bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024–08–18
23 papers selected by
Connor Rogerson, University of Cambridge
  1. Live-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation.
  2. Structural insights into the cooperative nucleosome recognition and chromatin opening by FOXA1 and GATA4.
  3. Multi-layered heterochromatin interaction as a switch for DIM2-mediated DNA methylation.
  4. HMGA1 orchestrates chromatin compartmentalization and sequesters genes into 3D networks coordinating senescence heterogeneity.
  5. Super enhancer acquisition drives expression of oncogenic PPP1R15B that regulates protein homeostasis in multiple myeloma.
  6. Comprehensive network modeling approaches unravel dynamic enhancer-promoter interactions across neural differentiation.
  7. bHLH transcription factors cooperate with chromatin remodelers to regulate cell fate decisions during Arabidopsis stomatal development.
  8. The C-terminal 4CXXC-type zinc finger domain of CDCA7 recognizes hemimethylated DNA and modulates activities of chromatin remodeling enzyme HELLS.
  9. Combinatorial regulatory states define cell fate diversity during embryogenesis.
  10. Quantitative analysis of cis-regulatory elements in transcription with KAS-ATAC-seq.
  11. SOX2 represses c-MYC transcription by altering the co-activator landscape of the c-MYC super-enhancer and promoter regions.
  12. MOCHA's advanced statistical modeling of scATAC-seq data enables functional genomic inference in large human cohorts.
  13. XL-DNase-Seq: Footprinting Analysis of Dynamic Transcription Factors.
  14. Prevalence of and gene regulatory constraints on transcriptional adaptation in single cells.
  15. Nuclear retention coupled with sequential polyadenylation dictates post-transcriptional m6A modification in the nucleus.
  16. Associating transcription factors to single-cell trajectories with DREAMIT.
  17. Conditional RNA interference in mammalian cells via RNA transactivation.
  18. Tracking live-cell single-molecule dynamics enables measurements of heterochromatin-associated protein-protein interactions.
  19. Decoding the genomic landscape of chromatin-associated biomolecular condensates.
  20. Protocol for establishing inducible CRISPRd system for blocking transcription factor-binding sites in human pluripotent stem cells.
  21. Arid1a-dependent canonical BAF complex suppresses inflammatory programs to drive efficient germinal center B cell responses.
  22. The molecular architecture of the nuclear basket.
  23. Histone H4 acetylation differentially modulates proliferation in adult oligodendrocyte progenitors.
  1. Mol Cell. 2024 Aug 08. pii: S1097-2765(24)00585-9. [Epub ahead of print]84(15): 2856-2869.e9
    Live-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation.
    Philip Versluis, Thomas G W Graham, Vincent Eng, Jonathan Ebenezer, Xavier Darzacq, Warren R Zipfel, John T Lis.
      RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
    Keywords:  DSIF; P-TEFb; PAF1; RNA Polymerase II; Spt6; heat shock; live-cell imaging
    DOI:  https://doi.org/10.1016/j.molcel.2024.07.009
  2. Mol Cell. 2024 Jul 31. pii: S1097-2765(24)00592-6. [Epub ahead of print]
    Structural insights into the cooperative nucleosome recognition and chromatin opening by FOXA1 and GATA4.
    Bing-Rui Zhou, Hanqiao Feng, Furong Huang, Iris Zhu, Stephanie Portillo-Ledesma, Dan Shi, Kenneth S Zaret, Tamar Schlick, David Landsman, Qianben Wang, Yawen Bai.
      Mouse FOXA1 and GATA4 are prototypes of pioneer factors, initiating liver cell development by binding to the N1 nucleosome in the enhancer of the ALB1 gene. Using cryoelectron microscopy (cryo-EM), we determined the structures of the free N1 nucleosome and its complexes with FOXA1 and GATA4, both individually and in combination. We found that the DNA-binding domains of FOXA1 and GATA4 mainly recognize the linker DNA and an internal site in the nucleosome, respectively, whereas their intrinsically disordered regions interact with the acidic patch on histone H2A-H2B. FOXA1 efficiently enhances GATA4 binding by repositioning the N1 nucleosome. In vivo DNA editing and bioinformatics analyses suggest that the co-binding mode of FOXA1 and GATA4 plays important roles in regulating genes involved in liver cell functions. Our results reveal the mechanism whereby FOXA1 and GATA4 cooperatively bind to the nucleosome through nucleosome repositioning, opening chromatin by bending linker DNA and obstructing nucleosome packing.
    Keywords:  FOXA1; GATA4; N1 nucleosome; cell fate determination; chromatin; chromatosome; enhancer; nucleosome; nucleosome array; pioneer transcription factor
    DOI:  https://doi.org/10.1016/j.molcel.2024.07.016
  3. Nat Commun. 2024 Aug 09. 15(1): 6815
    Multi-layered heterochromatin interaction as a switch for DIM2-mediated DNA methylation.
    Zengyu Shao, Jiuwei Lu, Nelli Khudaverdyan, Jikui Song.
      Functional crosstalk between DNA methylation, histone H3 lysine-9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1) is essential for proper heterochromatin assembly and genome stability. However, how repressive chromatin cues guide DNA methyltransferases for region-specific DNA methylation remains largely unknown. Here, we report structure-function characterizations of DNA methyltransferase Defective-In-Methylation-2 (DIM2) in Neurospora. The DNA methylation activity of DIM2 requires the presence of both H3K9me3 and HP1. Our structural study reveals a bipartite DIM2-HP1 interaction, leading to a disorder-to-order transition of the DIM2 target-recognition domain that is essential for substrate binding. Furthermore, the structure of DIM2-HP1-H3K9me3-DNA complex reveals a substrate-binding mechanism distinct from that for its mammalian orthologue DNMT1. In addition, the dual recognition of H3K9me3 peptide by the DIM2 RFTS and BAH1 domains allosterically impacts the DIM2-substrate binding, thereby controlling DIM2-mediated DNA methylation. Together, this study uncovers how multiple heterochromatin factors coordinately orchestrate an activity-switching mechanism for region-specific DNA methylation.
    DOI:  https://doi.org/10.1038/s41467-024-51246-4
  4. Nat Commun. 2024 Aug 12. 15(1): 6891
    HMGA1 orchestrates chromatin compartmentalization and sequesters genes into 3D networks coordinating senescence heterogeneity.
    Ioana Olan, Masami Ando-Kuri, Aled J Parry, Tetsuya Handa, Stefan Schoenfelder, Peter Fraser, Yasuyuki Ohkawa, Hiroshi Kimura, Masako Narita, Masashi Narita.
      HMGA1 is an abundant non-histone chromatin protein that has been implicated in embryonic development, cancer, and cellular senescence, but its specific role remains elusive. Here, we combine functional genomics approaches with graph theory to investigate how HMGA1 genomic deposition controls high-order chromatin networks in an oncogene-induced senescence model. While the direct role of HMGA1 in gene activation has been described previously, we find little evidence to support this. Instead, we show that the heterogeneous linear distribution of HMGA1 drives a specific 3D chromatin organization. HMGA1-dense loci form highly interactive networks, similar to, but independent of, constitutive heterochromatic loci. This, coupled with the exclusion of HMGA1-poor chromatin regions, leads to coordinated gene regulation through the repositioning of genes. In the absence of HMGA1, the whole process is largely reversed, but many regulatory interactions also emerge, amplifying the inflammatory senescence-associated secretory phenotype. Such HMGA1-mediated fine-tuning of gene expression contributes to the heterogeneous nature of senescence at the single-cell level. A similar 'buffer' effect of HMGA1 on inflammatory signalling is also detected in lung cancer cells. Our study reveals a mechanism through which HMGA1 modulates chromatin compartmentalization and gene regulation in senescence and beyond.
    DOI:  https://doi.org/10.1038/s41467-024-51153-8
  5. Nat Commun. 2024 Aug 09. 15(1): 6810
    Super enhancer acquisition drives expression of oncogenic PPP1R15B that regulates protein homeostasis in multiple myeloma.
    Sinan Xiong, Jianbiao Zhou, Tze King Tan, Tae-Hoon Chung, Tuan Zea Tan, Sabrina Hui-Min Toh, Nicole Xin Ning Tang, Yunlu Jia, Yi Xiang See, Melissa Jane Fullwood, Takaomi Sanda, Wee-Joo Chng.
      Multiple myeloma is a hematological malignancy arising from immunoglobulin-secreting plasma cells. It remains poorly understood how chromatin rewiring of regulatory elements contributes to tumorigenesis and therapy resistance in myeloma. Here we generate a high-resolution contact map of myeloma-associated super-enhancers by integrating H3K27ac ChIP-seq and HiChIP from myeloma cell lines, patient-derived myeloma cells and normal plasma cells. Our comprehensive transcriptomic and phenomic analyses prioritize candidate genes with biological and clinical implications in myeloma. We show that myeloma cells frequently acquire SE that transcriptionally activate an oncogene PPP1R15B, which encodes a regulatory subunit of the holophosphatase complex that dephosphorylates translation initiation factor eIF2α. Epigenetic silencing or knockdown of PPP1R15B activates pro-apoptotic eIF2α-ATF4-CHOP pathway, while inhibiting protein synthesis and immunoglobulin production. Pharmacological inhibition of PPP1R15B using Raphin1 potentiates the anti-myeloma effect of bortezomib. Our study reveals that myeloma cells are vulnerable to perturbation of PPP1R15B-dependent protein homeostasis, highlighting a promising therapeutic strategy.
    DOI:  https://doi.org/10.1038/s41467-024-50910-z
  6. Genome Biol. 2024 Aug 14. 25(1): 221
    Comprehensive network modeling approaches unravel dynamic enhancer-promoter interactions across neural differentiation.
    William DeGroat, Fumitaka Inoue, Tal Ashuach, Nir Yosef, Nadav Ahituv, Anat Kreimer.
       BACKGROUND: Increasing evidence suggests that a substantial proportion of disease-associated mutations occur in enhancers, regions of non-coding DNA essential to gene regulation. Understanding the structures and mechanisms of the regulatory programs this variation affects can shed light on the apparatuses of human diseases.
    RESULTS: We collect epigenetic and gene expression datasets from seven early time points during neural differentiation. Focusing on this model system, we construct networks of enhancer-promoter interactions, each at an individual stage of neural induction. These networks serve as the base for a rich series of analyses, through which we demonstrate their temporal dynamics and enrichment for various disease-associated variants. We apply the Girvan-Newman clustering algorithm to these networks to reveal biologically relevant substructures of regulation. Additionally, we demonstrate methods to validate predicted enhancer-promoter interactions using transcription factor overexpression and massively parallel reporter assays.
    CONCLUSIONS: Our findings suggest a generalizable framework for exploring gene regulatory programs and their dynamics across developmental processes; this includes a comprehensive approach to studying the effects of disease-associated variation on transcriptional networks. The techniques applied to our networks have been published alongside our findings as a computational tool, E-P-INAnalyzer. Our procedure can be utilized across different cellular contexts and disorders.
    Keywords:  Computational genomics; Functional genomics; Gene regulation; Neural development
    DOI:  https://doi.org/10.1186/s13059-024-03365-w
  7. PLoS Biol. 2024 Aug 16. 22(8): e3002770
    bHLH transcription factors cooperate with chromatin remodelers to regulate cell fate decisions during Arabidopsis stomatal development.
    Ao Liu, Andrea Mair, Juliana L Matos, Macy Vollbrecht, Shou-Ling Xu, Dominique C Bergmann.
      The development of multicellular organisms requires coordinated changes in gene expression that are often mediated by the interaction between transcription factors (TFs) and their corresponding cis-regulatory elements (CREs). During development and differentiation, the accessibility of CREs is dynamically modulated by the epigenome. How the epigenome, CREs, and TFs together exert control over cell fate commitment remains to be fully understood. In the Arabidopsis leaf epidermis, meristemoids undergo a series of stereotyped cell divisions, then switch fate to commit to stomatal differentiation. Newly created or reanalyzed scRNA-seq and ChIP-seq data confirm that stomatal development involves distinctive phases of transcriptional regulation and that differentially regulated genes are bound by the stomatal basic helix-loop-helix (bHLH) TFs. Targets of the bHLHs often reside in repressive chromatin before activation. MNase-seq evidence further suggests that the repressive state can be overcome and remodeled upon activation by specific stomatal bHLHs. We propose that chromatin remodeling is mediated through the recruitment of a set of physical interactors that we identified through proximity labeling-the ATPase-dependent chromatin remodeling SWI/SNF complex and the histone acetyltransferase HAC1. The bHLHs and chromatin remodelers localize to overlapping genomic regions in a hierarchical order. Furthermore, plants with stage-specific knockdown of the SWI/SNF components or HAC1 fail to activate specific bHLH targets and display stomatal development defects. Together, these data converge on a model for how stomatal TFs and epigenetic machinery cooperatively regulate transcription and chromatin remodeling during progressive fate specification.
    DOI:  https://doi.org/10.1371/journal.pbio.3002770
  8. Nucleic Acids Res. 2024 Aug 15. pii: gkae677. [Epub ahead of print]
    The C-terminal 4CXXC-type zinc finger domain of CDCA7 recognizes hemimethylated DNA and modulates activities of chromatin remodeling enzyme HELLS.
    Akeo Shinkai, Hideharu Hashimoto, Chikako Shimura, Hiroaki Fujimoto, Kei Fukuda, Naoki Horikoshi, Masaki Okano, Hitoshi Niwa, Erik W Debler, Hitoshi Kurumizaka, Yoichi Shinkai.
      The chromatin-remodeling enzyme helicase lymphoid-specific (HELLS) interacts with cell division cycle-associated 7 (CDCA7) on nucleosomes and is involved in the regulation of DNA methylation in higher organisms. Mutations in these genes cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, which also results in DNA hypomethylation of satellite repeat regions. We investigated the functional domains of human CDCA7 in HELLS using several mutant CDCA7 proteins. The central region is critical for binding to HELLS, activation of ATPase, and nucleosome sliding activities of HELLS-CDCA7. The N-terminal region tends to inhibit ATPase activity. The C-terminal 4CXXC-type zinc finger domain contributes to CpG and hemimethylated CpG DNA preference for DNA-dependent HELLS-CDCA7 ATPase activity. Furthermore, CDCA7 showed a binding preference to DNA containing hemimethylated CpG, and replication-dependent pericentromeric heterochromatin foci formation of CDCA7 with HELLS was observed in mouse embryonic stem cells; however, all these phenotypes were lost in the case of an ICF syndrome mutant of CDCA7 mutated in the zinc finger domain. Thus, CDCA7 most likely plays a role in the recruitment of HELLS, activates its chromatin remodeling function, and efficiently induces DNA methylation, especially at hemimethylated replication sites.
    DOI:  https://doi.org/10.1093/nar/gkae677
  9. Nat Commun. 2024 Aug 09. 15(1): 6841
    Combinatorial regulatory states define cell fate diversity during embryogenesis.
    Jonathan E Valencia, Isabelle S Peter.
      Cell fate specification occurs along invariant species-specific trajectories that define the animal body plan. This process is controlled by gene regulatory networks that regulate the expression of the limited set of transcription factors encoded in animal genomes. Here we globally assess the spatial expression of ~90% of expressed transcription factors during sea urchin development from embryo to larva to determine the activity of gene regulatory networks and their regulatory states during cell fate specification. We show that >200 embryonically expressed transcription factors together define >70 cell fates that recapitulate the morphological and functional organization of this organism. Most cell fate-specific regulatory states consist of ~15-40 transcription factors with similarity particularly among functionally related cell types regardless of developmental origin. Temporally, regulatory states change continuously during development, indicating that progressive changes in regulatory circuit activity determine cell fate specification. We conclude that the combinatorial expression of transcription factors provides molecular definitions that suffice for the unique specification of cell states in time and space during embryogenesis.
    DOI:  https://doi.org/10.1038/s41467-024-50822-y
  10. Nat Commun. 2024 Aug 10. 15(1): 6852
    Quantitative analysis of cis-regulatory elements in transcription with KAS-ATAC-seq.
    Ruitu Lyu, Yun Gao, Tong Wu, Chang Ye, Pingluan Wang, Chuan He.
      Cis-regulatory elements (CREs) are pivotal in orchestrating gene expression throughout diverse biological systems. Accurate identification and in-depth characterization of functional CREs are crucial for decoding gene regulation networks during cellular processes. In this study, we develop Kethoxal-Assisted Single-stranded DNA Assay for Transposase-Accessible Chromatin with Sequencing (KAS-ATAC-seq) to quantitatively analyze the transcriptional activity of CREs. A main advantage of KAS-ATAC-seq lies in its precise measurement of ssDNA levels within both proximal and distal ATAC-seq peaks, enabling the identification of transcriptional regulatory sequences. This feature is particularly adept at defining Single-Stranded Transcribing Enhancers (SSTEs). SSTEs are highly enriched with nascent RNAs and specific transcription factors (TFs) binding sites that define cellular identity. Moreover, KAS-ATAC-seq provides a detailed characterization and functional implications of various SSTE subtypes. Our analysis of CREs during mouse neural differentiation demonstrates that KAS-ATAC-seq can effectively identify immediate-early activated CREs in response to retinoic acid (RA) treatment. Our findings indicate that KAS-ATAC-seq provides more precise annotation of functional CREs in transcription. Future applications of KAS-ATAC-seq would help elucidate the intricate dynamics of gene regulation in diverse biological processes.
    DOI:  https://doi.org/10.1038/s41467-024-50680-8
  11. J Biol Chem. 2024 Aug 07. pii: S0021-9258(24)02143-4. [Epub ahead of print] 107642
    SOX2 represses c-MYC transcription by altering the co-activator landscape of the c-MYC super-enhancer and promoter regions.
    Briana D Ormsbee Golden, Daisy V Gonzalez, Gregory S Yochum, Donald W Coulter, Angie Rizzino.
      Our previous studies determined that elevating SOX2 in a wide range of tumor cells leads to a reversible state of tumor growth arrest. Efforts to understand how tumor cell growth is inhibited led to the discovery of a SOX2:MYC axis that is responsible for downregulating c-MYC (MYC) when SOX2 is elevated. Although we had determined that elevating SOX2 downregulates MYC transcription, the mechanism responsible was not determined. Given the challenges of targeting MYC clinically, we set out to identify how elevating SOX2 downregulates MYC transcription. In this study, we focused on the MYC promoter region and an upstream region of the MYC locus that contains a MYC super-enhancer encompassing five MYC enhancers and which is associated with several cancers. Here we report that BRD4 and p300 associate with each of the MYC enhancers in the upstream MYC super-enhancer as well as the MYC promoter region and that elevating SOX2 decreases the recruitment of BRD4 and p300 to these sites. Additionally, we determined that elevating SOX2 leads to increases in the association of SOX2 and H3K27me3 within the MYC super-enhancer and the promoter region of MYC. Importantly, we conclude that the increases in SOX2 within the MYC super-enhancer precipitate a cascade of events that culminates in the repression of MYC transcription. Together, our studies identify a novel molecular mechanism able to regulate MYC transcription in two distinctly different tumor types and provide new mechanistic insights into the molecular interrelationships between two master regulators, SOX2 and MYC, widely involved in multiple cancers.
    Keywords:  A-485; BRD4; RNA polymerase II; SOX2; c-MYC; colorectal cancer; medulloblastoma; p300; super-enhancer; transcription
    DOI:  https://doi.org/10.1016/j.jbc.2024.107642
  12. Nat Commun. 2024 Aug 09. 15(1): 6828
    MOCHA's advanced statistical modeling of scATAC-seq data enables functional genomic inference in large human cohorts.
    Samir Rachid Zaim, Mark-Phillip Pebworth, Imran McGrath, Lauren Okada, Morgan Weiss, Julian Reading, Julie L Czartoski, Troy R Torgerson, M Juliana McElrath, Thomas F Bumol, Peter J Skene, Xiao-Jun Li.
      Single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) is being increasingly used to study gene regulation. However, major analytical gaps limit its utility in studying gene regulatory programs in complex diseases. In response, MOCHA (Model-based single cell Open CHromatin Analysis) presents major advances over existing analysis tools, including: 1) improving identification of sample-specific open chromatin, 2) statistical modeling of technical drop-out with zero-inflated methods, 3) mitigation of false positives in single cell analysis, 4) identification of alternative transcription-starting-site regulation, and 5) modules for inferring temporal gene regulatory networks from longitudinal data. These advances, in addition to open chromatin analyses, provide a robust framework after quality control and cell labeling to study gene regulatory programs in human disease. We benchmark MOCHA with four state-of-the-art tools to demonstrate its advances. We also construct cross-sectional and longitudinal gene regulatory networks, identifying potential mechanisms of COVID-19 response. MOCHA provides researchers with a robust analytical tool for functional genomic inference from scATAC-seq data.
    DOI:  https://doi.org/10.1038/s41467-024-50612-6
  13. Methods Mol Biol. 2024 ;2846 243-261
    XL-DNase-Seq: Footprinting Analysis of Dynamic Transcription Factors.
    Kyu-Seon Oh, Mohammad Aqdas, Myong-Hee Sung.
      We have developed a novel method for genomic footprinting of transcription factors (TFs) that detects potential gene regulatory relationships from DNase-seq data at the nucleotide level. We introduce an assay termed cross-link (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. A mild cross-linking step in XL-DNase-seq improves the detection of DNase-based footprints of dynamic TFs. The footprint strengths and detectability depend on an optimal cross-linking procedure. This method may help extract novel gene regulatory circuits involving previously undetectable TFs. The XL-DNase-seq method is illustrated here for activated mouse macrophage-like cells, which share several features with inflammatory macrophages.
    Keywords:  Chromatin accessibility; DNase-seq; Genomic footprinting; High-throughput sequencing; Transcription factor footprinting
    DOI:  https://doi.org/10.1007/978-1-0716-4071-5_15
  14. Genome Biol. 2024 Aug 12. 25(1): 217
    Prevalence of and gene regulatory constraints on transcriptional adaptation in single cells.
    Ian A Mellis, Madeline E Melzer, Nicholas Bodkin, Yogesh Goyal.
       BACKGROUND: Cells and tissues have a remarkable ability to adapt to genetic perturbations via a variety of molecular mechanisms. Nonsense-induced transcriptional compensation, a form of transcriptional adaptation, has recently emerged as one such mechanism, in which nonsense mutations in a gene trigger upregulation of related genes, possibly conferring robustness at cellular and organismal levels. However, beyond a handful of developmental contexts and curated sets of genes, no comprehensive genome-wide investigation of this behavior has been undertaken for mammalian cell types and conditions. How the regulatory-level effects of inherently stochastic compensatory gene networks contribute to phenotypic penetrance in single cells remains unclear.
    RESULTS: We analyze existing bulk and single-cell transcriptomic datasets to uncover the prevalence of transcriptional adaptation in mammalian systems across diverse contexts and cell types. We perform regulon gene expression analyses of transcription factor target sets in both bulk and pooled single-cell genetic perturbation datasets. Our results reveal greater robustness in expression of regulons of transcription factors exhibiting transcriptional adaptation compared to those of transcription factors that do not. Stochastic mathematical modeling of minimal compensatory gene networks qualitatively recapitulates several aspects of transcriptional adaptation, including paralog upregulation and robustness to mutation. Combined with machine learning analysis of network features of interest, our framework offers potential explanations for which regulatory steps are most important for transcriptional adaptation.
    CONCLUSIONS: Our integrative approach identifies several putative hits-genes demonstrating possible transcriptional adaptation-to follow-up on experimentally and provides a formal quantitative framework to test and refine models of transcriptional adaptation.
    DOI:  https://doi.org/10.1186/s13059-024-03351-2
  15. Mol Cell. 2024 Aug 01. pii: S1097-2765(24)00593-8. [Epub ahead of print]
    Nuclear retention coupled with sequential polyadenylation dictates post-transcriptional m6A modification in the nucleus.
    Peng Tang, Jiayi Yang, Zonggui Chen, Chen Du, Yang Yang, Haiping Zhao, Li Huang, Guangnan Li, Feiyan Liu, Bei Dong, Ting Shan, Xichen Bao, Yu Zhou.
      N6-methyladenosine (m6A) modification is deemed to be co-transcriptionally installed on pre-mRNAs, thereby influencing various downstream RNA metabolism events. However, the causal relationship between m6A modification and RNA processing is often unclear, resulting in premature or even misleading generalizations on the function of m6A modification. Here, we develop 4sU-coupled m6A-level and isoform-characterization sequencing (4sU-m6A-LAIC-seq) and 4sU-GLORI to quantify the m6A levels for both newly synthesized and steady-state RNAs at transcript and single-base-resolution levels, respectively, which enable dissecting the relationship between m6A modification and alternative RNA polyadenylation. Unexpectedly, our results show that many m6A addition events occur post-transcriptionally, especially on transcripts with high m6A levels. Importantly, we find higher m6A levels on shorter 3' UTR isoforms, which likely result from sequential polyadenylation of longer 3' UTR isoforms with prolonged nuclear dwelling time. Therefore, m6A modification can also take place post-transcriptionally to intimately couple with other key RNA metabolism processes to establish and dynamically regulate epi-transcriptomics in mammalian cells.
    Keywords:  m(6)A modification; nuclear retention; post-transcriptional RNA processing; sequential polyadenylation
    DOI:  https://doi.org/10.1016/j.molcel.2024.07.017
  16. Genome Biol. 2024 Aug 14. 25(1): 220
    Associating transcription factors to single-cell trajectories with DREAMIT.
    Nathan D Maulding, Lucas Seninge, Joshua M Stuart.
      Inferring gene regulatory networks from single-cell RNA-sequencing trajectories has been an active area of research yet methods are still needed to identify regulators governing cell transitions. We developed DREAMIT (Dynamic Regulation of Expression Across Modules in Inferred Trajectories) to annotate transcription-factor activity along single-cell trajectory branches, using ensembles of relations to target genes. Using a benchmark representing several different tissues, as well as external validation with ATAC-Seq and Perturb-Seq data on hematopoietic cells, the method was found to have higher tissue-specific sensitivity and specificity over competing approaches.
    DOI:  https://doi.org/10.1186/s13059-024-03368-7
  17. Nat Commun. 2024 Aug 10. 15(1): 6855
    Conditional RNA interference in mammalian cells via RNA transactivation.
    Yu Zhou, Peike Sheng, Jiayi Li, Yudan Li, Mingyi Xie, Alexander A Green.
      RNA interference (RNAi) is a powerful tool for sequence-specific gene knockdown in therapeutic and research applications. However, spatiotemporal control of RNAi is required to decrease nonspecific targeting, potential toxicity, and allow targeting of essential genes. Herein we describe a class of de-novo-designed RNA switches that enable sequence-specific regulation of RNAi in mammalian cells. Using cis-repressing RNA elements, we engineer RNA devices that only initiate microRNA biogenesis when binding with cognate trigger RNAs. We demonstrate that this conditional RNAi system, termed Orthogonal RNA Interference induced by Trigger RNA (ORIENTR), provides up to 14-fold increases in artificial miRNA biogenesis upon activation in orthogonal libraries. We show that integration of ORIENTR triggers with dCas13d enhances dynamic range to up to 31-fold. We further demonstrate that ORIENTR can be applied to detect endogenous RNA signals and to conditionally knockdown endogenous genes, thus enabling regulatory possibilities including cell-type-specific RNAi and rewiring of transcriptional networks via RNA profile.
    DOI:  https://doi.org/10.1038/s41467-024-50600-w
  18. Nucleic Acids Res. 2024 Aug 15. pii: gkae692. [Epub ahead of print]
    Tracking live-cell single-molecule dynamics enables measurements of heterochromatin-associated protein-protein interactions.
    Ziyuan Chen, Melissa Seman, Yekaterina Fyodorova, Ali Farhat, Amanda Ames, Alexander Levashkevich, Saikat Biswas, Fengting Huang, Lydia Freddolino, Julie S Biteen, Kaushik Ragunathan.
      Visualizing and measuring molecular-scale interactions in living cells represents a major challenge, but recent advances in single-molecule super-resolution microscopy are bringing us closer to achieving this goal. Single-molecule super-resolution microscopy enables high-resolution and sensitive imaging of the positions and movement of molecules in living cells. HP1 proteins are important regulators of gene expression because they selectively bind and recognize H3K9 methylated (H3K9me) histones to form heterochromatin-associated protein complexes that silence gene expression, but several important mechanistic details of this process remain unexplored. Here, we extended live-cell single-molecule tracking studies in fission yeast to determine how HP1 proteins interact with their binding partners in the nucleus. We measured how genetic perturbations that affect H3K9me alter the diffusive properties of HP1 proteins and their binding partners, and we inferred their most likely interaction sites. Our results demonstrate that H3K9 methylation spatially restricts HP1 proteins and their interactors, thereby promoting ternary complex formation on chromatin while simultaneously suppressing off-chromatin binding. As opposed to being an inert platform to direct HP1 binding, our studies propose a novel function for H3K9me in promoting ternary complex formation by enhancing the specificity and stimulating the assembly of HP1-protein complexes in living cells.
    DOI:  https://doi.org/10.1093/nar/gkae692
  19. Nat Commun. 2024 Aug 13. 15(1): 6952
    Decoding the genomic landscape of chromatin-associated biomolecular condensates.
    Zhaowei Yu, Qi Wang, Qichen Zhang, Yawen Tian, Guo Yan, Jidong Zhu, Guangya Zhu, Yong Zhang.
      Biomolecular condensates play a significant role in chromatin activities, primarily by concentrating and compartmentalizing proteins and/or nucleic acids. However, their genomic landscapes and compositions remain largely unexplored due to a lack of dedicated computational tools for systematic identification in vivo. To address this, we develop CondSigDetector, a computational framework designed to detect condensate-like chromatin-associated protein co-occupancy signatures (CondSigs), to predict genomic loci and component proteins of distinct chromatin-associated biomolecular condensates. Applying this framework to mouse embryonic stem cells (mESC) and human K562 cells enable us to depict the high-resolution genomic landscape of chromatin-associated biomolecular condensates, and uncover both known and potentially unknown biomolecular condensates. Multi-omics analysis and experimental validation further verify the condensation properties of CondSigs. Additionally, our investigation sheds light on the impact of chromatin-associated biomolecular condensates on chromatin activities. Collectively, CondSigDetector provides an approach to decode the genomic landscape of chromatin-associated condensates, facilitating a deeper understanding of their biological functions and underlying mechanisms in cells.
    DOI:  https://doi.org/10.1038/s41467-024-51426-2
  20. STAR Protoc. 2024 Aug 11. pii: S2666-1667(24)00398-8. [Epub ahead of print]5(3): 103233
    Protocol for establishing inducible CRISPRd system for blocking transcription factor-binding sites in human pluripotent stem cells.
    Satoshi Matsui, Joseph R Shiley, Morgan Buckley, Hee-Woong Lim, Yueh-Chiang Hu, Christopher N Mayhew, Makiko Iwafuchi.
      Transcription factor (TF) gene knockout or knockdown experiments provide comprehensive downstream effects on gene regulation. However, distinguishing primary direct effects from secondary effects remains challenging. To assess the direct effect of TF binding events, we present a protocol for establishing a doxycycline (Dox)-inducible CRISPRd system in human pluripotent stem cells (hPSCs). We describe the steps for establishing CRISPRd host hPSCs, designing and preparing single-guide RNA (sgRNA) expression lentivirus vectors, generating CRISPRd hPSCs transduced with sgRNAs, and analyzing CRISPRd TF-block effects by chromatin immunoprecipitation (ChIP)-qPCR. For complete details on the use and execution of this protocol, please refer to Matsui et al.1.
    Keywords:  CRISPR; Cell Biology; Cell Differentiation; Cell culture; Cell isolation; ChIP; Chromatin immunoprecipitation; Flow Cytometry; Molecular Biology; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2024.103233
  21. Nat Immunol. 2024 Aug 14.
    Arid1a-dependent canonical BAF complex suppresses inflammatory programs to drive efficient germinal center B cell responses.
    Ajay Abraham, Daniela Samaniego-Castruita, Isabella Han, Prathyaya Ramesh, Mi Thao Tran, Jillian Paladino, Heather Kligfeld, Roxroy C Morgan, Rebecca L Schmitz, Rebecca M Southern, Ashima Shukla, Vipul Shukla.
      The mammalian Brg1/Brm-associated factor (BAF) complexes are major regulators of nucleosomal remodeling that are commonly mutated in several cancers, including germinal center (GC)-derived B cell lymphomas. However, the specific roles of different BAF complexes in GC B cell biology are not well understood. Here we show that the AT-rich interaction domain 1a (Arid1a) containing canonical BAF (cBAF) complex is required for maintenance of GCs and high-affinity antibody responses. While Arid1a-deficient B cells undergo initial activation, they fail to sustain the GC program. Arid1a establishes permissive chromatin landscapes for B cell activation and is concomitantly required to suppress inflammatory gene programs. The inflammatory signatures instigated by Arid1a deficiency promoted the recruitment of neutrophils and inflammatory monocytes. Dampening of inflammatory cues through interleukin-1β blockade or glucocorticoid receptor agonist partially rescued Arid1a-deficient GCs, highlighting a critical role for inflammation in impeding GCs. Our work reveals essential functions of Arid1a-dependent cBAF in promoting efficient GC responses.
    DOI:  https://doi.org/10.1038/s41590-024-01920-y
  22. Cell. 2024 Aug 05. pii: S0092-8674(24)00780-3. [Epub ahead of print]
    The molecular architecture of the nuclear basket.
    Digvijay Singh, Neelesh Soni, Joshua Hutchings, Ignacia Echeverria, Farhaz Shaikh, Madeleine Duquette, Sergey Suslov, Zhixun Li, Trevor van Eeuwen, Kelly Molloy, Yi Shi, Junjie Wang, Qiang Guo, Brian T Chait, Javier Fernandez-Martinez, Michael P Rout, Andrej Sali, Elizabeth Villa.
      The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.
    Keywords:  NPC; chromatin organization; cross-linking mass spectrometry; cryo-electron tomography; cryo-focused-ion-beam milling; in-cell structural biology; integrative modeling; mRNA transport; nuclear basket; nuclear pore complex; subtomogram analysis
    DOI:  https://doi.org/10.1016/j.cell.2024.07.020
  23. J Cell Biol. 2024 Nov 04. pii: e202308064. [Epub ahead of print]223(11):
    Histone H4 acetylation differentially modulates proliferation in adult oligodendrocyte progenitors.
    David K Dansu, Ipek Selcen, Sami Sauma, Emily Prentice, Dennis Huang, Meng Li, Sarah Moyon, Patrizia Casaccia.
      Adult oligodendrocyte progenitors (aOPCs) generate myelinating oligodendrocytes like neonatal progenitors (nOPCs), and they also display unique functional features. Here, using unbiased histone proteomics analysis and ChIP sequencing analysis of PDGFRα+ OPCs sorted from neonatal and adult Pdgfra-H2B-EGFP reporter mice, we identify the activating H4K8ac histone mark as enriched in the aOPCs. We detect increased occupancy of the H4K8ac activating mark at chromatin locations corresponding to genes related to the progenitor state (e.g., Hes5, Gpr17), metabolic processes (e.g., Txnip, Ptdgs), and myelin components (e.g., Cnp, Mog). aOPCs showed higher levels of transcripts related to lipid metabolism and myelin, and lower levels of transcripts related to cell cycle and proliferation compared with nOPCs. In addition, pharmacological inhibition of histone acetylation decreased the expression of the H4K8ac target genes in aOPCs and decreased their proliferation. Overall, this study identifies acetylation of the histone H4K8 as a regulator of the proliferative capacity of aOPCs.
    DOI:  https://doi.org/10.1083/jcb.202308064
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