bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024–04–21
twenty-six papers selected by
Connor Rogerson, University of Cambridge
  1. Detection of newly synthesized RNA reveals transcriptional reprogramming during ZGA and a role of Obox3 in totipotency acquisition.
  2. Physical and functional interaction among Irf8 enhancers during dendritic cell differentiation.
  3. A fine-scale Arabidopsis chromatin landscape reveals chromatin conformation-associated transcriptional dynamics.
  4. A critical role for Pol II CTD phosphorylation in heterochromatic gene activation.
  5. Nuclear receptor corepressors non-canonically drive glucocorticoid receptor-dependent activation of hepatic gluconeogenesis.
  6. Differentiation shifts from a reversible to an irreversible heterochromatin state at the DM1 locus.
  7. Engineering a synthetic gene circuit for high-performance inducible expression in mammalian systems.
  8. Combined and differential roles of ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells.
  9. FOXA1 regulates ribosomal RNA transcription in prostate cancer.
  10. Switch of ELF3 and ATF4 transcriptional axis programs the amino acid insufficiency-linked epithelial-to-mesenchymal transition.
  11. PU.1 and BCL11B sequentially cooperate with RUNX1 to anchor mSWI/SNF to poise the T cell effector landscape.
  12. Analysis of REST binding sites with canonical and non-canonical motifs in human cell lines.
  13. Simultaneous single-cell three-dimensional genome and gene expression profiling uncovers dynamic enhancer connectivity underlying olfactory receptor choice.
  14. Probabilistic association of differentially expressed genes with cis-regulatory elements.
  15. Von Hippel Lindau tumor suppressor controls m6A-dependent gene expression in renal tumorigenesis.
  16. RUNX1 regulates promoter activity in the absence of cognate DNA binding motifs.
  17. CHD4 and SMYD1 repress common transcriptional programs in the developing heart.
  18. Tissue-specific atlas of trans-models for gene regulation elucidates complex regulation patterns.
  19. m6ACali: machine learning-powered calibration for accurate m6A detection in MeRIP-Seq.
  20. Detection of ac4C in human mRNA is preserved upon data reassessment.
  21. An esophagus cell atlas reveals dynamic rewiring during active eosinophilic esophagitis and remission.
  22. Sustained ERK signaling promotes G2 cell cycle exit and primes cells for whole-genome duplication.
  23. SWI/SNF complex-mediated chromatin remodeling in Candida glabrata promotes immune evasion.
  24. RBM22 regulates RNA polymerase II 5' pausing, elongation rate, and termination by coordinating 7SK-P-TEFb complex and SPT5.
  25. No evidence for ac4C within human mRNA upon data reassessment.
  26. Dual-role transcription factors stabilize intermediate expression levels.
  1. Cell Rep. 2024 Apr 14. pii: S2211-1247(24)00446-7. [Epub ahead of print]43(4): 114118
    Detection of newly synthesized RNA reveals transcriptional reprogramming during ZGA and a role of Obox3 in totipotency acquisition.
    Mizuki Sakamoto, Aoi Ito, Sayaka Wakayama, Hiroyuki Sasaki, Teruhiko Wakayama, Takashi Ishiuchi.
      Zygotic genome activation (ZGA) after fertilization enables the maternal-to-zygotic transition. However, the global view of ZGA, particularly at initiation, is incompletely understood. Here, we develop a method to capture and sequence newly synthesized RNA in early mouse embryos, providing a view of transcriptional reprogramming during ZGA. Our data demonstrate that major ZGA gene activation begins earlier than previously thought. Furthermore, we identify a set of genes activated during minor ZGA, the promoters of which show enrichment of the Obox factor motif, and find that Obox3 or Obox5 overexpression in mouse embryonic stem cells activates ZGA genes. Notably, the expression of Obox factors is severely impaired in somatic cell nuclear transfer (SCNT) embryos, and restoration of Obox3 expression corrects the ZGA profile and greatly improves SCNT embryo development. Hence, our study reveals dynamic transcriptional reprogramming during ZGA and underscores the crucial role of Obox3 in facilitating totipotency acquisition.
    Keywords:  CP: Developmental biology; CP: Molecular biology; Obox3; mouse early development; newly synthesized RNA; somatic cell nuclear transfer; zygotic genome activation
    DOI:  https://doi.org/10.1016/j.celrep.2024.114118
  2. Cell Rep. 2024 Apr 12. pii: S2211-1247(24)00435-2. [Epub ahead of print]43(4): 114107
    Physical and functional interaction among Irf8 enhancers during dendritic cell differentiation.
    Takaya Yamasaki, Akira Nishiyama, Nagomi Kurogi, Koutarou Nishimura, Shion Nishida, Daisuke Kurotaki, Tatsuma Ban, Jordan A Ramilowski, Keiko Ozato, Atsushi Toyoda, Tomohiko Tamura.
      The production of type 1 conventional dendritic cells (cDC1s) requires high expression of the transcription factor IRF8. Three enhancers at the Irf8 3' region function in a differentiation stage-specific manner. However, whether and how these enhancers interact physically and functionally remains unclear. Here, we show that the Irf8 3' enhancers directly interact with each other and contact the Irf8 gene body during cDC1 differentiation. The +56 kb enhancer, which functions from multipotent progenitor stages, activates the other 3' enhancers through an IRF8-dependent transcription factor program, that is, in trans. Then, the +32 kb enhancer, which operates in cDC1-committed cells, reversely acts in cis on the other 3' enhancers to maintain the high expression of Irf8. Indeed, mice with compound heterozygous deletion of the +56 and +32 kb enhancers are unable to generate cDC1s. These results illustrate how multiple enhancers cooperate to induce a lineage-determining transcription factor gene during cell differentiation.
    Keywords:  CP: Immunology; dendritic cell; enhancer; higher-order chromatin structure; transcription factor
    DOI:  https://doi.org/10.1016/j.celrep.2024.114107
  3. Nat Commun. 2024 Apr 16. 15(1): 3253
    A fine-scale Arabidopsis chromatin landscape reveals chromatin conformation-associated transcriptional dynamics.
    Yueying Zhang, Qianli Dong, Zhen Wang, Qinzhe Liu, Haopeng Yu, Wenqing Sun, Jitender Cheema, Qiancheng You, Ling Ding, Xiaofeng Cao, Chuan He, Yiliang Ding, Huakun Zhang.
      Plants, as sessile organisms, deploy transcriptional dynamics for adapting to extreme growth conditions such as cold stress. Emerging evidence suggests that chromatin architecture contributes to transcriptional regulation. However, the relationship between chromatin architectural dynamics and transcriptional reprogramming in response to cold stress remains unclear. Here, we apply a chemical-crosslinking assisted proximity capture (CAP-C) method to elucidate the fine-scale chromatin landscape, revealing chromatin interactions within gene bodies closely associated with RNA polymerase II (Pol II) densities across initiation, pausing, and termination sites. We observe dynamic changes in chromatin interactions alongside Pol II activity alterations during cold stress, suggesting local chromatin dynamics may regulate Pol II activity. Notably, cold stress does not affect large-scale chromatin conformations. We further identify a comprehensive promoter-promoter interaction (PPI) network across the genome, potentially facilitating co-regulation of gene expression in response to cold stress. Our study deepens the understanding of chromatin conformation-associated gene regulation in plant response to cold.
    DOI:  https://doi.org/10.1038/s41467-024-47678-7
  4. Gene. 2024 Apr 12. pii: S0378-1119(24)00354-8. [Epub ahead of print] 148473
    A critical role for Pol II CTD phosphorylation in heterochromatic gene activation.
    Amoldeep S Kainth, Hesheng Zhang, David S Gross.
      How gene activation works in heterochromatin, and how the mechanism might differ from the one used in euchromatin, has been largely unexplored. Previous work has shown that in SIR-regulated heterochromatin of Saccharomyces cerevisiae, gene activation occurs in the absence of covalent histone modifications and other alterations of chromatin commonly associated with transcription.Here we demonstrate that such activation occurs in a substantial fraction of cells, consistent with frequent transcriptional bursting, and this raises the possibility that an alternative activation pathway might be used. We address one such possibility, Pol II CTD phosphorylation, and explore this idea using a natural telomere-linked gene, YFR057w, as a model. Unlike covalent histone modifications, we find that Ser2, Ser5 and Ser7 CTD phosphorylated Pol II is prevalent at the drug-induced heterochromatic gene. Particularly enriched relative to the euchromatic state is Ser2 phosphorylation. Consistent with a functional role for Ser2P, YFR057w is negligibly activated in cells deficient in the Ser2 CTD kinases Ctk1 and Bur1 even though the gene is strongly stimulated when it is placed in a euchromatic context. Collectively, our results are consistent with a critical role for Ser2 CTD phosphorylation in driving Pol II recruitment and transcription of a natural heterochromatic gene - an activity that may supplant the need for histone epigenetic modifications.
    Keywords:  Bur1; CTD code; ChIP; Ctk1; Euchromatin; Fluorescence Microscopy; Gene Activation; Heat Shock; Heterochromatin; Histone Code; Nucleosomes; RNA Polymerase II; Sir2/3/4-mediated Silent Chromatin; Transcription; Yeast
    DOI:  https://doi.org/10.1016/j.gene.2024.148473
  5. Nat Metab. 2024 Apr 15.
    Nuclear receptor corepressors non-canonically drive glucocorticoid receptor-dependent activation of hepatic gluconeogenesis.
    Amy K Hauck, Rashid Mehmood, Bryce J Carpenter, Maxwell T Frankfurter, Michael C Tackenberg, Shin-Ichi Inoue, Maria K Krieg, Fathima N Cassim Bawa, Mohit K Midha, Delaine M Zundell, Kirill Batmanov, Mitchell A Lazar.
      Nuclear receptor corepressors (NCoRs) function in multiprotein complexes containing histone deacetylase 3 (HDAC3) to alter transcriptional output primarily through repressive chromatin remodelling at target loci1-5. In the liver, loss of HDAC3 causes a marked hepatosteatosis largely because of de-repression of genes involved in lipid metabolism6,7; however, the individual roles and contribution of other complex members to hepatic and systemic metabolic regulation are unclear. Here we show that adult loss of both NCoR1 and NCoR2 (double knockout (KO)) in hepatocytes phenocopied the hepatomegalic fatty liver phenotype of HDAC3 KO. In addition, double KO livers exhibited a dramatic reduction in glycogen storage and gluconeogenic gene expression that was not observed with hepatic KO of individual NCoRs or HDAC3, resulting in profound fasting hypoglycaemia. This surprising HDAC3-independent activation function of NCoR1 and NCoR2 is due to an unexpected loss of chromatin accessibility on deletion of NCoRs that prevented glucocorticoid receptor binding and stimulatory effect on gluconeogenic genes. These studies reveal an unanticipated, non-canonical activation function of NCoRs that is required for metabolic health.
    DOI:  https://doi.org/10.1038/s42255-024-01029-4
  6. Nat Commun. 2024 Apr 16. 15(1): 3270
    Differentiation shifts from a reversible to an irreversible heterochromatin state at the DM1 locus.
    Tayma Handal, Sarah Juster, Manar Abu Diab, Shira Yanovsky-Dagan, Fouad Zahdeh, Uria Aviel, Roni Sarel-Gallily, Shir Michael, Ester Bnaya, Shulamit Sebban, Yosef Buganim, Yotam Drier, Vincent Mouly, Stefan Kubicek, Walther J A A van den Broek, Derick G Wansink, Silvina Epsztejn-Litman, Rachel Eiges.
      Epigenetic defects caused by hereditary or de novo mutations are implicated in various human diseases. It remains uncertain whether correcting the underlying mutation can reverse these defects in patient cells. Here we show by the analysis of myotonic dystrophy type 1 (DM1)-related locus that in mutant human embryonic stem cells (hESCs), DNA methylation and H3K9me3 enrichments are completely abolished by repeat excision (CTG2000 expansion), whereas in patient myoblasts (CTG2600 expansion), repeat deletion fails to do so. This distinction between undifferentiated and differentiated cells arises during cell differentiation, and can be reversed by reprogramming of gene-edited myoblasts. We demonstrate that abnormal methylation in DM1 is distinctively maintained in the undifferentiated state by the activity of the de novo DNMTs (DNMT3b in tandem with DNMT3a). Overall, the findings highlight a crucial difference in heterochromatin maintenance between undifferentiated (sequence-dependent) and differentiated (sequence-independent) cells, thus underscoring the role of differentiation as a locking mechanism for repressive epigenetic modifications at the DM1 locus.
    DOI:  https://doi.org/10.1038/s41467-024-47217-4
  7. Nat Commun. 2024 Apr 17. 15(1): 3311
    Engineering a synthetic gene circuit for high-performance inducible expression in mammalian systems.
    Giuliano De Carluccio, Virginia Fusco, Diego di Bernardo.
      Inducible gene expression systems can be used to control the expression of a gene of interest by means of a small-molecule. One of the most common designs involves engineering a small-molecule responsive transcription factor (TF) and its cognate promoter, which often results in a compromise between minimal uninduced background expression (leakiness) and maximal induced expression. Here, we focus on an alternative strategy using quantitative synthetic biology to mitigate leakiness while maintaining high expression, without modifying neither the TF nor the promoter. Through mathematical modelling and experimental validations, we design the CASwitch, a mammalian synthetic gene circuit based on combining two well-known network motifs: the Coherent Feed-Forward Loop (CFFL) and the Mutual Inhibition (MI). The CASwitch combines the CRISPR-Cas endoribonuclease CasRx with the state-of-the-art Tet-On3G inducible gene system to achieve high performances. To demonstrate the potentialities of the CASwitch, we apply it to three different scenarios: enhancing a whole-cell biosensor, controlling expression of a toxic gene and inducible production of Adeno-Associated Virus (AAV) vectors.
    DOI:  https://doi.org/10.1038/s41467-024-47592-y
  8. Nat Commun. 2024 Apr 16. 15(1): 3266
    Combined and differential roles of ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells.
    Naoki Kubo, Ryuji Uehara, Shuhei Uemura, Hiroaki Ohishi, Kenjiro Shirane, Hiroyuki Sasaki.
      DNA methyltransferase 3A (DNMT3A) and its catalytically inactive cofactor DNA methyltransferase 3-Like (DNMT3L) proteins form functional heterotetramers to deposit DNA methylation in mammalian germ cells. While both proteins have an ATRX-DNMT3-DNMT3L (ADD) domain that recognizes histone H3 tail unmethylated at lysine-4 (H3K4me0), the combined and differential roles of the domains in the two proteins have not been fully defined in vivo. Here we investigate DNA methylation landscapes in female and male germ cells derived from mice with loss-of-function amino acid substitutions in the ADD domains of DNMT3A and/or DNMT3L. Mutations in either the DNMT3A-ADD or the DNMT3L-ADD domain moderately decrease global CG methylation levels, but to different degrees, in both germ cells. Furthermore, when the ADD domains of both DNMT3A and DNMT3L lose their functions, the CG methylation levels are much more reduced, especially in oocytes, comparable to the impact of the Dnmt3a/3L knockout. In contrast, aberrant accumulation of non-CG methylation occurs at thousands of genomic regions in the double mutant oocytes and spermatozoa. These results highlight the critical role of the ADD-H3K4me0 binding in proper CG and non-CG methylation in germ cells and the various impacts of the ADD domains of the two proteins.
    DOI:  https://doi.org/10.1038/s41467-024-47699-2
  9. Prostate. 2024 Apr 17.
    FOXA1 regulates ribosomal RNA transcription in prostate cancer.
    Tianwei Jia, Chenxu Liu, Ping Guo, Yaning Xu, Wenzheng Wang, Xiaoyu Liu, Song Wang, Xianglin Zhang, Haiyang Guo.
       BACKGROUND: Ribosome biogenesis is excessively activated in tumor cells, yet it is little known whether oncogenic transcription factors (TFs) are involved in the ribosomal RNA (rRNA) transactivation.
    METHODS: Nucleolar proteomics data and large-scale immunofluorescence were re-analyzed to jointly identify the proteins localized at nucleolus. RNA-Seq data of five prostate cancer (PCa) cohorts were combined and integrated with multi-dimensional data to define the upregulated nucleolar TFs in PCa tissues. Then, ChIP-Seq data of PCa cell lines and two PCa clinical cohorts were re-analyzed to reveal the TF binding patterns at ribosomal DNA (rDNA) repeats. The TF binding at rDNA was validated by ChIP-qPCR. The effect of the TF on rRNA transcription was determined by rDNA luciferase reporter, nascent RNA synthesis, and global protein translation assays.
    RESULTS: In this study, we reveal the role of oncogenic TF FOXA1 in regulating rRNA transcription within nucleolar organization regions. By analyzing human TFs in prostate cancer clinical datasets and nucleolar proteomics data, we identified that FOXA1 is partially localized in the nucleolus and correlated with global protein translation. Our extensive FOXA1 ChIP-Seq analysis provides robust evidence of FOXA1 binding across rDNA repeats in prostate cancer cell lines, primary tumors, and castration-resistant variants. Notably, FOXA1 occupancy at rDNA repeats correlates with histone modifications associated with active transcription, namely H3K27ac and H3K4me3. Reducing FOXA1 expression results in decreased transactivation at rDNA, subsequently diminishing global protein synthesis.
    CONCLUSIONS: Our results suggest FOXA1 regulates aberrant ribosome biogenesis downstream of oncogenic signaling in prostate cancer.
    Keywords:  FOXA1; prostate cancer; ribosomal RNA
    DOI:  https://doi.org/10.1002/pros.24714
  10. Mol Ther. 2024 Apr 15. pii: S1525-0016(24)00242-9. [Epub ahead of print]
    Switch of ELF3 and ATF4 transcriptional axis programs the amino acid insufficiency-linked epithelial-to-mesenchymal transition.
    Jianxiang Lin, Linjun Hou, Xin Zhao, Jingli Zhong, Yilv Lv, Xiaohua Jiang, Bo Ye, Yunbo Qiao.
      Epithelial-to-mesenchymal transition (EMT) that endows cancer cells with increased invasive and migratory capacity enables cancer dissemination and metastasis. This process is tightly associated with metabolic reprogramming acquired for rewiring cell status and signaling pathways for survival in dietary insufficiency conditions. However, it remains largely unclear how transcription factor (TF)-mediated transcriptional programs are modulated during the EMT process. Here we reveal that depletion of a key epithelial TF, ELF3, triggers a TGFβ signaling activation-like mesenchymal transcriptomic profile and metastatic features linked to the aminoacyl-tRNA biogenesis pathway. Moreover, the transcriptome alterations elicited by ELF3 depletion perfectly resemble an ATF4-dependent weak response to amino acid starvation. Intriguingly, we observe an exclusive enrichment of ELF3 and ATF4 in epithelial and TGFβ-induced or ELF3 depletion-elicited mesenchymal enhancers, respectively, with rare co-binding on altered enhancers. We also find that the upregulation of aminoacyl-tRNA synthetases and some mesenchymal genes upon amino acid deprivation is diminished in ATF4-depleted cells. In sum, the loss of ELF3 binding on epithelial enhancers and the gain of ATF4 binding on the enhancers of mesenchymal factors and amino acid-deprivation responsive genes facilitate the loss of epithelial cell features and the gain of TGFβ signaling-associated mesenchymal signatures, which further promote lung cancer cell metastasis.
    DOI:  https://doi.org/10.1016/j.ymthe.2024.04.025
  11. Nat Immunol. 2024 Apr 17.
    PU.1 and BCL11B sequentially cooperate with RUNX1 to anchor mSWI/SNF to poise the T cell effector landscape.
    Noah Gamble, Alexandra Bradu, Jason A Caldwell, Joshua McKeever, Olubusayo Bolonduro, Ebru Ermis, Caroline Kaiser, YeEun Kim, Benjamin Parks, Sandy Klemm, William J Greenleaf, Gerald R Crabtree, Andrew S Koh.
      Adaptive immunity relies on specialized effector functions elicited by lymphocytes, yet how antigen recognition activates appropriate effector responses through nonspecific signaling intermediates is unclear. Here we examined the role of chromatin priming in specifying the functional outputs of effector T cells and found that most of the cis-regulatory landscape active in effector T cells was poised early in development before the expression of the T cell antigen receptor. We identified two principal mechanisms underpinning this poised landscape: the recruitment of the nucleosome remodeler mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) by the transcription factors RUNX1 and PU.1 to establish chromatin accessibility at T effector loci; and a 'relay' whereby the transcription factor BCL11B succeeded PU.1 to maintain occupancy of the chromatin remodeling complex mSWI/SNF together with RUNX1, after PU.1 silencing during lineage commitment. These mechanisms define modes by which T cells acquire the potential to elicit specialized effector functions early in their ontogeny and underscore the importance of integrating extrinsic cues to the developmentally specified intrinsic program.
    DOI:  https://doi.org/10.1038/s41590-024-01807-y
  12. BMC Med Genomics. 2024 Apr 17. 17(Suppl 1): 92
    Analysis of REST binding sites with canonical and non-canonical motifs in human cell lines.
    Jaejoon Choi, Eunjung Alice Lee.
       BACKGROUND: Repressor element 1 (RE1) silencing transcription factor (REST) is a transcriptional repressor abundantly expressed in aging human brains. It is known to regulate genes associated with oxidative stress, inflammation, and neurological disorders by binding to a canonical form of sequence motif and its non-canonical variations. Although analysis of genomic sequence motifs is crucial to understand transcriptional regulation by transcription factors (TFs), a comprehensive characterization of various forms of RE1 motifs in human cell lines has not been performed.
    RESULTS: Here, we analyzed 23 ENCODE REST ChIP-seq datasets from diverse human cell lines and identified a non-redundant set of 68,975 loci with ChIP-seq peaks. Our systematic characterization of these binding sites revealed that the canonical form of REST binding motif was found primarily in ChIP-seq peaks shared across multiple cell lines, while non-canonical forms of motifs were identified in both cell-line-specific binding sites and those shared across cell lines. Remarkably, we observed a notable prevalence of non-canonical motifs that corresponded to half segments of the canonical motif. Furthermore, our analysis unveiled the presence of cell-line-specific REST binding patterns, as evidenced by the clustering of ChIP-seq experiments according to their respective cell lines. This observation underscores the cell-line specificity of REST binding at certain genomic loci, implying intricate cell-line-specific regulatory mechanisms.
    CONCLUSIONS: Overall, our study provides a comprehensive characterization of REST binding motifs in human cell lines and genome-wide RE1 motif profiles. These findings contribute to a deeper understanding of REST-mediated transcriptional regulation and highlight the importance of considering cell-line-specific effects in future investigations.
    DOI:  https://doi.org/10.1186/s12920-024-01860-4
  13. Nat Methods. 2024 Apr 15.
    Simultaneous single-cell three-dimensional genome and gene expression profiling uncovers dynamic enhancer connectivity underlying olfactory receptor choice.
    Honggui Wu, Jiankun Zhang, Fanchong Jian, Jinxin Phaedo Chen, Yinghui Zheng, Longzhi Tan, X Sunney Xie.
      The simultaneous measurement of three-dimensional (3D) genome structure and gene expression of individual cells is critical for understanding a genome's structure-function relationship, yet this is challenging for existing methods. Here we present 'Linking mRNA to Chromatin Architecture (LiMCA)', which jointly profiles the 3D genome and transcriptome with exceptional sensitivity and from low-input materials. Combining LiMCA and our high-resolution scATAC-seq assay, METATAC, we successfully characterized chromatin accessibility, as well as paired 3D genome structures and gene expression information, of individual developing olfactory sensory neurons. We expanded the repertoire of known olfactory receptor (OR) enhancers and discovered unexpected rules of their dynamics: OR genes and their enhancers are most accessible during early differentiation. Furthermore, we revealed the dynamic spatial relationship between ORs and enhancers behind stepwise OR expression. These findings offer valuable insights into how 3D connectivity of ORs and enhancers dynamically orchestrate the 'one neuron-one receptor' selection process.
    DOI:  https://doi.org/10.1038/s41592-024-02239-0
  14. Genome Res. 2024 Apr 17. pii: gr.278598.123. [Epub ahead of print]
    Probabilistic association of differentially expressed genes with cis-regulatory elements.
    Brian S Roberts, Ashlyn G Anderson, Chris Partridge, Gregory M Cooper, Richard M Myers.
      Differential gene expression in response to perturbations is mediated at least in part by changes in binding of transcription factors (TFs) and other proteins at specific genomic regions. Association of these cis-regulatory elements (CREs) with their target genes is a challenging task that is essential to address many biological and mechanistic questions. Many current approaches rely on chromatin conformation capture techniques or single-cell correlational methods to establish CRE-to-gene associations. These methods can be effective but have limitations, including resolution, gaps in detectable association distances, and cost. As an alternative, we have developed DegCre, a nonparametric method that evaluates correlations between measurements of perturbation-induced differential gene expression and differential regulatory signal at CREs to score possible CRE-to-gene associations. It has several unique features, including the ability to: use any type of CRE activity measurement; yield probabilistic scores for CRE-to-gene pairs; and assess CRE-to-gene pairings across a wide range of sequence distances. We apply DegCre to six data sets, each employing different perturbations and containing a variety of regulatory signal measurements, including chromatin openness, histone modifications, and TF occupancy. To test their efficacy, we compare DegCre associations to Hi-C loop calls and CRISPR-validated CRE-to-gene associations, establishing good performance by DegCre that is comparable or superior to competing methods. DegCre is a novel approach to the association of CREs to genes from a perturbation-differential perspective, with strengths that are complementary to existing approaches and allow for new insights into gene regulation.
    DOI:  https://doi.org/10.1101/gr.278598.123
  15. J Clin Invest. 2024 Apr 15. pii: e175703. [Epub ahead of print]134(8):
    Von Hippel Lindau tumor suppressor controls m6A-dependent gene expression in renal tumorigenesis.
    Cheng Zhang, Miaomiao Yu, Austin J Hepperla, Zhao Zhang, Rishi Raj, Hua Zhong, Jin Zhou, Lianxin Hu, Jun Fang, Hongyi Liu, Qian Liang, Liwei Jia, Chengheng Liao, Sichuan Xi, Jeremy M Simon, Kexin Xu, Zhijie Liu, Yunsun Nam, Payal Kapur, Qing Zhang.
      N6-Methyladenosine (m6A) is the most abundant posttranscriptional modification, and its contribution to cancer evolution has recently been appreciated. Renal cancer is the most common adult genitourinary cancer, approximately 85% of which is accounted for by the clear cell renal cell carcinoma (ccRCC) subtype characterized by VHL loss. However, it is unclear whether VHL loss in ccRCC affects m6A patterns. In this study, we demonstrate that VHL binds and promotes METTL3/METTL14 complex formation while VHL depletion suppresses m6A modification, which is distinctive from its canonical E3 ligase role. m6A RNA immunoprecipitation sequencing (RIP-Seq) coupled with RNA-Seq allows us to identify a selection of genes whose expression may be regulated by VHL-m6A signaling. Specifically, PIK3R3 is identified to be a critical gene whose mRNA stability is regulated by VHL in a m6A-dependent but HIF-independent manner. Functionally, PIK3R3 depletion promotes renal cancer cell growth and orthotopic tumor growth while its overexpression leads to decreased tumorigenesis. Mechanistically, the VHL-m6A-regulated PIK3R3 suppresses tumor growth by restraining PI3K/AKT activity. Taken together, we propose a mechanism by which VHL regulates m6A through modulation of METTL3/METTL14 complex formation, thereby promoting PIK3R3 mRNA stability and protein levels that are critical for regulating ccRCC tumorigenesis.
    Keywords:  Oncology; Tumor suppressors
    DOI:  https://doi.org/10.1172/JCI175703
  16. J Cell Biochem. 2024 Apr 15.
    RUNX1 regulates promoter activity in the absence of cognate DNA binding motifs.
    Alex M Woodworth, Kristine Hardy, Phillippa C Taberlay, Joanne L Dickinson, Adele F Holloway.
      Runt-related transcription factor 1 (RUNX1) plays an important role in normal haematopoietic cell development and function, and its function is frequently disrupted in leukaemia. RUNX1 is widely recognised as a sequence-specific DNA binding factor that recognises the motif 5'-TG(T/C)GGT-3' in promoter and enhancer regions of its target genes. Moreover, RUNX1 fusion proteins, such as RUNX1-ETO formed by the t(8;21) translocation, retain the ability to recognise and bind to this sequence to elicit atypical gene regulatory effects on bona fide RUNX1 targets. However, our analysis of publicly available RUNX1 chromatin immunoprecipitation sequencing (ChIP-Seq) data has provided evidence challenging this dogma, revealing that this motif-specific model of RUNX1 recruitment and function is incomplete. Our analyses revealed that the majority of RUNX1 genomic localisation occurs outside of promoters, that 20% of RUNX1 binding sites lack consensus RUNX motifs, and that binding in the absence of a cognate binding site is more common in promoter regions compared to distal sites. Reporter assays demonstrate that RUNX1 can drive promoter activity in the absence of a recognised DNA binding motif, in contrast to RUNX1-ETO. RUNX1-ETO supresses activity when it is recruited to promoters containing a sequence specific motif, while interestingly, it binds but does not repress promoters devoid of a RUNX1 recognition site. These data suggest that RUNX1 regulation of target genes occurs through multiple mechanisms depending on genomic location, the type of regulatory element and mode of recruitment.
    Keywords:  RUNX1; RUNX1‐ETO; gene regulation; promoter activation; transcriptional regulation
    DOI:  https://doi.org/10.1002/jcb.30570
  17. Development. 2024 Apr 15. pii: dev.202505. [Epub ahead of print]
    CHD4 and SMYD1 repress common transcriptional programs in the developing heart.
    Wei Shi, Lauren K Wasson, Kerry M Dorr, Zachary L Robbe, Caralynn M Wilczewski, Austin J Hepperla, Ian J Davis, Christine E Seidman, Jonathan G Seidman, Frank L Conlon.
      Regulation of chromatin states is essential for proper temporal and spatial gene expression. Chromatin states are modulated by remodeling complexes composed of components that have enzymatic activities. CHD4 is the catalytic core of the Nucleosome Remodeling and Deacetylase (NuRD) complex that represses gene transcription. However, it remains to be determined how CHD4, a ubiquitous enzyme that remodels chromatin structure, functions in cardiomyocytes to maintain heart development. Particularly, there exists controversy as to whether other proteins besides the NuRD components interact with CHD4 in the heart. Using quantitative proteomics, we identified that CHD4 interacts with SMYD1, a striated muscle-restricted histone methyltransferase that is essential for cardiomyocyte differentiation and cardiac morphogenesis. Comprehensive transcriptomic and chromatin accessibility studies of Smyd1 and Chd4 null embryonic hearts revealed that SMYD1 and CHD4 repress a group of common genes and pathways that included glycolysis, response to hypoxia, and angiogenesis. Our study reveals a novel mechanism by which CHD4 functions during heart development, and we have revealed an uncharacterized mechanism regarding how SMYD1 represses cardiac transcription in the developing heart.
    Keywords:  CHD4; Chromatin accessibility; SMYD1; Transcription
    DOI:  https://doi.org/10.1242/dev.202505
  18. BMC Genomics. 2024 Apr 17. 25(1): 377
    Tissue-specific atlas of trans-models for gene regulation elucidates complex regulation patterns.
    Robert Dagostino, Assaf Gottlieb.
       BACKGROUND: Deciphering gene regulation is essential for understanding the underlying mechanisms of healthy and disease states. While the regulatory networks formed by transcription factors (TFs) and their target genes has been mostly studied with relation to cis effects such as in TF binding sites, we focused on trans effects of TFs on the expression of their transcribed genes and their potential mechanisms.
    RESULTS: We provide a comprehensive tissue-specific atlas, spanning 49 tissues of TF variations affecting gene expression through computational models considering two potential mechanisms, including combinatorial regulation by the expression of the TFs, and by genetic variants within the TF. We demonstrate that similarity between tissues based on our discovered genes corresponds to other types of tissue similarity. The genes affected by complex TF regulation, and their modelled TFs, were highly enriched for pharmacogenomic functions, while the TFs themselves were also enriched in several cancer and metabolic pathways. Additionally, genes that appear in multiple clusters are enriched for regulation of immune system while tissue clusters include cluster-specific genes that are enriched for biological functions and diseases previously associated with the tissues forming the cluster. Finally, our atlas exposes multilevel regulation across multiple tissues, where TFs regulate other TFs through the two tested mechanisms.
    CONCLUSIONS: Our tissue-specific atlas provides hierarchical tissue-specific trans genetic regulations that can be further studied for association with human phenotypes.
    Keywords:  Transcription factor polymorphism; Transcriptional regulation; Transcriptome imputation
    DOI:  https://doi.org/10.1186/s12864-024-10317-y
  19. Nucleic Acids Res. 2024 Apr 18. pii: gkae280. [Epub ahead of print]
    m6ACali: machine learning-powered calibration for accurate m6A detection in MeRIP-Seq.
    Haokai Ye, Tenglong Li, Daniel J Rigden, Zhen Wei.
      We present m6ACali, a novel machine-learning framework aimed at enhancing the accuracy of N6-methyladenosine (m6A) epitranscriptome profiling by reducing the impact of non-specific antibody enrichment in MeRIP-Seq. The calibration model serves as a genomic feature-based classifier that refines the identification of m6A sites, distinguishing those genuinely present from those that can be detected in in-vitro transcribed (IVT) control experiments. We find that m6ACali effectively identifies non-specific binding peaks reported by exomePeak2 and MACS2 in novel MeRIP-Seq datasets without the need for paired IVT controls. The model interpretation revealed that off-target antibody binding sites commonly occur at short exons and short mRNAs, originating from high read coverage regions that share the motif sequence with true m6A sites. We also reveal that the ML strategy can efficiently adjust differentially methylated peaks and other antibody-dependent, base-resolution m6A detection techniques. As a result, m6ACali offers a promising method for the universal enhancement of m6A profiles generated by MeRIP-Seq experiments, elevating the benchmark for omics-level m6A data integration.
    DOI:  https://doi.org/10.1093/nar/gkae280
  20. Mol Cell. 2024 Apr 18. pii: S1097-2765(24)00228-4. [Epub ahead of print]84(8): 1611-1625.e3
    Detection of ac4C in human mRNA is preserved upon data reassessment.
    Hamid Beiki, David Sturgill, Daniel Arango, Sebastien Relier, Sarah Schiffers, Shalini Oberdoerffer.
      We recently reported the distribution of N4-acetylcytidine (ac4C) in HeLa mRNA at base resolution through chemical reduction and the induction of C:T mismatches in sequencing (RedaC:T-seq). Our results contradicted an earlier report from Schwartz and colleagues utilizing a similar method termed ac4C-seq. Here, we revisit both datasets and reaffirm our findings. Through RedaC:T-seq reanalysis, we establish a low basal error rate at unmodified nucleotides that is not skewed to any specific mismatch type and a prominent increase in C:T substitutions as the dominant mismatch type in both treated wild-type replicates, with a high degree of reproducibility across replicates. In contrast, through ac4C-seq reanalysis, we uncover significant data quality issues including insufficient depth, with one wild-type replicate yielding 2.7 million reads, inconsistencies in reduction efficiencies between replicates, and an overall increase in mismatches involving thymine that could obscure ac4C detection. These analyses bolster the detection of ac4C in HeLa mRNA through RedaC:T-seq.
    Keywords:  NAT10; ac4C; acetylcytidine; epitranscriptome
    DOI:  https://doi.org/10.1016/j.molcel.2024.03.018
  21. Nat Commun. 2024 Apr 18. 15(1): 3344
    An esophagus cell atlas reveals dynamic rewiring during active eosinophilic esophagitis and remission.
    Jiarui Ding, John J Garber, Amiko Uchida, Ariel Lefkovith, Grace T Carter, Praveen Vimalathas, Lauren Canha, Michael Dougan, Kyle Staller, Joseph Yarze, Toni M Delorey, Orit Rozenblatt-Rosen, Orr Ashenberg, Daniel B Graham, Jacques Deguine, Aviv Regev, Ramnik J Xavier.
      Coordinated cell interactions within the esophagus maintain homeostasis, and disruption can lead to eosinophilic esophagitis (EoE), a chronic inflammatory disease with poorly understood pathogenesis. We profile 421,312 individual cells from the esophageal mucosa of 7 healthy and 15 EoE participants, revealing 60 cell subsets and functional alterations in cell states, compositions, and interactions that highlight previously unclear features of EoE. Active disease displays enrichment of ALOX15+ macrophages, PRDM16+ dendritic cells expressing the EoE risk gene ATP10A, and cycling mast cells, with concomitant reduction of TH17 cells. Ligand-receptor expression uncovers eosinophil recruitment programs, increased fibroblast interactions in disease, and IL-9+IL-4+IL-13+ TH2 and endothelial cells as potential mast cell interactors. Resolution of inflammation-associated signatures includes mast and CD4+ TRM cell contraction and cell type-specific downregulation of eosinophil chemoattractant, growth, and survival factors. These cellular alterations in EoE and remission advance our understanding of eosinophilic inflammation and opportunities for therapeutic intervention.
    DOI:  https://doi.org/10.1038/s41467-024-47647-0
  22. Dev Cell. 2024 Apr 11. pii: S1534-5807(24)00200-4. [Epub ahead of print]
    Sustained ERK signaling promotes G2 cell cycle exit and primes cells for whole-genome duplication.
    Adler Guerrero Zuniga, Timothy J Aikin, Connor McKenney, Yovel Lendner, Alain Phung, Paul W Hook, Amy Meltzer, Winston Timp, Sergi Regot.
      Whole-genome duplication (WGD) is a frequent event in cancer evolution that fuels chromosomal instability. WGD can result from mitotic errors or endoreduplication, yet the molecular mechanisms that drive WGD remain unclear. Here, we use live single-cell analysis to characterize cell-cycle dynamics upon aberrant Ras-ERK signaling. We find that sustained ERK signaling in human cells leads to reactivation of the APC/C in G2, resulting in tetraploid G0-like cells that are primed for WGD. This process is independent of DNA damage or p53 but dependent on p21. Transcriptomics analysis and live-cell imaging showed that constitutive ERK activity promotes p21 expression, which is necessary and sufficient to inhibit CDK activity and which prematurely activates the anaphase-promoting complex (APC/C). Finally, either loss of p53 or reduced ERK signaling allowed for endoreduplication, completing a WGD event. Thus, sustained ERK signaling-induced G2 cell cycle exit represents an alternative path to WGD.
    Keywords:  cell cycle dynamics; oncogenesis; signaling dynamics; whole-genome duplication
    DOI:  https://doi.org/10.1016/j.devcel.2024.03.032
  23. iScience. 2024 Apr 19. 27(4): 109607
    SWI/SNF complex-mediated chromatin remodeling in Candida glabrata promotes immune evasion.
    Kundan Kumar, Aditi Pareek, Rupinder Kaur.
      Immune evasion is critical for fungal virulence. However, how the human opportunistic pathogen Candida glabrata (Cg) accomplishes this is unknown. Here, we present the first genome-wide nucleosome map of the macrophage-internalized Cg consisting of ∼12,000 dynamic and 70,000 total nucleosomes. We demonstrate that CgSnf2 (SWI/SNF chromatin remodeling complex-ATPase subunit)-mediated chromatin reorganization in macrophage-internalized Cg upregulates and downregulates the immunosuppressive seven-gene mannosyltransferase-cluster (CgMT-C) and immunostimulatory cell surface adhesin-encoding EPA1 gene, respectively. Consistently, EPA1 overexpression and CgMT-C deletion elevated IL-1β (pro-inflammatory cytokine) production and diminished Cg proliferation in macrophages. Further, Cgsnf2Δ had higher Epa1 surface expression, and evoked increased IL-1β secretion, and was killed in macrophages. Akt-, p38-, NF-κB- or NLRP3 inflammasome-inhibition partially reversed increased IL-1β secretion in Cgsnf2Δ-infected macrophages. Importantly, macrophages responded to multiple Candida pathogens via NF-κB-dependent IL-1β production, underscoring NF-κB signaling's role in fungal diseases. Altogether, our findings directly link the nucleosome positioning-based chromatin remodeling to fungal immunomodulatory molecule expression.
    Keywords:  Biological sciences; Immunology; Microbiology; Molecular biology; Mycology
    DOI:  https://doi.org/10.1016/j.isci.2024.109607
  24. Genome Biol. 2024 Apr 19. 25(1): 102
    RBM22 regulates RNA polymerase II 5' pausing, elongation rate, and termination by coordinating 7SK-P-TEFb complex and SPT5.
    Xian Du, Wenying Qin, Chunyu Yang, Lin Dai, Mingkui San, Yingdan Xia, Siyu Zhou, Mengyang Wang, Shuang Wu, Shaorui Zhang, Huiting Zhou, Fangshu Li, Fang He, Jingfeng Tang, Jia-Yu Chen, Yu Zhou, Rui Xiao.
       BACKGROUND: Splicing factors are vital for the regulation of RNA splicing, but some have also been implicated in regulating transcription. The underlying molecular mechanisms of their involvement in transcriptional processes remain poorly understood.
    RESULTS: Here, we describe a direct role of splicing factor RBM22 in coordinating multiple steps of RNA Polymerase II (RNAPII) transcription in human cells. The RBM22 protein widely occupies the RNAPII-transcribed gene locus in the nucleus. Loss of RBM22 promotes RNAPII pause release, reduces elongation velocity, and provokes transcriptional readthrough genome-wide, coupled with production of transcripts containing sequences from downstream of the gene. RBM22 preferentially binds to the hyperphosphorylated, transcriptionally engaged RNAPII and coordinates its dynamics by regulating the homeostasis of the 7SK-P-TEFb complex and the association between RNAPII and SPT5 at the chromatin level.
    CONCLUSIONS: Our results uncover the multifaceted role of RBM22 in orchestrating the transcriptional program of RNAPII and provide evidence implicating a splicing factor in both RNAPII elongation kinetics and termination control.
    Keywords:  5′ pausing; RBM22; RNA polymerase II; Transcription elongation; Transcription termination
    DOI:  https://doi.org/10.1186/s13059-024-03242-6
  25. Mol Cell. 2024 Apr 18. pii: S1097-2765(24)00227-2. [Epub ahead of print]84(8): 1601-1610.e2
    No evidence for ac4C within human mRNA upon data reassessment.
    Joseph Georgeson, Schraga Schwartz.
      Cytidine acetylation (ac4C) of RNA is a post-transcriptional modification catalyzed by Nat10. Recently, an approach termed RedaC:T was employed to map ac4C in human mRNA, relying on detection of C>T mutations in WT but not in Nat10-KO cells. RedaC:T suggested widespread ac4C presence. Here, we reanalyze RedaC:T data. We find that mismatch signatures are not reproducible, as C>T mismatches are nearly exclusively present in only one of two biological replicates. Furthermore, all mismatch types-not only C>T-are highly enriched in WT samples, inconsistent with an acetylation signature. We demonstrate that the originally observed enrichment in mutations in one of the WT samples is due to its low complexity, resulting in the technical amplification of all classes of mismatch counts. Removal of duplicate reads abolishes the skewed mismatch patterns. These analyses account for the irreproducible mismatch patterns across samples while failing to find evidence for acetylation of RedaC:T sites.
    Keywords:  N4-acetylcytidine; NAT10; RNA; RNA modifications; ac4C; acetylation; epitranscriptome; mRNA
    DOI:  https://doi.org/10.1016/j.molcel.2024.03.017
  26. Cell. 2024 Apr 10. pii: S0092-8674(24)00314-3. [Epub ahead of print]
    Dual-role transcription factors stabilize intermediate expression levels.
    Jinnan He, Xiangru Huo, Gaofeng Pei, Zeran Jia, Yiming Yan, Jiawei Yu, Haozhi Qu, Yunxin Xie, Junsong Yuan, Yuan Zheng, Yanyan Hu, Minglei Shi, Kaiqiang You, Tingting Li, Tianhua Ma, Michael Q Zhang, Sheng Ding, Pilong Li, Yinqing Li.
      Precise control of gene expression levels is essential for normal cell functions, yet how they are defined and tightly maintained, particularly at intermediate levels, remains elusive. Here, using a series of newly developed sequencing, imaging, and functional assays, we uncover a class of transcription factors with dual roles as activators and repressors, referred to as condensate-forming level-regulating dual-action transcription factors (TFs). They reduce high expression but increase low expression to achieve stable intermediate levels. Dual-action TFs directly exert activating and repressing functions via condensate-forming domains that compartmentalize core transcriptional unit selectively. Clinically relevant mutations in these domains, which are linked to a range of developmental disorders, impair condensate selectivity and dual-action TF activity. These results collectively address a fundamental question in expression regulation and demonstrate the potential of level-regulating dual-action TFs as powerful effectors for engineering controlled expression levels.
    Keywords:  CRISPR gene modulation; biomolecular condensate; condensate occupancy sequencing; condensate selectivity; developmental program; dual transcriptional regulation; intrinsically disordered regions; noise reduction; primed expression; stablized expression variation
    DOI:  https://doi.org/10.1016/j.cell.2024.03.023
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