bims-crepig 2024-07-07 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2024–07–07
twenty-six papers selected by
Connor Rogerson, University of Cambridge

cBAF generates subnucleosomes that expand OCT4 binding and function beyond DNA motifs at enhancers.
nucMACC: An MNase-seq pipeline to identify structurally altered nucleosomes in the genome.
An activity-specificity trade-off encoded in human transcription factors.
Cellular zinc status alters chromatin accessibility and binding of p53 to DNA.
Defining a chromatin architecture that supports transcription at RNA polymerase II promoters.
ChAHP2 and ChAHP control diverse retrotransposons by complementary activities.
DNA sequence and chromatin differentiate sequence-specific transcription factor binding in the human malaria parasite Plasmodium falciparum.
A single-cell chromatin accessibility dataset of human primed and naïve pluripotent stem cell-derived teratoma.
Exploring the reciprocity between pioneer factors and development.
SETDB1 regulates short interspersed nuclear elements and chromatin loop organization in mouse neural precursor cells.
Direct neuronal reprogramming of mouse astrocytes is associated with multiscale epigenome remodeling and requires Yy1.
A transient transcriptional activation governs unpolarized-to-polarized morphogenesis during embryo implantation.
DNA break induces rapid transcription repression mediated by proteasome-dependent RNAPII removal.
Proteome-scale tagging and functional screening in mammalian cells by ORFtag.
An IDR-dependent mechanism for nuclear receptor control of Mediator interaction with RNA polymerase II.
TBP facilitates RNA Polymerase I transcription following mitosis.
Interface-guided phenotyping of coding variants in the transcription factor RUNX1.
Downstream-of-gene (DoG) transcripts contribute to an imbalance in the cancer cell transcriptome.
Chromatin context-dependent effects of epigenetic drugs on CRISPR-Cas9 editing.
Nucleoporin Elys attaches peripheral chromatin to the nuclear pores in interphase nuclei.
YAP represses the TEAD-NF-κB complex and inhibits the growth of clear cell renal cell carcinoma.
TWEAK/Fn14 signalling driven super-enhancer reprogramming promotes pro-metastatic metabolic rewiring in triple-negative breast cancer.
Epithelial cells maintain memory of prior infection with Streptococcus pneumoniae through di-methylation of histone H3.
The activity of early-life gene regulatory elements is hijacked in aging through pervasive AP-1-linked chromatin opening.
Nucleosomes play a dual role in regulating transcription dynamics.
CTCF mutation at R567 causes developmental disorders via 3D genome rearrangement and abnormal neurodevelopment.

Nat Struct Mol Biol. 2024 Jul 02.

cBAF generates subnucleosomes that expand OCT4 binding and function beyond DNA motifs at enhancers.

Marina C Nocente, Anida Mesihovic Karamitsos, Emilie Drouineau, Manon Soleil, Waad Albawardi, Cécile Dulary, Florence Ribierre, Hélène Picaud, Olivier Alibert, Joël Acker, Marie Kervella, Jean-Christophe Aude, Nick Gilbert, Françoise Ochsenbein, Sophie Chantalat, Matthieu Gérard.

The canonical BRG/BRM-associated factor (cBAF) complex is essential for chromatin opening at enhancers in mammalian cells. However, the nature of the open chromatin remains unclear. Here, we show that, in addition to producing histone-free DNA, cBAF generates stable hemisome-like subnucleosomal particles containing the four core histones associated with 50-80 bp of DNA. Our genome-wide analysis indicates that cBAF makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4-subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude compared to the region occupied on histone-free DNA. We propose that cBAF-dependent subnucleosomes orchestrate a molecular mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs.

DOI: https://doi.org/10.1038/s41594-024-01344-0
Sci Adv. 2024 Jul 05. 10(27): eadm9740

nucMACC: An MNase-seq pipeline to identify structurally altered nucleosomes in the genome.

Sara Wernig-Zorc, Fabian Kugler, Leo Schmutterer, Patrick Räß, Clemens Hausmann, Simon Holzinger, Gernot Längst, Uwe Schwartz.

Micrococcal nuclease sequencing is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Data analysis is complex due to the AT-dependent sequence bias of the endonuclease and the requirement for high sequencing depth. Here, we present the nucleosome-based MNase accessibility (nucMACC) pipeline unveiling the regulatory chromatin landscape by measuring nucleosome accessibility and stability. The nucMACC pipeline represents a systematic and genome-wide approach for detecting unstable ("fragile") nucleosomes. We have characterized the regulatory nucleosome landscape in Drosophila melanogaster, Saccharomyces cerevisiae, and mammals. Two functionally distinct sets of promoters were characterized, one associated with an unstable nucleosome and the other being nucleosome depleted. We show that unstable nucleosomes present intermediate states of nucleosome remodeling, preparing inducible genes for transcriptional activation in response to stimuli or stress. The presence of unstable nucleosomes correlates with RNA polymerase II proximal pausing. The nucMACC pipeline offers unparalleled precision and depth in nucleosome research and is a valuable tool for future nucleosome studies.

DOI: https://doi.org/10.1126/sciadv.adm9740
Nat Cell Biol. 2024 Jul 05.

An activity-specificity trade-off encoded in human transcription factors.

Julian Naderi, Alexandre P Magalhaes, Gözde Kibar, Gregoire Stik, Yaotian Zhang, Sebastian D Mackowiak, Hannah M Wieler, Francesca Rossi, Rene Buschow, Marie Christou-Kent, Marc Alcoverro-Bertran, Thomas Graf, Martin Vingron, Denes Hnisz.

Transcription factors (TFs) control specificity and activity of gene transcription, but whether a relationship between these two features exists is unclear. Here we provide evidence for an evolutionary trade-off between the activity and specificity in human TFs encoded as submaximal dispersion of aromatic residues in their intrinsically disordered protein regions. We identified approximately 500 human TFs that encode short periodic blocks of aromatic residues in their intrinsically disordered regions, resembling imperfect prion-like sequences. Mutation of periodic aromatic residues reduced transcriptional activity, whereas increasing the aromatic dispersion of multiple human TFs enhanced transcriptional activity and reprogramming efficiency, promoted liquid-liquid phase separation in vitro and more promiscuous DNA binding in cells. Together with recent work on enhancer elements, these results suggest an important evolutionary role of suboptimal features in transcriptional control. We propose that rational engineering of amino acid features that alter phase separation may be a strategy to optimize TF-dependent processes, including cellular reprogramming.

DOI: https://doi.org/10.1038/s41556-024-01411-0
Life Sci Alliance. 2024 Sep;pii: e202402638. [Epub ahead of print]7(9):

Cellular zinc status alters chromatin accessibility and binding of p53 to DNA.

Daniel Ocampo, Leah J Damon, Lynn Sanford, Samuel E Holtzen, Taylor Jones, Mary A Allen, Robin D Dowell, Amy E Palmer.

Zn2+ is an essential metal required by approximately 850 human transcription factors. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Using ATAC-seq to profile regions of accessible chromatin coupled with transcription factor enrichment analysis, we examined how increases and decreases in the labile zinc pool affect chromatin accessibility and transcription factor enrichment. We found 685 transcription factor motifs were differentially enriched, corresponding to 507 unique transcription factors. The pattern of perturbation and the types of transcription factors were notably different at promoters versus intergenic regions, with zinc-finger transcription factors strongly enriched in intergenic regions in elevated Zn2+ To test whether ATAC-seq and transcription factor enrichment analysis predictions correlate with changes in transcription factor binding, we used ChIP-qPCR to profile six p53 binding sites. We found that for five of the six targets, p53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc alter chromatin accessibility and transcription factor binding to DNA.

DOI: https://doi.org/10.26508/lsa.202402638
J Biol Chem. 2024 Jun 28. pii: S0021-9258(24)02016-7. [Epub ahead of print] 107515

Defining a chromatin architecture that supports transcription at RNA polymerase II promoters.

Michael J Fisher, Donal S Luse.

  Mammalian RNA polymerase II preinitiation complexes assemble adjacent to a nucleosome whose proximal edge (NPE) is typically 40-50 bp downstream of the transcription start site (TSS). At active promoters, that +1 nucleosome is universally modified by trimethylation on lysine 4 of histone H3 (H3K4me3). The Pol II preinitiation complex only extends 35 bp beyond the TSS, but nucleosomal templates with an NPE at +51 are nearly inactive in vitro with promoters that lack a TATA element and thus depend on TFIID for promoter recognition. Significantly, this inhibition is relieved when the +1 nucleosome contains H3K4me3, which can interact with TFIID subunits. Here we show that H3K4me3 templates with both TATA and TATA-less promoters are active with +35 NPEs when transcription is driven by TFIID. Templates with +20 NPE are also active but at reduced levels compared to +35 and +51 NPEs, consistent with a general inhibition of promoter function when the proximal nucleosome encroaches on the preinitation complex. Remarkably, dinucleosome templates support transcription when H3K4me3 is only present in the distal nucleosome, suggesting that TFIID-H3K4me3 interaction does not require modification of the +1 nucleosome. Transcription reactions performed with an alternative protocol that retains most nuclear factors results primarily in early termination, with a minority of complexes successfully traversing the first nucleosome. In such reactions the +1 nucleosome does not substantially affect the level of termination even with an NPE of +20, indicating that a nucleosome barrier is not a major driver of early termination by Pol II.

Keywords:  RNA polymerase II; TFIID; histone methylation; nucleosome; promoter

DOI:  https://doi.org/10.1016/j.jbc.2024.107515
Genes Dev. 2024 Jul 03.

ChAHP2 and ChAHP control diverse retrotransposons by complementary activities.

Josip Ahel, Aparna Pandey, Michaela Schwaiger, Fabio Mohn, Anja Basters, Georg Kempf, Aude Andriollo, Lucas Kaaij, Daniel Hess, Marc Bühler.

  Retrotransposon control in mammals is an intricate process that is effectuated by a broad network of chromatin regulatory pathways. We previously discovered ChAHP, a protein complex with repressive activity against short interspersed element (SINE) retrotransposons that is composed of the transcription factor ADNP, chromatin remodeler CHD4, and HP1 proteins. Here we identify ChAHP2, a protein complex homologous to ChAHP, in which ADNP is replaced by ADNP2. ChAHP2 is predominantly targeted to endogenous retroviruses (ERVs) and long interspersed elements (LINEs) via HP1β-mediated binding of H3K9 trimethylated histones. We further demonstrate that ChAHP also binds these elements in a manner mechanistically equivalent to that of ChAHP2 and distinct from DNA sequence-specific recruitment at SINEs. Genetic ablation of ADNP2 alleviates ERV and LINE1 repression, which is synthetically exacerbated by additional depletion of ADNP. Together, our results reveal that the ChAHP and ChAHP2 complexes function to control both nonautonomous and autonomous retrotransposons by complementary activities, further adding to the complexity of mammalian transposon control.

Keywords:  ADNP; ADNP2; CHD4; ChAHP; ChAHP2; HP1; retrotransposon

DOI:  https://doi.org/10.1101/gad.351769.124
Nucleic Acids Res. 2024 Jul 05. pii: gkae585. [Epub ahead of print]

DNA sequence and chromatin differentiate sequence-specific transcription factor binding in the human malaria parasite Plasmodium falciparum.

Victoria A Bonnell, Yuning Zhang, Alan S Brown, John Horton, Gabrielle A Josling, Tsu-Pei Chiu, Remo Rohs, Shaun Mahony, Raluca Gordân, Manuel Llinás.

Development of the malaria parasite, Plasmodium falciparum, is regulated by a limited number of sequence-specific transcription factors (TFs). However, the mechanisms by which these TFs recognize genome-wide binding sites is largely unknown. To address TF specificity, we investigated the binding of two TF subsets that either bind CACACA or GTGCAC DNA sequence motifs and further characterized two additional ApiAP2 TFs, PfAP2-G and PfAP2-EXP, which bind unique DNA motifs (GTAC and TGCATGCA). We also interrogated the impact of DNA sequence and chromatin context on P. falciparum TF binding by integrating high-throughput in vitro and in vivo binding assays, DNA shape predictions, epigenetic post-translational modifications, and chromatin accessibility. We found that DNA sequence context minimally impacts binding site selection for paralogous CACACA-binding TFs, while chromatin accessibility, epigenetic patterns, co-factor recruitment, and dimerization correlate with differential binding. In contrast, GTGCAC-binding TFs prefer different DNA sequence context in addition to chromatin dynamics. Finally, we determined that TFs that preferentially bind divergent DNA motifs may bind overlapping genomic regions due to low-affinity binding to other sequence motifs. Our results demonstrate that TF binding site selection relies on a combination of DNA sequence and chromatin features, thereby contributing to the complexity of P. falciparum gene regulatory mechanisms.

DOI: https://doi.org/10.1093/nar/gkae585
Sci Data. 2024 Jul 02. 11(1): 725

A single-cell chromatin accessibility dataset of human primed and naïve pluripotent stem cell-derived teratoma.

Jinxiu Li, Lixin Fu, Yunpan Li, Wei Sun, Yao Yi, Wenqi Jia, Haiwei Li, Hao Liu, Pengcheng Guo, Yang Wang, Yue Shen, Xiuqing Zhang, Yuan Lv, Baoming Qin, Wenjuan Li, Chuanyu Liu, Longqi Liu, Md Abdul Mazid, Yiwei Lai, Miguel A Esteban, Yu Jiang, Liang Wu.

Teratoma, due to its remarkable ability to differentiate into multiple cell lineages, is a valuable model for studying human embryonic development. The similarity of the gene expression and chromatin accessibility patterns in these cells to those observed in vivo further underscores its potential as a research tool. Notably, teratomas derived from human naïve (pre-implantation epiblast-like) pluripotent stem cells (PSCs) have larger embryonic cell diversity and contain extraembryonic lineages, making them more suitable to study developmental processes. However, the cell type-specific epigenetic profiles of naïve PSC teratomas have not been yet characterized. Using single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), we analyzed 66,384 cell profiles from five teratomas derived from human naïve PSCs and their post-implantation epiblast-like (primed) counterparts. We observed 17 distinct cell types from both embryonic and extraembryonic lineages, resembling the corresponding cell types in human fetal tissues. Additionally, we identified key transcription factors specific to different cell types. Our dataset provides a resource for investigating gene regulatory programs in a relevant model of human embryonic development.

DOI: https://doi.org/10.1038/s41597-024-03558-9
Development. 2024 Jul 01. pii: dev201921. [Epub ahead of print]151(13):

Exploring the reciprocity between pioneer factors and development.

Meghan M Freund, Melissa M Harrison, Eliana F Torres-Zelada.

  Development is regulated by coordinated changes in gene expression. Control of these changes in expression is largely governed by the binding of transcription factors to specific regulatory elements. However, the packaging of DNA into chromatin prevents the binding of many transcription factors. Pioneer factors overcome this barrier owing to unique properties that enable them to bind closed chromatin, promote accessibility and, in so doing, mediate binding of additional factors that activate gene expression. Because of these properties, pioneer factors act at the top of gene-regulatory networks and drive developmental transitions. Despite the ability to bind target motifs in closed chromatin, pioneer factors have cell type-specific chromatin occupancy and activity. Thus, developmental context clearly shapes pioneer-factor function. Here, we discuss this reciprocal interplay between pioneer factors and development: how pioneer factors control changes in cell fate and how cellular environment influences pioneer-factor binding and activity.

Keywords:  Chromatin; Gene-regulatory network; Pioneer factor

DOI:  https://doi.org/10.1242/dev.201921
Genome Biol. 2024 Jul 03. 25(1): 175

SETDB1 regulates short interspersed nuclear elements and chromatin loop organization in mouse neural precursor cells.

Daijing Sun, Yueyan Zhu, Wenzhu Peng, Shenghui Zheng, Jie Weng, Shulong Dong, Jiaqi Li, Qi Chen, Chuanhui Ge, Liyong Liao, Yuhao Dong, Yun Liu, Weida Meng, Yan Jiang.

   BACKGROUND: Transposable elements play a critical role in maintaining genome architecture during neurodevelopment. Short Interspersed Nuclear Elements (SINEs), a major subtype of transposable elements, are known to harbor binding sites for the CCCTC-binding factor (CTCF) and pivotal in orchestrating chromatin organization. However, the regulatory mechanisms controlling the activity of SINEs in the developing brain remains elusive.
RESULTS: In our study, we conduct a comprehensive genome-wide epigenetic analysis in mouse neural precursor cells using ATAC-seq, ChIP-seq, whole genome bisulfite sequencing, in situ Hi-C, and RNA-seq. Our findings reveal that the SET domain bifurcated histone lysine methyltransferase 1 (SETDB1)-mediated H3K9me3, in conjunction with DNA methylation, restricts chromatin accessibility on a selective subset of SINEs in neural precursor cells. Mechanistically, loss of Setdb1 increases CTCF access to these SINE elements and contributes to chromatin loop reorganization. Moreover, de novo loop formation contributes to differential gene expression, including the dysregulation of genes enriched in mitotic pathways. This leads to the disruptions of cell proliferation in the embryonic brain after genetic ablation of Setdb1 both in vitro and in vivo.
CONCLUSIONS: In summary, our study sheds light on the epigenetic regulation of SINEs in mouse neural precursor cells, suggesting their role in maintaining chromatin organization and cell proliferation during neurodevelopment.

Keywords:  Chromatin loop; DNA methylation; H3K9me3; Neurodevelopment; SETDB1; Transposable elements

DOI:  https://doi.org/10.1186/s13059-024-03327-2
Nat Neurosci. 2024 Jul 02.

Direct neuronal reprogramming of mouse astrocytes is associated with multiscale epigenome remodeling and requires Yy1.

Allwyn Pereira, Jeisimhan Diwakar, Giacomo Masserdotti, Sude Beşkardeş, Tatiana Simon, Younju So, Lucía Martín-Loarte, Franziska Bergemann, Lakshmy Vasan, Tamas Schauer, Anna Danese, Riccardo Bocchi, Maria Colomé-Tatché, Carol Schuurmans, Anna Philpott, Tobias Straub, Boyan Bonev, Magdalena Götz.

Direct neuronal reprogramming is a promising approach to regenerate neurons from local glial cells. However, mechanisms of epigenome remodeling and co-factors facilitating this process are unclear. In this study, we combined single-cell multiomics with genome-wide profiling of three-dimensional nuclear architecture and DNA methylation in mouse astrocyte-to-neuron reprogramming mediated by Neurogenin2 (Ngn2) and its phosphorylation-resistant form (PmutNgn2), respectively. We show that Ngn2 drives multilayered chromatin remodeling at dynamic enhancer-gene interaction sites. PmutNgn2 leads to higher reprogramming efficiency and enhances epigenetic remodeling associated with neuronal maturation. However, the differences in binding sites or downstream gene activation cannot fully explain this effect. Instead, we identified Yy1, a transcriptional co-factor recruited by direct interaction with Ngn2 to its target sites. Upon deletion of Yy1, activation of neuronal enhancers, genes and ultimately reprogramming are impaired without affecting Ngn2 binding. Thus, our work highlights the key role of interactors of proneural factors in direct neuronal reprogramming.

DOI: https://doi.org/10.1038/s41593-024-01677-5
Mol Cell. 2024 Jun 27. pii: S1097-2765(24)00483-0. [Epub ahead of print]

A transient transcriptional activation governs unpolarized-to-polarized morphogenesis during embryo implantation.

Xuehui Lyu, Yingzi Cui, Yinfei Kong, Min Yang, Hui Shen, Shuyun Liao, Shiyu Li, Chenrui An, Haoyi Wang, Zhe Zhang, Jennie Ong, Yan Li, Peng Du.

  During implantation, embryos undergo an unpolarized-to-polarized transition to initiate postimplantation morphogenesis. However, the underlying molecular mechanism is unknown. Here, we identify a transient transcriptional activation governing embryonic morphogenesis and pluripotency transition during implantation. In naive pluripotent embryonic stem cells (ESCs), which represent preimplantation embryos, we find that the microprocessor component DGCR8 can recognize stem-loop structures within nascent mRNAs to sequester transcriptional coactivator FLII to suppress transcription directly. When mESCs exit from naive pluripotency, the ERK/RSK/P70S6K pathway rapidly activates, leading to FLII phosphorylation and disruption of DGCR8/FLII interaction. Phosphorylated FLII can bind to transcription factor JUN, activating cell migration-related genes to establish poised pluripotency akin to implanting embryos. Resequestration of FLII by DGCR8 drives poised ESCs into formative pluripotency. In summary, we identify a DGCR8/FLII/JUN-mediated transient transcriptional activation mechanism. Disruption of this mechanism inhibits naive-poised-formative pluripotency transition and the corresponding unpolarized-to-polarized transition during embryo implantation, which are conserved in mice and humans.

Keywords:  DGCR8; ERK; FLII; JUN; embryo implantation; miRNA independent; nascent RNA; pluripotency; stem cells; transcription regulation

DOI:  https://doi.org/10.1016/j.molcel.2024.06.005
Cell Rep. 2024 Jul 01. pii: S2211-1247(24)00749-6. [Epub ahead of print]43(7): 114420

DNA break induces rapid transcription repression mediated by proteasome-dependent RNAPII removal.

Shuaixin He, Zhiyuan Huang, Yang Liu, Taekjip Ha, Bin Wu.

  A DNA double-strand break (DSB) jeopardizes genome integrity and endangers cell viability. Actively transcribed genes are particularly detrimental if broken and need to be repressed. However, it remains elusive how fast the repression is initiated and how far it influences the neighboring genes on the chromosome. We adopt a recently developed, very fast CRISPR to generate a DSB at a specific genomic locus with precise timing, visualize transcription in live cells, and measure the RNA polymerase II (RNAPII) occupancy near the broken site. We observe that a single DSB represses the transcription of the damaged gene in minutes, which coincides with the recruitment of a damage repair protein. Transcription repression propagates bi-directionally along the chromosome from the DSB for hundreds of kilobases, and proteasome is evoked to remove RNAPII in this process. Our method builds a foundation to measure the rapid kinetic events around a single DSB and elucidate the molecular mechanism.

Keywords:  CP: Molecular biology; CRISPR-Cas9; DNA damage repair; proteasome; single molecule; transcription

DOI:  https://doi.org/10.1016/j.celrep.2024.114420
Nat Methods. 2024 Jul 05.

Proteome-scale tagging and functional screening in mammalian cells by ORFtag.

Filip Nemčko, Moritz Himmelsbach, Vincent Loubiere, Ramesh Yelagandula, Michaela Pagani, Nina Fasching, Julius Brennecke, Ulrich Elling, Alexander Stark, Stefan L Ameres.

The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs). We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and posttranscriptional regulators in mouse embryonic stem cells. Each screen recovers known and identifies new regulators, including long ORFs inaccessible by other methods. Among other hits, we find that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.

DOI: https://doi.org/10.1038/s41592-024-02339-x
Mol Cell. 2024 Jun 27. pii: S1097-2765(24)00484-2. [Epub ahead of print]

An IDR-dependent mechanism for nuclear receptor control of Mediator interaction with RNA polymerase II.

Haiyan Zhao, Jiaqin Li, Yufei Xiang, Sohail Malik, Supriya V Vartak, Giovana M B Veronezi, Natalie Young, McKayla Riney, Jens Kalchschmidt, Andrea Conte, Seol Kyoung Jung, Srinivas Ramachandran, Robert G Roeder, Yi Shi, Rafael Casellas, Francisco J Asturias.

  The essential Mediator (MED) coactivator complex plays a well-understood role in regulation of basal transcription in all eukaryotes, but the mechanism underlying its role in activator-dependent transcription remains unknown. We investigated modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) by antagonistic effects of the MED26 subunit and the CDK8 kinase module (CKM). Biochemical analysis of CKM-MED showed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction. This restriction is eliminated by nuclear receptor (NR) binding to CKM-MED, which enables CTD binding in a MED26-dependent manner. Cryoelectron microscopy (cryo-EM) and crosslinking-mass spectrometry (XL-MS) revealed that the structural basis for modulation of CTD interaction with MED relates to a large intrinsically disordered region (IDR) in CKM subunit MED13 that blocks MED26 and CTD interaction with MED but is repositioned upon NR binding. Hence, NRs can control transcription initiation by priming CKM-MED for MED26-dependent RNA Pol II interaction.

Keywords:  CDK8 kinase module; MED26; Mediator complex; RNA polymerase II CTD; activation; eukaryotic transcription; nuclear receptors; transcription regulation

DOI:  https://doi.org/10.1016/j.molcel.2024.06.006
RNA Biol. 2024 Jan;21(1): 42-51

TBP facilitates RNA Polymerase I transcription following mitosis.

James Z J Kwan, Thomas F Nguyen, Sheila S Teves.

  The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated. In this report, we show that in murine embryonic stem cells TBP localizes onto Pol I promoters, whereas the TBP paralog TRF2 only weakly associates to the Spacer Promoter of rDNA, suggesting that it may not be able to replace TBP for Pol I transcription. Importantly, acute TBP depletion does not fully disrupt Pol I occupancy or activity on ribosomal RNA genes, but TBP binding in mitosis leads to efficient Pol I reactivation following cell division. These findings provide a more nuanced role for TBP in Pol I transcription in murine embryonic stem cells.

Keywords:  Biological sciences; Cell biology; RNA polymerase I; TATA-box binding protein; embryonic stem cells; genome profiling; mitotic Bookmarking; transcription initiation

DOI:  https://doi.org/10.1080/15476286.2024.2375097
Cell Rep. 2024 Jul 04. pii: S2211-1247(24)00765-4. [Epub ahead of print]43(7): 114436

Interface-guided phenotyping of coding variants in the transcription factor RUNX1.

Kivilcim Ozturk, Rebecca Panwala, Jeanna Sheen, Kyle Ford, Nathan Jayne, Andrew Portell, Dong-Er Zhang, Stephan Hutter, Torsten Haferlach, Trey Ideker, Prashant Mali, Hannah Carter.

  Single-gene missense mutations remain challenging to interpret. Here, we deploy scalable functional screening by sequencing (SEUSS), a Perturb-seq method, to generate mutations at protein interfaces of RUNX1 and quantify their effect on activities of downstream cellular programs. We evaluate single-cell RNA profiles of 115 mutations in myelogenous leukemia cells and categorize them into three functionally distinct groups, wild-type (WT)-like, loss-of-function (LoF)-like, and hypomorphic, that we validate in orthogonal assays. LoF-like variants dominate the DNA-binding site and are recurrent in cancer; however, recurrence alone does not predict functional impact. Hypomorphic variants share characteristics with LoF-like but favor protein interactions, promoting gene expression indicative of nerve growth factor (NGF) response and cytokine recruitment of neutrophils. Accessible DNA near differentially expressed genes frequently contains RUNX1-binding motifs. Finally, we reclassify 16 variants of uncertain significance and train a classifier to predict 103 more. Our work demonstrates the potential of targeting protein interactions to better define the landscape of phenotypes reachable by missense mutations.

Keywords:  CP: Genomics; CP: Molecular biology; Perturb-seq; RNA-seq; cancer; coding variant; interface; protein-protein interaction; single-cell; transcription factor

DOI:  https://doi.org/10.1016/j.celrep.2024.114436
Sci Adv. 2024 Jul 05. 10(27): eadh9613

Downstream-of-gene (DoG) transcripts contribute to an imbalance in the cancer cell transcriptome.

Kouki Abe, Brian Maunze, Pedro-Avila Lopez, Jessica Xu, Nefertiti Muhammad, Guang-Yu Yang, David Katz, Yaping Liu, Shannon M Lauberth.

Downstream-of-gene (DoG) transcripts are an emerging class of noncoding RNAs. However, it remains largely unknown how DoG RNA production is regulated and whether alterations in DoG RNA signatures exist in major cancers. Here, through transcriptomic analyses of matched tumors and nonneoplastic tissues and cancer cell lines, we reveal a comprehensive catalog of DoG RNA signatures. Through separate lines of evidence, we support the biological importance of DoG RNAs in carcinogenesis. First, we show tissue-specific and stage-specific differential expression of DoG RNAs in tumors versus paired normal tissues with their respective host genes involved in tumor-promoting versus tumor-suppressor pathways. Second, we identify that differential DoG RNA expression is associated with poor patient survival. Third, we identify that DoG RNA induction is a consequence of treating colon cancer cells with the topoisomerase I (TOP1) poison camptothecin and following TOP1 depletion. Our results underlie the significance of DoG RNAs and TOP1-dependent regulation of DoG RNAs in diversifying and modulating the cancer transcriptome.

DOI: https://doi.org/10.1126/sciadv.adh9613
Nucleic Acids Res. 2024 Jul 02. pii: gkae570. [Epub ahead of print]

Chromatin context-dependent effects of epigenetic drugs on CRISPR-Cas9 editing.

Ruben Schep, Max Trauernicht, Xabier Vergara, Anoek Friskes, Ben Morris, Sebastian Gregoricchio, Stefano G Manzo, Wilbert Zwart, Roderick L Beijersbergen, René H Medema, Bas van Steensel.

The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break in different chromatin environments. We then measured Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 58 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g. PCI-24781), while others were only effective in euchromatin and H3K27me3-marked regions (e.g. apicidin). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a resource to improve Cas9 editing more selectively at the desired location.

DOI: https://doi.org/10.1093/nar/gkae570
Commun Biol. 2024 Jun 29. 7(1): 783

Nucleoporin Elys attaches peripheral chromatin to the nuclear pores in interphase nuclei.

Semen A Doronin, Artem A Ilyin, Anna D Kononkova, Mikhail A Solovyev, Oxana M Olenkina, Valentina V Nenasheva, Elena A Mikhaleva, Sergey A Lavrov, Anna Y Ivannikova, Ruslan A Simonov, Anna A Fedotova, Ekaterina E Khrameeva, Sergey V Ulianov, Sergey V Razin, Yuri Y Shevelyov.

Transport of macromolecules through the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs) consisting of nucleoporins (Nups). Elys/Mel-28 is the Nup that binds and connects the decondensing chromatin with the reassembled NPCs at the end of mitosis. Whether Elys links chromatin with the NE during interphase is unknown. Here, using DamID-seq, we identified Elys binding sites in Drosophila late embryos and divided them into those associated with nucleoplasmic or with NPC-linked Elys. These Elys binding sites are located within active or inactive chromatin, respectively. Strikingly, Elys knockdown in S2 cells results in peripheral chromatin displacement from the NE, in decondensation of NE-attached chromatin, and in derepression of genes within. It also leads to slightly more compact active chromatin regions. Our findings indicate that NPC-linked Elys, together with the nuclear lamina, anchors peripheral chromatin to the NE, whereas nucleoplasmic Elys decompacts active chromatin.

DOI: https://doi.org/10.1038/s42003-024-06495-w
Sci Signal. 2024 Jul 02. 17(843): eadk0231

YAP represses the TEAD-NF-κB complex and inhibits the growth of clear cell renal cell carcinoma.

Zhongbo Li, Peng Su, Miao Yu, Xufeng Zhang, Yaning Xu, Tianwei Jia, Penghe Yang, Chenmiao Zhang, Yanan Sun, Xin Li, Huijie Yang, Yinlu Ding, Ting Zhuang, Haiyang Guo, Jian Zhu.

The Hippo pathway is generally understood to inhibit tumor growth by phosphorylating the transcriptional cofactor YAP to sequester it to the cytoplasm and reduce the formation of YAP-TEAD transcriptional complexes. Aberrant activation of YAP occurs in various cancers. However, we found a tumor-suppressive function of YAP in clear cell renal cell carcinoma (ccRCC). Using cell cultures, xenografts, and patient-derived explant models, we found that the inhibition of upstream Hippo-pathway kinases MST1 and MST2 or expression of a constitutively active YAP mutant impeded ccRCC proliferation and decreased gene expression mediated by the transcription factor NF-κB. Mechanistically, the NF-κB subunit p65 bound to the transcriptional cofactor TEAD to facilitate NF-κB-target gene expression that promoted cell proliferation. However, by competing for TEAD, YAP disrupted its interaction with NF-κB and prompted the dissociation of p65 from target gene promoters, thereby inhibiting NF-κB transcriptional programs. This cross-talk between the Hippo and NF-κB pathways in ccRCC suggests that targeting the Hippo-YAP axis in an atypical manner-that is, by activating YAP-may be a strategy for slowing tumor growth in patients.

DOI: https://doi.org/10.1126/scisignal.adk0231
Nat Commun. 2024 Jul 05. 15(1): 5638

TWEAK/Fn14 signalling driven super-enhancer reprogramming promotes pro-metastatic metabolic rewiring in triple-negative breast cancer.

Nicholas Sim, Jean-Michel Carter, Kamalakshi Deka, Benita Kiat Tee Tan, Yirong Sim, Suet-Mien Tan, Yinghui Li.

Triple Negative Breast Cancer (TNBC) is the most aggressive breast cancer subtype suffering from limited targeted treatment options. Following recent reports correlating Fibroblast growth factor-inducible 14 (Fn14) receptor overexpression in Estrogen Receptor (ER)-negative breast cancers with metastatic events, we show that Fn14 is specifically overexpressed in TNBC patients and associated with poor survival. We demonstrate that constitutive Fn14 signalling rewires the transcriptomic and epigenomic landscape of TNBC, leading to enhanced tumour growth and metastasis. We further illustrate that such mechanisms activate TNBC-specific super enhancers (SE) to drive the transcriptional activation of cancer dependency genes via chromatin looping. In particular, we uncover the SE-driven upregulation of Nicotinamide phosphoribosyltransferase (NAMPT), which promotes NAD+ and ATP metabolic reprogramming critical for filopodia formation and metastasis. Collectively, our study details the complex mechanistic link between TWEAK/Fn14 signalling and TNBC metastasis, which reveals several vulnerabilities which could be pursued for the targeted treatment of TNBC patients.

DOI: https://doi.org/10.1038/s41467-024-50071-z
Nat Commun. 2024 Jul 02. 15(1): 5545

Epithelial cells maintain memory of prior infection with Streptococcus pneumoniae through di-methylation of histone H3.

Christine Chevalier, Claudia Chica, Justine Matheau, Adrien Pain, Michael G Connor, Melanie A Hamon.

Epithelial cells are the first point of contact for bacteria entering the respiratory tract. Streptococcus pneumoniae is an obligate human pathobiont of the nasal mucosa, carried asymptomatically but also the cause of severe pneumoniae. The role of the epithelium in maintaining homeostatic interactions or mounting an inflammatory response to invasive S. pneumoniae is currently poorly understood. However, studies have shown that chromatin modifications, at the histone level, induced by bacterial pathogens interfere with the host transcriptional program and promote infection. Here, we uncover a histone modification induced by S. pneumoniae infection maintained for at least 9 days upon clearance of bacteria with antibiotics. Di-methylation of histone H3 on lysine 4 (H3K4me2) is induced in an active manner by bacterial attachment to host cells. We show that infection establishes a unique epigenetic program affecting the transcriptional response of epithelial cells, rendering them more permissive upon secondary infection. Our results establish H3K4me2 as a unique modification induced by infection, distinct from H3K4me3 or me1, which localizes to enhancer regions genome-wide. Therefore, this study reveals evidence that bacterial infection leaves a memory in epithelial cells after bacterial clearance, in an epigenomic mark, thereby altering cellular responses to subsequent infections and promoting infection.

DOI: https://doi.org/10.1038/s41467-024-49347-1
Cell Metab. 2024 Jun 28. pii: S1550-4131(24)00231-6. [Epub ahead of print]

The activity of early-life gene regulatory elements is hijacked in aging through pervasive AP-1-linked chromatin opening.

Ralph Patrick, Marina Naval-Sanchez, Nikita Deshpande, Yifei Huang, Jingyu Zhang, Xiaoli Chen, Ying Yang, Kanupriya Tiwari, Mohammadhossein Esmaeili, Minh Tran, Amin R Mohamed, Binxu Wang, Di Xia, Jun Ma, Jacqueline Bayliss, Kahlia Wong, Michael L Hun, Xuan Sun, Benjamin Cao, Denny L Cottle, Tara Catterall, Hila Barzilai-Tutsch, Robin-Lee Troskie, Zhian Chen, Andrea F Wise, Sheetal Saini, Ye Mon Soe, Snehlata Kumari, Matthew J Sweet, Helen E Thomas, Ian M Smyth, Anne L Fletcher, Konstantin Knoblich, Matthew J Watt, Majid Alhomrani, Walaa Alsanie, Kylie M Quinn, Tobias D Merson, Ann P Chidgey, Sharon D Ricardo, Di Yu, Thierry Jardé, Seth W Cheetham, Christophe Marcelle, Susan K Nilsson, Quan Nguyen, Melanie D White, Christian M Nefzger.

  A mechanistic connection between aging and development is largely unexplored. Through profiling age-related chromatin and transcriptional changes across 22 murine cell types, analyzed alongside previous mouse and human organismal maturation datasets, we uncovered a transcription factor binding site (TFBS) signature common to both processes. Early-life candidate cis-regulatory elements (cCREs), progressively losing accessibility during maturation and aging, are enriched for cell-type identity TFBSs. Conversely, cCREs gaining accessibility throughout life have a lower abundance of cell identity TFBSs but elevated activator protein 1 (AP-1) levels. We implicate TF redistribution toward these AP-1 TFBS-rich cCREs, in synergy with mild downregulation of cell identity TFs, as driving early-life cCRE accessibility loss and altering developmental and metabolic gene expression. Such remodeling can be triggered by elevating AP-1 or depleting repressive H3K27me3. We propose that AP-1-linked chromatin opening drives organismal maturation by disrupting cell identity TFBS-rich cCREs, thereby reprogramming transcriptome and cell function, a mechanism hijacked in aging through ongoing chromatin opening.

Keywords:  AP-1; CTCF; aging; cell identity; chromatin; development; maturation; polycomb repressive complex 2; redistribution; transcription factors

DOI:  https://doi.org/10.1016/j.cmet.2024.06.006
Proc Natl Acad Sci U S A. 2024 Jul 09. 121(28): e2319772121

Nucleosomes play a dual role in regulating transcription dynamics.

Sumitabha Brahmachari, Shubham Tripathi, José N Onuchic, Herbert Levine.

  Transcription has a mechanical component, as the translocation of the transcription machinery or RNA polymerase (RNAP) on DNA or chromatin is dynamically coupled to the chromatin torsion. This posits chromatin mechanics as a possible regulator of eukaryotic transcription, however, the modes and mechanisms of this regulation are elusive. Here, we first take a statistical mechanics approach to model the torsional response of topology-constrained chromatin. Our model recapitulates the experimentally observed weaker torsional stiffness of chromatin compared to bare DNA and proposes structural transitions of nucleosomes into chirally distinct states as the driver of the contrasting torsional mechanics. Coupling chromatin mechanics with RNAP translocation in stochastic simulations, we reveal a complex interplay of DNA supercoiling and nucleosome dynamics in governing RNAP velocity. Nucleosomes play a dual role in controlling the transcription dynamics. The steric barrier aspect of nucleosomes in the gene body counteracts transcription via hindering RNAP motion, whereas the chiral transitions facilitate RNAP motion via driving a low restoring torque upon twisting the DNA. While nucleosomes with low dissociation rates are typically transcriptionally repressive, highly dynamic nucleosomes offer less of a steric barrier and enhance the transcription elongation dynamics of weakly transcribed genes via buffering DNA twist. We use the model to predict transcription-dependent levels of DNA supercoiling in segments of the budding yeast genome that are in accord with available experimental data. The model unveils a paradigm of DNA supercoiling-mediated interaction between genes and makes testable predictions that will guide experimental design.

Keywords:  nucleosomes; supercoiling; transcription

DOI:  https://doi.org/10.1073/pnas.2319772121
Nat Commun. 2024 Jul 01. 15(1): 5524

CTCF mutation at R567 causes developmental disorders via 3D genome rearrangement and abnormal neurodevelopment.

Jie Zhang, Gongcheng Hu, Yuli Lu, Huawei Ren, Yin Huang, Yulin Wen, Binrui Ji, Diyang Wang, Haidong Wang, Huisheng Liu, Ning Ma, Lingling Zhang, Guangjin Pan, Yibo Qu, Hua Wang, Wei Zhang, Zhichao Miao, Hongjie Yao.

The three-dimensional genome structure organized by CTCF is required for development. Clinically identified mutations in CTCF have been linked to adverse developmental outcomes. Nevertheless, the underlying mechanism remains elusive. In this investigation, we explore the regulatory roles of a clinically relevant R567W point mutation, located within the 11th zinc finger of CTCF, by introducing this mutation into both murine models and human embryonic stem cell-derived cortical organoid models. Mice with homozygous CTCFR567W mutation exhibit growth impediments, resulting in postnatal mortality, and deviations in brain, heart, and lung development at the pathological and single-cell transcriptome levels. This mutation induces premature stem-like cell exhaustion, accelerates the maturation of GABAergic neurons, and disrupts neurodevelopmental and synaptic pathways. Additionally, it specifically hinders CTCF binding to peripheral motifs upstream to the core consensus site, causing alterations in local chromatin structure and gene expression, particularly at the clustered protocadherin locus. Comparative analysis using human cortical organoids mirrors the consequences induced by this mutation. In summary, this study elucidates the influence of the CTCFR567W mutation on human neurodevelopmental disorders, paving the way for potential therapeutic interventions.

DOI: https://doi.org/10.1038/s41467-024-49684-1