainly through changes in gene regulation. Although many genetic variants have been linked to target genes in cis, the trans-regulatory cascade mediating their effects remains largely uncharacterized. Mapping trans-regulators based on natural genetic variation has been challenging due to small effects, but experimental perturbations offer a complementary approach. Using CRISPR, we knocked out 84 genes in primary CD4+ T cells, targeting inborn error of immunity (IEI) disease transcription factors (TFs) and TFs without immune disease association. We developed a novel gene network inference method called linear latent causal Bayes (LLCB) to estimate the network from perturbation data and observed 211 regulatory connections between genes. We characterized programs affected by the TFs, which we associated with immune genome-wide association study (GWAS) genes, finding that JAK-STAT family members are regulated by KMT2A, an epigenetic regulator. These analyses reveal the trans-regulatory cascades linking GWAS genes to signaling pathways.

Keywords:  CRISPR; GWAS; RNA-seq; T cells; gene regulatory networks; immunology; inbornb errors of immunity; network inference

DOI:  https://doi.org/10.1016/j.xgen.2024.100671
NAR Genom Bioinform. 2024 Sep;6(4): lqae141

MoNETA: MultiOmics Network Embedding for SubType Analysis.

Giovanni Scala, Luigi Ferraro, Aurora Brandi, Yan Guo, Barbara Majello, Michele Ceccarelli.

Cells are complex systems whose behavior emerges from a huge number of reactions taking place within and among different molecular districts. The availability of bulk and single-cell omics data fueled the creation of multi-omics systems biology models capturing the dynamics within and between omics layers. Powerful modeling strategies are needed to cope with the increased amount of data to be interrogated and the relative research questions. Here, we present MultiOmics Network Embedding for SubType Analysis (MoNETA) for fast and scalable identification of relevant multi-omics relationships between biological entities at the bulk and single-cells level. We apply MoNETA to show how glioma subtypes previously described naturally emerge with our approach. We also show how MoNETA can be used to identify cell types in five multi-omic single-cell datasets.

DOI: https://doi.org/10.1093/nargab/lqae141
Genome Biol. 2024 Oct 18. 25(1): 275

Scalable identification of lineage-specific gene regulatory networks from metacells with NetID.

Weixu Wang, Yichen Wang, Ruiqi Lyu, Dominic Grün.

The identification of gene regulatory networks (GRNs) is crucial for understanding cellular differentiation. Single-cell RNA sequencing data encode gene-level covariations at high resolution, yet data sparsity and high dimensionality hamper accurate and scalable GRN reconstruction. To overcome these challenges, we introduce NetID leveraging homogenous metacells while avoiding spurious gene-gene correlations. Benchmarking demonstrates superior performance of NetID compared to imputation-based methods. By incorporating cell fate probability information, NetID facilitates the prediction of lineage-specific GRNs and recovers known network motifs governing bone marrow hematopoiesis, making it a powerful toolkit for deciphering gene regulatory control of cellular differentiation from large-scale single-cell transcriptome data.

DOI: https://doi.org/10.1186/s13059-024-03418-0