bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2021‒06‒27
twenty-six papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit
  1. A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker.
  2. Phase separation drives aberrant chromatin looping and cancer development.
  3. The HIF complex recruits the histone methyltransferase SET1B to activate specific hypoxia-inducible genes.
  4. Nascent RNA antagonizes the interaction of a set of regulatory proteins with chromatin.
  5. MUC1-C activates the PBAF chromatin remodeling complex in integrating redox balance with progression of human prostate cancer stem cells.
  6. Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome.
  7. A transcriptional switch governs fibroblast activation in heart disease.
  8. Two distinct mechanisms of RNA polymerase II elongation stimulation in vivo.
  9. Chromatin accessibility profiling in Neurospora crassa reveals molecular features associated with accessible and inaccessible chromatin.
  10. Runx1 shapes the chromatin landscape via a cascade of direct and indirect targets.
  11. Transcriptional super-enhancers control cancer stemness and metastasis genes in squamous cell carcinoma.
  12. Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells.
  13. Accurate prediction of cis-regulatory modules reveals a prevalent regulatory genome of humans.
  14. The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22.
  15. Single-cell ATAC-seq reveals GATA2-dependent priming defect in myeloid and a maturation bottleneck in lymphoid lineages.
  16. Nuclear ADP-ribosylation drives IFNγ-dependent STAT1α enhancer formation in macrophages.
  17. Epithelial cell plasticity drives endoderm formation during gastrulation.
  18. Genome-wide binding analysis of 195 DNA binding proteins reveals "reservoir" promoters and human specific SVA-repeat family regulation.
  19. Genomic imprinting in mouse blastocysts is predominantly associated with H3K27me3.
  20. An enhancer screen identifies new suppressors of small-RNA-mediated epigenetic gene silencing.
  21. KEAP1 deficiency drives glucose dependency and sensitizes lung cancer cells and tumors to GLUT inhibition.
  22. Dissecting regulatory pathways for transcription recovery following DNA damage reveals a non-canonical function of the histone chaperone HIRA.
  23. The EMT transcription factor Snai1 maintains myocardial wall integrity by repressing intermediate filament gene expression.
  24. In silico analysis of the transcriptional regulatory logic of neuronal identity specification throughout the C. elegans nervous system.
  25. The MLL3/4 H3K4 methyltransferase complex in establishing an active enhancer landscape.
  26. The combined action of CTCF and its testis-specific paralog BORIS is essential for spermatogenesis.
  1. Nat Commun. 2021 06 21. 12(1): 3806
    A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker.
    Yavor K Bozhilov, Damien J Downes, Jelena Telenius, A Marieke Oudelaar, Emmanuel N Olivier, Joanne C Mountford, Jim R Hughes, Richard J Gibbons, Douglas R Higgs.
      Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements.
    DOI:  https://doi.org/10.1038/s41467-021-23980-6
  2. Nature. 2021 Jun 23.
    Phase separation drives aberrant chromatin looping and cancer development.
    Jeong Hyun Ahn, Eric S Davis, Timothy A Daugird, Shuai Zhao, Ivana Yoseli Quiroga, Hidetaka Uryu, Jie Li, Aaron J Storey, Yi-Hsuan Tsai, Daniel P Keeley, Samuel G Mackintosh, Ricky D Edmondson, Stephanie D Byrum, Ling Cai, Alan J Tackett, Deyou Zheng, Wesley R Legant, Douglas H Phanstiel, Gang Greg Wang.
      The development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human haematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains the nucleoporin IDR-tandemly dispersed repeats of phenylalanine and glycine residues1,2. However, how unstructured IDRs contribute to oncogenesis remains unclear. Here we show that IDRs contained within NUP98-HOXA9, a homeodomain-containing transcription factor chimera recurrently detected in leukaemias1,2, are essential for establishing liquid-liquid phase separation (LLPS) puncta of chimera and for inducing leukaemic transformation. Notably, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera transcription factors, but also is required for the formation of a broad 'super-enhancer'-like binding pattern typically seen at leukaemogenic genes, which potentiates transcriptional activation. An artificial HOX chimera, created by replacing the phenylalanine and glycine repeats of NUP98 with an unrelated LLPS-forming IDR of the FUS protein3,4, had similar enhancing effects on the genome-wide binding and target gene activation of the chimera. Deeply sequenced Hi-C revealed that phase-separated NUP98-HOXA9 induces CTCF-independent chromatin loops that are enriched at proto-oncogenes. Together, this report describes a proof-of-principle example in which cancer acquires mutation to establish oncogenic transcription factor condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumourous transformation. As LLPS-competent molecules are frequently implicated in diseases1,2,4-7, this mechanism can potentially be generalized to many malignant and pathological settings.
    DOI:  https://doi.org/10.1038/s41586-021-03662-5
  3. Nat Genet. 2021 Jun 21.
    The HIF complex recruits the histone methyltransferase SET1B to activate specific hypoxia-inducible genes.
    Brian M Ortmann, Natalie Burrows, Ian T Lobb, Esther Arnaiz, Niek Wit, Peter S J Bailey, Louise H Jordon, Olivia Lombardi, Ana Peñalver, James McCaffrey, Rachel Seear, David R Mole, Peter J Ratcliffe, Patrick H Maxwell, James A Nathan.
      Hypoxia-inducible transcription factors (HIFs) are fundamental to cellular adaptation to low oxygen levels, but it is unclear how they interact with chromatin and activate their target genes. Here, we use genome-wide mutagenesis to identify genes involved in HIF transcriptional activity, and define a requirement for the histone H3 lysine 4 (H3K4) methyltransferase SET1B. SET1B loss leads to a selective reduction in transcriptional activation of HIF target genes, resulting in impaired cell growth, angiogenesis and tumor establishment in SET1B-deficient xenografts. Mechanistically, we show that SET1B accumulates on chromatin in hypoxia, and is recruited to HIF target genes by the HIF complex. The selective induction of H3K4 trimethylation at HIF target loci is both HIF- and SET1B-dependent and, when impaired, correlates with decreased promoter acetylation and gene expression. Together, these findings show SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated.
    DOI:  https://doi.org/10.1038/s41588-021-00887-y
  4. Mol Cell. 2021 Jun 17. pii: S1097-2765(21)00410-X. [Epub ahead of print]
    Nascent RNA antagonizes the interaction of a set of regulatory proteins with chromatin.
    Lenka Skalska, Victoria Begley, Manuel Beltran, Saulius Lukauskas, Garima Khandelwal, Peter Faull, Amandeep Bhamra, Manuel Tavares, Rachel Wellman, Andrey Tvardovskiy, Benjamin M Foster, Igor Ruiz de Los Mozos, Javier Herrero, Silvia Surinova, Ambrosius P Snijders, Till Bartke, Richard G Jenner.
      A number of regulatory factors are recruited to chromatin by specialized RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonized the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors, including BAF, NuRD, EHMT1, and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA, and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonizing the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.
    Keywords:  RNA polymerase II; RNAse; chromatin; histone modification; nascent RNA; nucleosome; nucleosome remodeller; pre-mRNA; transcription factor; transcriptional elongation
    DOI:  https://doi.org/10.1016/j.molcel.2021.05.026
  5. Oncogene. 2021 Jun 23.
    MUC1-C activates the PBAF chromatin remodeling complex in integrating redox balance with progression of human prostate cancer stem cells.
    Masayuki Hagiwara, Atsushi Fushimi, Nami Yamashita, Atrayee Bhattacharya, Hasan Rajabi, Mark D Long, Yota Yasumizu, Mototsugu Oya, Song Liu, Donald Kufe.
      The polybromo-associated PBAF (SWI/SNF) chromatin remodeling complex, which includes PBRM1, ARID2, and BRD7, regulates cell differentiation and genomic integrity. MUC1-C is an oncogenic protein that drives lineage plasticity in prostate cancer (PC) progression. The present work demonstrates that MUC1-C induces PBRM1, ARID2, and BRD7 expression by the previously unrecognized E2F1-mediated activation of their respective promoters. The functional significance of the MUC1-C→PBAF pathway is supported by demonstrating involvement of MUC1-C in associating with nuclear PBAF and driving the NRF2 antioxidant gene transcriptome in PC cells. Mechanistically, MUC1-C forms a complex with NRF2 and PBRM1 on the NRF2 target SLC7A11 gene that encodes the xCT cystine-glutamate antiporter, increases chromatin accessibility and induces SLC7A11/xCT expression. We also show that MUC1-C and PBRM1 are necessary for induction of other NRF2 target genes, including G6PD and PGD that regulate the pentose phosphate pathway. Our results further demonstrate that MUC1-C integrates activation of PBRM1 with the regulation of antioxidant genes, ROS levels, pluripotency factor expression and the cancer stem cell (CSC) state. These findings reveal a role for MUC1-C in regulating PBAF, redox balance and lineage plasticity of PC CSC progression. Our findings also uncover involvement of MUC1-C in integrating the PBAF and BAF pathways in cancer.
    DOI:  https://doi.org/10.1038/s41388-021-01899-y
  6. Nat Commun. 2021 06 23. 12(1): 3885
    Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome.
    Sara Lago, Matteo Nadai, Filippo M Cernilogar, Maryam Kazerani, Helena Domíniguez Moreno, Gunnar Schotta, Sara N Richter.
      Cell identity is maintained by activation of cell-specific gene programs, regulated by epigenetic marks, transcription factors and chromatin organization. DNA G-quadruplex (G4)-folded regions in cells were reported to be associated with either increased or decreased transcriptional activity. By G4-ChIP-seq/RNA-seq analysis on liposarcoma cells we confirmed that G4s in promoters are invariably associated with high transcription levels in open chromatin. Comparing G4 presence, location and transcript levels in liposarcoma cells to available data on keratinocytes, we showed that the same promoter sequences of the same genes in the two cell lines had different G4-folding state: high transcript levels consistently associated with G4-folding. Transcription factors AP-1 and SP1, whose binding sites were the most significantly represented in G4-folded sequences, coimmunoprecipitated with their G4-folded promoters. Thus, G4s and their associated transcription factors cooperate to determine cell-specific transcriptional programs, making G4s to strongly emerge as new epigenetic regulators of the transcription machinery.
    DOI:  https://doi.org/10.1038/s41467-021-24198-2
  7. Nature. 2021 Jun 23.
    A transcriptional switch governs fibroblast activation in heart disease.
    Michael Alexanian, Pawel F Przytycki, Rudi Micheletti, Arun Padmanabhan, Lin Ye, Joshua G Travers, Barbara Gonzalez-Teran, Ana Catarina Silva, Qiming Duan, Sanjeev S Ranade, Franco Felix, Ricardo Linares-Saldana, Li Li, Clara Youngna Lee, Nandhini Sadagopan, Angelo Pelonero, Yu Huang, Gaia Andreoletti, Rajan Jain, Timothy A McKinsey, Michael G Rosenfeld, Casey A Gifford, Katherine S Pollard, Saptarsi M Haldar, Deepak Srivastava.
      In diseased organs, stress-activated signalling cascades alter chromatin, thereby triggering maladaptive cell state transitions. Fibroblast activation is a common stress response in tissues that worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains unclear1,2. Pharmacological inhibition of bromodomain and extra-terminal domain (BET) proteins alleviates cardiac dysfunction3-7, providing a tool to interrogate and modulate cardiac cell states as a potential therapeutic approach. Here we use single-cell epigenomic analyses of hearts dynamically exposed to BET inhibitors to reveal a reversible transcriptional switch that underlies the activation of fibroblasts. Resident cardiac fibroblasts demonstrated robust toggling between the quiescent and activated state in a manner directly correlating with BET inhibitor exposure and cardiac function. Single-cell chromatin accessibility revealed previously undescribed DNA elements, the accessibility of which dynamically correlated with cardiac performance. Among the most dynamic elements was an enhancer that regulated the transcription factor MEOX1, which was specifically expressed in activated fibroblasts, occupied putative regulatory elements of a broad fibrotic gene program and was required for TGFβ-induced fibroblast activation. Selective CRISPR inhibition of the single most dynamic cis-element within the enhancer blocked TGFβ-induced Meox1 activation. We identify MEOX1 as a central regulator of fibroblast activation associated with cardiac dysfunction and demonstrate its upregulation after activation of human lung, liver and kidney fibroblasts. The plasticity and specificity of BET-dependent regulation of MEOX1 in tissue fibroblasts provide previously unknown trans- and cis-targets for treating fibrotic disease.
    DOI:  https://doi.org/10.1038/s41586-021-03674-1
  8. Mol Cell. 2021 Jun 17. pii: S1097-2765(21)00441-X. [Epub ahead of print]
    Two distinct mechanisms of RNA polymerase II elongation stimulation in vivo.
    Kristina Žumer, Kerstin C Maier, Lucas Farnung, Martin G Jaeger, Petra Rus, Georg Winter, Patrick Cramer.
      Transcription by RNA polymerase II (RNA Pol II) relies on the elongation factors PAF1 complex (PAF), RTF1, and SPT6. Here, we use rapid factor depletion and multi-omics analysis to investigate how these elongation factors influence RNA Pol II elongation activity in human cells. Whereas depletion of PAF subunits PAF1 and CTR9 has little effect on cellular RNA synthesis, depletion of RTF1 or SPT6 strongly compromises RNA Pol II activity, albeit in fundamentally different ways. RTF1 depletion decreases RNA Pol II velocity, whereas SPT6 depletion impairs RNA Pol II progression through nucleosomes. These results show that distinct elongation factors stimulate either RNA Pol II velocity or RNA Pol II progression through chromatin in vivo. Further analysis provides evidence for two distinct barriers to early elongation: the promoter-proximal pause site and the +1 nucleosome. It emerges that the first barrier enables loading of elongation factors that are required to overcome the second and subsequent barriers to transcription.
    Keywords:  CTR9; PAF1; RNA polymerase II; RTF1; SPT6; dTAG; dTAG7; transcription; transcription elongation
    DOI:  https://doi.org/10.1016/j.molcel.2021.05.028
  9. BMC Genomics. 2021 Jun 19. 22(1): 459
    Chromatin accessibility profiling in Neurospora crassa reveals molecular features associated with accessible and inaccessible chromatin.
    Aileen R Ferraro, Abigail J Ameri, Zefu Lu, Masayuki Kamei, Robert J Schmitz, Zachary A Lewis.
      BACKGROUND: Regulation of chromatin accessibility and transcription are tightly coordinated processes. Studies in yeast and higher eukaryotes have described accessible chromatin regions, but little work has been done in filamentous fungi.RESULTS: Here we present a genome-scale characterization of accessible chromatin regions in Neurospora crassa, which revealed characteristic molecular features of accessible and inaccessible chromatin. We present experimental evidence of inaccessibility within heterochromatin regions in Neurospora, and we examine features of both accessible and inaccessible chromatin, including the presence of histone modifications, types of transcription, transcription factor binding, and relative nucleosome turnover rates. Chromatin accessibility is not strictly correlated with expression level. Accessible chromatin regions in the model filamentous fungus Neurospora are characterized the presence of H3K27 acetylation and commonly associated with pervasive non-coding transcription. Conversely, methylation of H3 lysine-36 catalyzed by ASH1 is correlated with inaccessible chromatin within promoter regions.
    CONCLUSIONS: In N. crassa, H3K27 acetylation is the most predictive histone modification for open chromatin. Conversely, our data show that H3K36 methylation is a key marker of inaccessible chromatin in gene-rich regions of the genome. Our data are consistent with an expanded role for H3K36 methylation in intergenic regions of filamentous fungi compared to the model yeasts, S. cerevisiae and S. pombe, which lack homologs of the ASH1 methyltransferase.
    Keywords:  ATAC-seq; Chromatin accessibility; Fungi; H3K36me3
    DOI:  https://doi.org/10.1186/s12864-021-07774-0
  10. PLoS Genet. 2021 Jun;17(6): e1009574
    Runx1 shapes the chromatin landscape via a cascade of direct and indirect targets.
    Matthew R Hass, Daniel Brissette, Sreeja Parameswaran, Mario Pujato, Omer Donmez, Leah C Kottyan, Matthew T Weirauch, Raphael Kopan.
      Runt-related transcription factor 1 (Runx1) can act as both an activator and a repressor. Here we show that CRISPR-mediated deletion of Runx1 in mouse metanephric mesenchyme-derived mK4 cells results in large-scale genome-wide changes to chromatin accessibility and gene expression. Open chromatin regions near down-regulated loci enriched for Runx sites in mK4 cells lose chromatin accessibility in Runx1 knockout cells, despite remaining Runx2-bound. Unexpectedly, regions near upregulated genes are depleted of Runx sites and are instead enriched for Zeb transcription factor binding sites. Re-expressing Zeb2 in Runx1 knockout cells restores suppression, and CRISPR mediated deletion of Zeb1 and Zeb2 phenocopies the gained expression and chromatin accessibility changes seen in Runx1KO due in part to subsequent activation of factors like Grhl2. These data confirm that Runx1 activity is uniquely needed to maintain open chromatin at many loci, and demonstrate that Zeb proteins are required and sufficient to maintain Runx1-dependent genome-scale repression.
    DOI:  https://doi.org/10.1371/journal.pgen.1009574
  11. Nat Commun. 2021 Jun 25. 12(1): 3974
    Transcriptional super-enhancers control cancer stemness and metastasis genes in squamous cell carcinoma.
    Jiaqiang Dong, Jiong Li, Yang Li, Zhikun Ma, Yongxin Yu, Cun-Yu Wang.
      Cancer stem cells (CSCs) play a critical role in invasive growth and metastasis of human head and neck squamous cell carcinoma (HNSCC). Although significant progress has been made in understanding the self-renewal and pro-tumorigenic potentials of CSCs, a key challenge remains on how to eliminate CSCs and halt metastasis effectively. Here we show that super-enhancers (SEs) play a critical role in the transcription of cancer stemness genes as well as pro-metastatic genes, thereby controlling their tumorigenic potential and metastasis. Mechanistically, we find that bromodomain-containing protein 4 (BRD4) recruits Mediators and NF-κB p65 to form SEs at cancer stemness genes such as TP63, MET and FOSL1, in addition to oncogenic transcripts. In vivo lineage tracing reveals that disrupting SEs by BET inhibitors potently inhibited CSC self-renewal and eliminated CSCs in addition to elimination of proliferating non-stem tumor cells in a mouse model of HNSCC. Moreover, disrupting SEs also inhibits the invasive growth and lymph node metastasis of human CSCs isolated from human HNSCC. Taken together, our results suggest that targeting SEs may serve as an effective therapy for HNSCC by eliminating CSCs.
    DOI:  https://doi.org/10.1038/s41467-021-24137-1
  12. Genome Biol. 2021 Jun 22. 22(1): 186
    Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells.
    Atsushi Onodera, Edahí González-Avalos, Chan-Wang Jerry Lio, Romain O Georges, Alfonso Bellacosa, Toshinori Nakayama, Anjana Rao.
      BACKGROUND: TET enzymes mediate DNA demethylation by oxidizing 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since these oxidized methylcytosines (oxi-mCs) are not recognized by the maintenance methyltransferase DNMT1, DNA demethylation can occur through "passive," replication-dependent dilution when cells divide. A distinct, replication-independent ("active") mechanism of DNA demethylation involves excision of 5fC and 5caC by the DNA repair enzyme thymine DNA glycosylase (TDG), followed by base excision repair.RESULTS: Here by analyzing inducible gene-disrupted mice, we show that DNA demethylation during primary T cell differentiation occurs mainly through passive replication-dependent dilution of all three oxi-mCs, with only a negligible contribution from TDG. In addition, by pyridine borane sequencing (PB-seq), a simple recently developed method that directly maps 5fC/5caC at single-base resolution, we detect the accumulation of 5fC/5caC in TDG-deleted T cells. We also quantify the occurrence of concordant demethylation within and near enhancer regions in the Il4 locus. In an independent system that does not involve cell division, macrophages treated with liposaccharide accumulate 5hmC at enhancers and show altered gene expression without DNA demethylation; loss of TET enzymes disrupts gene expression, but loss of TDG has no effect. We also observe that mice with long-term (1 year) deletion of Tdg are healthy and show normal survival and hematopoiesis.
    CONCLUSIONS: We have quantified the relative contributions of TET and TDG to cell differentiation and DNA demethylation at representative loci in proliferating T cells. We find that TET enzymes regulate T cell differentiation and DNA demethylation primarily through passive dilution of oxi-mCs. In contrast, while we observe a low level of active, replication-independent DNA demethylation mediated by TDG, this process does not appear to be essential for immune cell activation or differentiation.
    DOI:  https://doi.org/10.1186/s13059-021-02384-1
  13. NAR Genom Bioinform. 2021 Jun;3(2): lqab052
    Accurate prediction of cis-regulatory modules reveals a prevalent regulatory genome of humans.
    Pengyu Ni, Zhengchang Su.
      cis-regulatory modules(CRMs) formed by clusters of transcription factor (TF) binding sites (TFBSs) are as important as coding sequences in specifying phenotypes of humans. It is essential to categorize all CRMs and constituent TFBSs in the genome. In contrast to most existing methods that predict CRMs in specific cell types using epigenetic marks, we predict a largely cell type agonistic but more comprehensive map of CRMs and constituent TFBSs in the gnome by integrating all available TF ChIP-seq datasets. Our method is able to partition 77.47% of genome regions covered by available 6092 datasets into a CRM candidate (CRMC) set (56.84%) and a non-CRMC set (43.16%). Intriguingly, the predicted CRMCs are under strong evolutionary constraints, while the non-CRMCs are largely selectively neutral, strongly suggesting that the CRMCs are likely cis-regulatory, while the non-CRMCs are not. Our predicted CRMs are under stronger evolutionary constraints than three state-of-the-art predictions (GeneHancer, EnhancerAtlas and ENCODE phase 3) and substantially outperform them for recalling VISTA enhancers and non-coding ClinVar variants. We estimated that the human genome might encode about 1.47M CRMs and 68M TFBSs, comprising about 55% and 22% of the genome, respectively; for both of which, we predicted 80%. Therefore, the cis-regulatory genome appears to be more prevalent than originally thought.
    DOI:  https://doi.org/10.1093/nargab/lqab052
  14. EMBO J. 2021 Jun 22. e102509
    The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22.
    Timothy J Stanek, Victoria J Gennaro, Mason A Tracewell, Daniela Di Marcantonio, Kristen L Pauley, Sabrina Butt, Christopher McNair, Feng Wang, Andrew V Kossenkov, Karen E Knudsen, Tauseef Butt, Stephen M Sykes, Steven B McMahon.
      The SAGA coactivator complex is essential for eukaryotic transcription and comprises four distinct modules, one of which contains the ubiquitin hydrolase USP22. In yeast, the USP22 ortholog deubiquitylates H2B, resulting in Pol II Ser2 phosphorylation and subsequent transcriptional elongation. In contrast to this H2B-associated role in transcription, we report here that human USP22 contributes to the early stages of stimulus-responsive transcription, where USP22 is required for pre-initiation complex (PIC) stability. Specifically, USP22 maintains long-range enhancer-promoter contacts and controls loading of Mediator tail and general transcription factors (GTFs) onto promoters, with Mediator core recruitment being USP22-independent. In addition, we identify Mediator tail subunits MED16 and MED24 and the Pol II subunit RBP1 as potential non-histone substrates of USP22. Overall, these findings define a role for human SAGA within the earliest steps of transcription.
    Keywords:  SAGA; USP22; epigenetic; pre-initiation complex; transcription
    DOI:  https://doi.org/10.15252/embj.2019102509
  15. Blood Adv. 2021 Jul 13. 5(13): 2673-2686
    Single-cell ATAC-seq reveals GATA2-dependent priming defect in myeloid and a maturation bottleneck in lymphoid lineages.
    Serine Avagyan, Margaret C Weber, Sai Ma, Meera Prasad, William P Mannherz, Song Yang, Jason D Buenrostro, Leonard I Zon.
      Germline heterozygous mutations in GATA2 are associated with a syndrome characterized by cytopenias, atypical infections, and increased risk of hematologic malignancies. Here, we generated a zebrafish mutant of gata2b that recapitulated the myelomonocytopenia and B-cell lymphopenia of GATA2 deficiency syndrome. Using single-cell assay for transposase accessible chromatin with sequencing of marrow cells, we showed that loss of gata2b led to contrasting alterations in chromosome accessibility in early myeloid and lymphoid progenitors, associated with defects in gene expression. Within the myeloid lineage in gata2b mutant zebrafish, we identified an attenuated myeloid differentiation with reduced transcriptional priming and skewing away from the monocytic program. In contrast, in early lymphoid progenitors, gata2b loss led to accumulation of B-lymphoid transcription factor accessibility coupled with increased expression of the B-cell lineage-specification program. However, gata2b mutant zebrafish had incomplete B-cell lymphopoiesis with loss of lineage-specific transcription factor accessibility in differentiating B cells, in the context of aberrantly reduced oxidative metabolic pathways. Our results establish that transcriptional events in early progenitors driven by Gata2 are required to complete normal differentiation.
    DOI:  https://doi.org/10.1182/bloodadvances.2020002992
  16. Nat Commun. 2021 06 24. 12(1): 3931
    Nuclear ADP-ribosylation drives IFNγ-dependent STAT1α enhancer formation in macrophages.
    Rebecca Gupte, Tulip Nandu, W Lee Kraus.
      STAT1α is a key transcription factor driving pro-inflammatory responses in macrophages. We found that the interferon gamma (IFNγ)-regulated transcriptional program in macrophages is controlled by ADP-ribosylation (ADPRylation) of STAT1α, a post-translational modification resulting in the site-specific covalent attachment of ADP-ribose moieties. PARP-1, the major nuclear poly(ADP-ribose) polymerase (PARP), supports IFNγ-stimulated enhancer formation by regulating the genome-wide binding and IFNγ-dependent transcriptional activation of STAT1α. It does so by ADPRylating STAT1α on specific residues in its DNA-binding domain (DBD) and transcription activation (TA) domain. ADPRylation of the DBD controls STAT1α binding to its cognate DNA elements, whereas ADPRylation of the TA domain regulates enhancer activation by modulating STAT1α phosphorylation and p300 acetyltransferase activity. Loss of ADPRylation at either site leads to diminished IFNγ-dependent transcription and downstream pro-inflammatory responses. We conclude that PARP-1-mediated ADPRylation of STAT1α drives distinct enhancer activation mechanisms and is a critical regulator of inflammatory responses in macrophages.
    DOI:  https://doi.org/10.1038/s41467-021-24225-2
  17. Nat Cell Biol. 2021 Jun 24.
    Epithelial cell plasticity drives endoderm formation during gastrulation.
    Katharina Scheibner, Silvia Schirge, Ingo Burtscher, Maren Büttner, Michael Sterr, Dapeng Yang, Anika Böttcher, Ansarullah, Martin Irmler, Johannes Beckers, Filippo M Cernilogar, Gunnar Schotta, Fabian J Theis, Heiko Lickert.
      It is generally accepted that epiblast cells ingress into the primitive streak by epithelial-to-mesenchymal transition (EMT) to give rise to the mesoderm; however, it is less clear how the endoderm acquires an epithelial fate. Here, we used embryonic stem cell and mouse embryo knock-in reporter systems to combine time-resolved lineage labelling with high-resolution single-cell transcriptomics. This allowed us to resolve the morphogenetic programs that segregate the mesoderm from the endoderm germ layer. Strikingly, while the mesoderm is formed by classical EMT, the endoderm is formed independent of the key EMT transcription factor Snail1 by mechanisms of epithelial cell plasticity. Importantly, forkhead box transcription factor A2 (Foxa2) acts as an epithelial gatekeeper and EMT suppressor to shield the endoderm from undergoing a mesenchymal transition. Altogether, these results not only establish the morphogenetic details of germ layer formation, but also have broader implications for stem cell differentiation and cancer metastasis.
    DOI:  https://doi.org/10.1038/s41556-021-00694-x
  18. PLoS One. 2021 ;16(6): e0237055
    Genome-wide binding analysis of 195 DNA binding proteins reveals "reservoir" promoters and human specific SVA-repeat family regulation.
    Michael J Smallegan, Soraya Shehata, Savannah F Spradlin, Alison Swearingen, Graycen Wheeler, Arpan Das, Giulia Corbet, Benjamin Nebenfuehr, Daniel Ahrens, Devin Tauber, Shelby Lennon, Kevin Choi, Thao Huynh, Tom Wieser, Kristen Schneider, Michael Bradshaw, Joel Basken, Maria Lai, Timothy Read, Matt Hynes-Grace, Dan Timmons, Jon Demasi, John L Rinn.
      A key aspect in defining cell state is the complex choreography of DNA binding events in a given cell type, which in turn establishes a cell-specific gene-expression program. Here we wanted to take a deep analysis of DNA binding events and transcriptional output of a single cell state (K562 cells). To this end we re-analyzed 195 DNA binding proteins contained in ENCODE data. We used standardized analysis pipelines, containerization, and literate programming with R Markdown for reproducibility and rigor. Our approach validated many findings from previous independent studies, underscoring the importance of ENCODE's goals in providing these reproducible data resources. We also had several new findings including: (i) 1,362 promoters, which we refer to as 'reservoirs,' that are defined by having up to 111 different DNA binding-proteins localized on one promoter, yet do not have any expression of steady-state RNA (ii) Reservoirs do not overlap super-enhancer annotations and distinct have distinct properties from super-enhancers. (iii) The human specific SVA repeat element may have been co-opted for enhancer regulation and is highly transcribed in PRO-seq and RNA-seq. Collectively, this study performed by the students of a CU Boulder computational biology class (BCHM 5631 -Spring 2020) demonstrates the value of reproducible findings and how resources like ENCODE that prioritize data standards can foster new findings with existing data in a didactic environment.
    DOI:  https://doi.org/10.1371/journal.pone.0237055
  19. Nat Commun. 2021 06 21. 12(1): 3804
    Genomic imprinting in mouse blastocysts is predominantly associated with H3K27me3.
    Laura Santini, Florian Halbritter, Fabian Titz-Teixeira, Toru Suzuki, Maki Asami, Xiaoyan Ma, Julia Ramesmayer, Andreas Lackner, Nick Warr, Florian Pauler, Simon Hippenmeyer, Ernest Laue, Matthias Farlik, Christoph Bock, Andreas Beyer, Anthony C F Perry, Martin Leeb.
      In mammalian genomes, differentially methylated regions (DMRs) and histone marks including trimethylation of histone 3 lysine 27 (H3K27me3) at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. However, neither parent-of-origin-specific transcription nor imprints have been comprehensively mapped at the blastocyst stage of preimplantation development. Here, we address this by integrating transcriptomic and epigenomic approaches in mouse preimplantation embryos. We find that seventy-one genes exhibit previously unreported parent-of-origin-specific expression in blastocysts (nBiX: novel blastocyst-imprinted expressed). Uniparental expression of nBiX genes disappears soon after implantation. Micro-whole-genome bisulfite sequencing (µWGBS) of individual uniparental blastocysts detects 859 DMRs. We further find that 16% of nBiX genes are associated with a DMR, whereas most are associated with parentally-biased H3K27me3, suggesting a role for Polycomb-mediated imprinting in blastocysts. nBiX genes are clustered: five clusters contained at least one published imprinted gene, and five clusters exclusively contained nBiX genes. These data suggest that early development undergoes a complex program of stage-specific imprinting involving different tiers of regulation.
    DOI:  https://doi.org/10.1038/s41467-021-23510-4
  20. PLoS Genet. 2021 Jun 22. 17(6): e1009645
    An enhancer screen identifies new suppressors of small-RNA-mediated epigenetic gene silencing.
    Yukiko Shimada, Sarah H Carl, Merle Skribbe, Valentin Flury, Tahsin Kuzdere, Georg Kempf, Marc Bühler.
      Small non-protein coding RNAs are involved in pathways that control the genome at the level of chromatin. In Schizosaccharomyces pombe, small interfering RNAs (siRNAs) are required for the faithful propagation of heterochromatin that is found at peri-centromeric repeats. In contrast to repetitive DNA, protein-coding genes are refractory to siRNA-mediated heterochromatin formation, unless siRNAs are expressed in mutant cells. Here we report the identification of 20 novel mutant alleles that enable de novo formation of heterochromatin at a euchromatic protein-coding gene by using trans-acting siRNAs as triggers. For example, a single amino acid substitution in the pre-mRNA cleavage factor Yth1 enables siRNAs to trigger silent chromatin formation with unparalleled efficiency. Our results are consistent with a kinetic nascent transcript processing model for the inhibition of small-RNA-directed de novo formation of heterochromatin and lay a foundation for further mechanistic dissection of cellular activities that counteract epigenetic gene silencing.
    DOI:  https://doi.org/10.1371/journal.pgen.1009645
  21. iScience. 2021 Jun 25. 24(6): 102649
    KEAP1 deficiency drives glucose dependency and sensitizes lung cancer cells and tumors to GLUT inhibition.
    Pranavi Koppula, Kellen Olszewski, Yilei Zhang, Lavanya Kondiparthi, Xiaoguang Liu, Guang Lei, Molina Das, Bingliang Fang, Masha V Poyurovsky, Boyi Gan.
      Metabolic reprogramming in cancer cells can create metabolic liabilities. KEAP1-mutant lung cancer is refractory to most current therapies. Here we show that KEAP1 deficiency promotes glucose dependency in lung cancer cells, and KEAP1-mutant/deficient lung cancer cells are more vulnerable to glucose deprivation than their WT counterparts. Mechanistically, KEAP1 inactivation in lung cancer cells induces constitutive activation of NRF2 transcription factor and aberrant expression of NRF2 target cystine transporter SLC7A11; under glucose limitation, high cystine uptake in KEAP1-inactivated lung cancer cells stimulates toxic intracellular disulfide buildup, NADPH depletion, and cell death, which can be rescued by genetic ablation of NRF2-SLC7A11 axis or treatments inhibiting disulfide accumulation. Finally, we show that KEAP1-inactivated lung cancer cells or xenograft tumors are sensitive to glucose transporter inhibitor. Together, our results reveal that KEAP1 deficiency induces glucose dependency in lung cancer cells and uncover a therapeutically relevant metabolic liability.
    Keywords:  cancer; cell biology; physiology
    DOI:  https://doi.org/10.1016/j.isci.2021.102649
  22. Nat Commun. 2021 06 22. 12(1): 3835
    Dissecting regulatory pathways for transcription recovery following DNA damage reveals a non-canonical function of the histone chaperone HIRA.
    Déborah Bouvier, Juliette Ferrand, Odile Chevallier, Michelle T Paulsen, Mats Ljungman, Sophie E Polo.
      Transcription restart after a genotoxic challenge is a fundamental yet poorly understood process. Here, we dissect the interplay between transcription and chromatin restoration after DNA damage by focusing on the human histone chaperone complex HIRA, which is required for transcription recovery post UV. We demonstrate that HIRA is recruited to UV-damaged chromatin via the ubiquitin-dependent segregase VCP to deposit new H3.3 histones. However, this local activity of HIRA is dispensable for transcription recovery. Instead, we reveal a genome-wide function of HIRA in transcription restart that is independent of new H3.3 and not restricted to UV-damaged loci. HIRA coordinates with ASF1B to control transcription restart by two independent pathways: by stabilising the associated subunit UBN2 and by reducing the expression of the transcription repressor ATF3. Thus, HIRA primes UV-damaged chromatin for transcription restart at least in part by relieving transcription inhibition rather than by depositing new H3.3 as an activating bookmark.
    DOI:  https://doi.org/10.1038/s41467-021-24153-1
  23. Elife. 2021 Jun 21. pii: e66143. [Epub ahead of print]10
    The EMT transcription factor Snai1 maintains myocardial wall integrity by repressing intermediate filament gene expression.
    Alessandra Gentile, Anabela Bensimon-Brito, Rashmi Priya, Hans-Martin Maischein, Janett Piesker, Stefan Guenther, Felix Gunawan, Didier Yr Stainier.
      The transcription factor Snai1, a well-known regulator of epithelial-to-mesenchymal transition, has been implicated in early cardiac morphogenesis as well as in cardiac valve formation. However, a role for Snai1 in regulating other aspects of cardiac morphogenesis has not been reported. Using genetic, transcriptomic, and chimeric analyses in zebrafish, we find that Snai1b is required in cardiomyocytes for myocardial wall integrity. Loss of snai1b increases the frequency of cardiomyocyte extrusion away from the cardiac lumen. Extruding cardiomyocytes exhibit increased actomyosin contractility basally as revealed by enrichment of p-myosin and α-catenin epitope α-18, as well as disrupted intercellular junctions. Transcriptomic analysis of wild-type and snai1b mutant hearts revealed the dysregulation of intermediate filament genes, including desmin b (desmb) upregulation. Cardiomyocyte-specific desmb overexpression caused increased cardiomyocyte extrusion, recapitulating the snai1b mutant phenotype. Altogether, these results indicate that Snai1 maintains the integrity of the myocardial epithelium, at least in part by repressing desmb expression.
    Keywords:  cardiac development; cell biology; cell extrusion; developmental biology; tissue integrity; zebrafish
    DOI:  https://doi.org/10.7554/eLife.66143
  24. Elife. 2021 Jun 24. pii: e64906. [Epub ahead of print]10
    In silico analysis of the transcriptional regulatory logic of neuronal identity specification throughout the C. elegans nervous system.
    Lori Glenwinkel, Seth R Taylor, Kasper Langebeck-Jensen, Laura Pereira, Molly B Reilly, Manasa Basavaraju, Ibnul Rafi, Eviatar Yemini, Roger Pocock, Nenad Sestan, Marc Hammarlund, David M Miller, Oliver Hobert.
      The generation of the enormous diversity of neuronal cell types in a differentiating nervous system entails the activation of neuron type-specific gene batteries. To examine the regulatory logic that controls the expression of neuron type-specific gene batteries, we interrogate single cell expression profiles of all 118 neuron classes of the Caenorhabditis elegans nervous system for the presence of DNA binding motifs of 136 neuronally expressed C. elegans transcription factors. Using a phylogenetic footprinting pipeline, we identify cis-regulatory motif enrichments among neuron class-specific gene batteries and we identify cognate transcription factors for 117 of the 118 neuron classes. In addition to predicting novel regulators of neuronal identities, our nervous system-wide analysis at single cell resolution supports the hypothesis that many transcription factors directly co-regulate the cohort of effector genes that define a neuron type, thereby corroborating the concept of so-called terminal selectors of neuronal identity. Our analysis provides a blueprint for how individual components of an entire nervous system are genetically specified.
    Keywords:  C. elegans; cis-regulatory analysis; neuronal differentiation; neuroscience; transcription factor
    DOI:  https://doi.org/10.7554/eLife.64906
  25. Biochem Soc Trans. 2021 Jun 22. pii: BST20191164. [Epub ahead of print]
    The MLL3/4 H3K4 methyltransferase complex in establishing an active enhancer landscape.
    Lan-Hsin Wang, Marvin Angelo E Aberin, Sean Wu, Shu-Ping Wang.
      Enhancers are cis-regulatory elements that play essential roles in tissue-specific gene expression during development. Enhancer function in the expression of developmental genes requires precise regulation, while deregulation of enhancer function could be the main cause of tissue-specific cancer development. MLL3/KMT2C and MLL4/KMT2D are two paralogous histone modifiers that belong to the SET1/MLL (also named COMPASS) family of lysine methyltransferases and play critical roles in enhancer-regulated gene activation. Importantly, large-scale DNA sequencing studies have revealed that they are amongst the most frequently mutated genes associated with human cancers. MLL3 and MLL4 form identical multi-protein complexes for modifying mono-methylation of histone H3 lysine 4 (H3K4) at enhancers, which together with the p300/CBP-mediated H3K27 acetylation can generate an active enhancer landscape for long-range target gene activation. Recent studies have provided a better understanding of the possible mechanisms underlying the roles of MLL3/MLL4 complexes in enhancer regulation. Moreover, accumulating studies offer new insights into our knowledge of the potential role of MLL3/MLL4 in cancer development. In this review, we summarize recent evidence on the molecular mechanisms of MLL3/MLL4 in the regulation of active enhancer landscape and long-range gene expression, and discuss their clinical implications in human cancers.
    Keywords:  H3K4 histone lysine methyltransferase; cancer; enhancer; epigenetics; gene expression and regulation
    DOI:  https://doi.org/10.1042/BST20191164
  26. Nat Commun. 2021 06 22. 12(1): 3846
    The combined action of CTCF and its testis-specific paralog BORIS is essential for spermatogenesis.
    Samuel Rivero-Hinojosa, Elena M Pugacheva, Sungyun Kang, Claudia Fabiola Méndez-Catalá, Alexander L Kovalchuk, Alexander V Strunnikov, Dmitri Loukinov, Jeannie T Lee, Victor V Lobanenkov.
      CTCF is a key organizer of the 3D genome. Its specialized paralog, BORIS, heterodimerizes with CTCF but is expressed only in male germ cells and in cancer states. Unexpectedly, BORIS-null mice have only minimal germ cell defects. To understand the CTCF-BORIS relationship, mouse models with varied CTCF and BORIS levels were generated. Whereas Ctcf+/+Boris+/+, Ctcf+/-Boris+/+, and Ctcf+/+Boris-/- males are fertile, Ctcf+/-Boris-/- (Compound Mutant; CM) males are sterile. Testes with combined depletion of both CTCF and BORIS show reduced size, defective meiotic recombination, increased apoptosis, and malformed spermatozoa. Although CM germ cells exhibit only 25% of CTCF WT expression, chromatin binding of CTCF is preferentially lost from CTCF-BORIS heterodimeric sites. Furthermore, CM testes lose the expression of a large number of spermatogenesis genes and gain the expression of developmentally inappropriate genes that are "toxic" to fertility. Thus, a combined action of CTCF and BORIS is required to both repress pre-meiotic genes and activate post-meiotic genes for a complete spermatogenesis program.
    DOI:  https://doi.org/10.1038/s41467-021-24140-6
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