bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2020‒06‒28
thirty-two papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit

  1. Cell Rep. 2020 Jun 23. pii: S2211-1247(20)30784-1. [Epub ahead of print]31(12): 107803
    Yang M, Lee JH, Zhang Z, De La Rosa R, Bi M, Tan Y, Liao Y, Hong J, Du B, Wu Y, Scheirer J, Hong T, Li W, Fei T, Hsieh CL, Liu Z, Li W, Rosenfeld MG, Xu K.
      The function of enhancer RNAs (eRNAs) in transcriptional regulation remains obscure. By analyzing the genome-wide nascent transcript profiles in breast cancer cells, we identify a special group of eRNAs that are essential for estrogen-induced transcriptional repression. Using eRNAs of TM4SF1 and EFEMP1 as the paradigms, we find that these RNA molecules not only stabilize promoter-enhancer interactions but also recruit liganded estrogen receptor α (ERα) to particular enhancer regions, facilitate the formation of a functional transcriptional complex, and cause gene silencing. Interestingly, ERα is shown to directly bind with eRNAs by its DNA-binding domain. These eRNAs help with the formation of a specific ERα-centered transcriptional complex and promote the association of the histone demethylase KDM2A, which dismisses RNA polymerase II from designated enhancers and suppresses the transcription of target genes. Our work demonstrates a complete mechanism underlying the action of eRNAs in modulating and refining the locus-specific transcriptional program.
    Keywords:  ERα signaling; enhancer RNA; enhancer activity regulation; transcriptional repression
  2. EBioMedicine. 2020 Jun 20. pii: S2352-3964(20)30210-3. [Epub ahead of print]57 102835
    Li L, Li F, Xia Y, Yang X, Lv Q, Fang F, Wang Q, Bu W, Wang Y, Zhang K, Wu Y, Shen J, Jiang M.
      BACKGROUND: Little is known about whether UVB can directly influence epigenetic regulatory pathways to induce cutaneous squamous cell carcinoma (CSCC). This study aimed to identify epigenetic-regulated signalling pathways through global methylation and gene expression profiling and to elucidate their function in CSCC development.METHODS: Global DNA methylation profiling by reduced representation bisulfite sequencing (RRBS) and genome-wide gene expression analysis by RNA sequencing (RNA-seq) in eight pairs of matched CSCC and adjacent normal skin tissues were used to investigate the potential candidate gene(s). Clinical samples, animal models, cell lines, and UVB irradiation were applied to validate the mechanism and function of the genes of interest.
    FINDINGS: We identified the downregulation of the TGF-β/BMP-SMAD-ID4 signalling pathway in CSCC and increased methylation of inhibitor of DNA binding/differentiation 4 (ID4). In normal human and mouse skin tissues and cutaneous cell lines, UVB exposure induced ID4 DNA methylation, upregulated DNMT1 and downregulated ten-eleven translocation (TETs). Similarly, we detected the upregulation of DNMT1 and downregulation of TETs accompanying ID4 DNA methylation in CSCC tissues. Silencing of DNMT1 and overexpression of TET1 and TET2 in A431 and Colo16 cells led to increased ID4 expression. Finally, we showed that overexpression of ID4 reduced cell proliferation, migration, and invasion, and increased apoptosis in CSCC cell lines and reduced tumourigenesis in mouse models.
    INTERPRETATION: The results indicate that ID4 is downregulated by UVB irradiation via DNA methylation. ID4 acts as a tumour suppressor gene in CSCC development.
    FUNDING: CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-3-021, 2017-I2M-1-017), the Natural Science Foundation of Jiangsu Province (BK20191136), and the Fundamental Research Funds for the Central Universities (3332019104).
    Keywords:  Cutaneous squamous cell carcinoma; DNA (cytosine-5-)-methyltransferase; DNA-binding proteins; Methylation; Ten-eleven translocation; Ultraviolet rays
  3. Nat Struct Mol Biol. 2020 Jun 22.
    Eckersley-Maslin MA, Parry A, Blotenburg M, Krueger C, Ito Y, Franklin VNR, Narita M, D'Santos CS, Reik W.
      How the epigenetic landscape is established in development is still being elucidated. Here, we uncover developmental pluripotency associated 2 and 4 (DPPA2/4) as epigenetic priming factors that establish a permissive epigenetic landscape at a subset of developmentally important bivalent promoters characterized by low expression and poised RNA-polymerase. Differentiation assays reveal that Dppa2/4 double knockout mouse embryonic stem cells fail to exit pluripotency and differentiate efficiently. DPPA2/4 bind both H3K4me3-marked and bivalent gene promoters and associate with COMPASS- and Polycomb-bound chromatin. Comparing knockout and inducible knockdown systems, we find that acute depletion of DPPA2/4 results in rapid loss of H3K4me3 from key bivalent genes, while H3K27me3 is initially more stable but lost following extended culture. Consequently, upon DPPA2/4 depletion, these promoters gain DNA methylation and are unable to be activated upon differentiation. Our findings uncover a novel epigenetic priming mechanism at developmental promoters, poising them for future lineage-specific activation.
  4. Mol Cell. 2020 Jun 22. pii: S1097-2765(20)30357-9. [Epub ahead of print]
    Lerner J, Gomez-Garcia PA, McCarthy RL, Liu Z, Lakadamyali M, Zaret KS.
      Enzymatic probes of chromatin structure reveal accessible versus inaccessible chromatin states, while super-resolution microscopy reveals a continuum of chromatin compaction states. Characterizing histone H2B movements by single-molecule tracking (SMT), we resolved chromatin domains ranging from low to high mobility and displaying different subnuclear localizations patterns. Heterochromatin constituents correlated with the lowest mobility chromatin, whereas transcription factors varied widely with regard to their respective mobility with low- or high-mobility chromatin. Pioneer transcription factors, which bind nucleosomes, can access the low-mobility chromatin domains, whereas weak or non-nucleosome binding factors are excluded from the domains and enriched in higher mobility domains. Nonspecific DNA and nucleosome binding accounted for most of the low mobility of strong nucleosome interactor FOXA1. Our analysis shows how the parameters of the mobility of chromatin-bound factors, but not their diffusion behaviors or SMT-residence times within chromatin, distinguish functional characteristics of different chromatin-interacting proteins.
  5. Sci Rep. 2020 Jun 23. 10(1): 10150
    Gontarz P, Fu S, Xing X, Liu S, Miao B, Bazylianska V, Sharma A, Madden P, Cates K, Yoo A, Moszczynska A, Wang T, Zhang B.
      ATAC-seq is widely used to measure chromatin accessibility and identify open chromatin regions (OCRs). OCRs usually indicate active regulatory elements in the genome and are directly associated with the gene regulatory network. The identification of differential accessibility regions (DARs) between different biological conditions is critical in determining the differential activity of regulatory elements. Differential analysis of ATAC-seq shares many similarities with differential expression analysis of RNA-seq data. However, the distribution of ATAC-seq signal intensity is different from that of RNA-seq data, and higher sensitivity is required for DARs identification. Many different tools can be used to perform differential analysis of ATAC-seq data, but a comprehensive comparison and benchmarking of these methods is still lacking. Here, we used simulated datasets to systematically measure the sensitivity and specificity of six different methods. We further discussed the statistical and signal density cut-offs in the differential analysis of ATAC-seq by applying them to real data. Batch effects are very common in high-throughput sequencing experiments. We illustrated that batch-effect correction can dramatically improve sensitivity in the differential analysis of ATAC-seq data. Finally, we developed a user-friendly package, BeCorrect, to perform batch effect correction and visualization of corrected ATAC-seq signals in a genome browser.
  6. Nat Protoc. 2020 Jun 26.
    Font-Tello A, Kesten N, Xie Y, Taing L, Varešlija D, Young LS, Hamid AA, Van Allen EM, Sweeney CJ, Gjini E, Lako A, Hodi FS, Bellmunt J, Brown M, Cejas P, Long HW.
      Fixed-tissue ChIP-seq for H3K27 acetylation (H3K27ac) profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65 °C in a higher concentration of detergent and a minimized sonication step, to produce robust genome-wide H3K27ac maps from clinical samples. FiTAc-seq generates high-quality enhancer landscapes and super-enhancer (SE) annotation in numerous archived FFPE samples from distinct tumor types. This approach will be of great interest for both basic and clinical researchers. The entire protocol from FFPE blocks to sequence-ready library can be accomplished within 4 d.
  7. Nat Commun. 2020 Jun 24. 11(1): 3199
    Yagi M, Kabata M, Tanaka A, Ukai T, Ohta S, Nakabayashi K, Shimizu M, Hata K, Meissner A, Yamamoto T, Yamada Y.
      De novo establishment of DNA methylation is accomplished by DNMT3A and DNMT3B. Here, we analyze de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from DNA-hypomethylated 2i/L ES cells with genetic ablation of Dnmt3a or Dnmt3b. We identify 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b knockout (KO) 2i-MEFs, respectively. We find that Dnmt3a is exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes, while Dnmt3b has a dominant role on the X chromosome. Consistent with this, tissue-specific DNA methylation at PcG target genes is substantially reduced in Dnmt3a KO embryos. Finally, we find that human patients with DNMT3 mutations exhibit reduced DNA methylation at regions that are hypomethylated in Dnmt3 KO 2i-MEFs. In conclusion, here we report a set of unique de novo DNA methylation target sites for both DNMT3 enzymes during mammalian development that overlap with hypomethylated sites in human patients.
  8. Mol Cell. 2020 Jun 16. pii: S1097-2765(20)30386-5. [Epub ahead of print]
    Zhang K, Wu DY, Zheng H, Wang Y, Sun QR, Liu X, Wang LY, Xiong WJ, Wang Q, Rhodes JDP, Xu K, Li L, Lin Z, Yu G, Xia W, Huang B, Du Z, Yao Y, Nasmyth KA, Klose RJ, Miao YL, Xie W.
      Somatic cell nuclear transfer (SCNT) can reprogram a somatic nucleus to a totipotent state. However, the re-organization of 3D chromatin structure in this process remains poorly understood. Using low-input Hi-C, we revealed that, during SCNT, the transferred nucleus first enters a mitotic-like state (premature chromatin condensation). Unlike fertilized embryos, SCNT embryos show stronger topologically associating domains (TADs) at the 1-cell stage. TADs become weaker at the 2-cell stage, followed by gradual consolidation. Compartments A/B are markedly weak in 1-cell SCNT embryos and become increasingly strengthened afterward. By the 8-cell stage, somatic chromatin architecture is largely reset to embryonic patterns. Unexpectedly, we found cohesin represses minor zygotic genome activation (ZGA) genes (2-cell-specific genes) in pluripotent and differentiated cells, and pre-depleting cohesin in donor cells facilitates minor ZGA and SCNT. These data reveal multi-step reprogramming of 3D chromatin architecture during SCNT and support dual roles of cohesin in TAD formation and minor ZGA repression.
    Keywords:  Hi-C; SCNT; TAD; chromatin reprogramming; cohesin; early embryo; minor ZGA; somatic cell nuclear transfer; three-dimensional chromatin structure
  9. Nat Struct Mol Biol. 2020 Jun 22.
    Gretarsson KH, Hackett JA.
      Early mammalian development entails genome-wide epigenome remodeling, including DNA methylation erasure and reacquisition, which facilitates developmental competence. To uncover the mechanisms that orchestrate DNA methylation dynamics, we coupled a single-cell ratiometric DNA methylation reporter with unbiased CRISPR screening in murine embryonic stem cells (ESCs). We identify key genes and regulatory pathways that drive global DNA hypomethylation, and characterize roles for Cop1 and Dusp6. We also identify Dppa2 and Dppa4 as essential safeguards of focal epigenetic states. In their absence, developmental genes and evolutionarily young LINE1 elements, which are specifically bound by DPPA2, lose H3K4me3 and gain ectopic de novo DNA methylation in pluripotent cells. Consequently, lineage-associated genes and LINE1 acquire a repressive epigenetic memory, which renders them incompetent for activation during future lineage specification. Dppa2/4 thereby sculpt the pluripotent epigenome by facilitating H3K4me3 and bivalency to counteract de novo methylation, a function co-opted by evolutionarily young LINE1 to evade epigenetic decommissioning.
  10. Genomics. 2020 Jun 20. pii: S0888-7543(19)30258-7. [Epub ahead of print]
    Nie Y, Shu C, Sun X.
      Transcription factors (TFs) cooperatively bind to specific DNA sequences to control chromatin and gene transcription in eukaryotes. Here, we searched canonical binding, co-binding and tethered binding regions of a TF within ChIP-seq peaks, and investigated the effect of TF-TF cooperation in human GM12878 and K562 cells. We found that TFs except for CTCF and SPI1 showed a large proportion of co-binding and tethered binding regions, and TFs frequently co-binding with other TFs would also frequently tether other TFs to their binding positions. We further observed lower in vivo nucleosome occupancy, higher in vitro nucleosome occupancy and higher levels of H2A.Z, H3K27ac, H3K9ac, H3K4me1, H3K4me2 and H3K4me3 within distal co-binding regions where other TFs were recruited. In addition, target genes for proximal co-binding regions where other TFs were recruited showed significantly higher expression levels. These results indicated that TF-TF cooperation directly associate with the chromatin structure and gene transcription.
    Keywords:  Chromatin; Co-binding; Tethered binding; Transcription factor
  11. Nucleic Acids Res. 2020 Jun 25. pii: gkaa529. [Epub ahead of print]
    Shukla R, Mjoseng HK, Thomson JP, Kling S, Sproul D, Dunican DS, Ramsahoye B, Wongtawan T, Treindl F, Templin MF, Adams IR, Pennings S, Meehan RR.
      Mouse embryonic stem cells (mESCs) cultured with MEK/ERK and GSK3β (2i) inhibitors transition to ground state pluripotency. Gene expression changes, redistribution of histone H3K27me3 profiles and global DNA hypomethylation are hallmarks of 2i exposure, but it is unclear whether epigenetic alterations are required to achieve and maintain ground state or occur as an outcome of 2i signal induced changes. Here we show that ESCs with three epitypes, WT, constitutively methylated, or hypomethylated, all undergo comparable morphological, protein expression and transcriptome changes independently of global alterations of DNA methylation levels or changes in H3K27me3 profiles. Dazl and Fkbp6 expression are induced by 2i in all three epitypes, despite exhibiting hypermethylated promoters in constitutively methylated ESCs. We identify a number of activated gene promoters that undergo 2i dependent loss of H3K27me3 in all three epitypes, however genetic and pharmaceutical inhibition experiments show that H3K27me3 is not required for their silencing in non-2i conditions. By separating and defining their contributions, our data suggest that repressive epigenetic systems play minor roles in mESC self-renewal and naïve ground state establishment by core sets of dominant pluripotency associated transcription factor networks, which operate independently from these epigenetic processes.
  12. Cell Rep. 2020 Jun 23. pii: S2211-1247(20)30762-2. [Epub ahead of print]31(12): 107782
    Cheng Y, Liao S, Xu G, Hu J, Guo D, Du F, Contreras A, Cai KQ, Peri S, Wang Y, Corney DC, Noronha AM, Chau LQ, Zhou G, Wiest DL, Bellacosa A, Wechsler-Reya RJ, Zhao Y, Yang ZJ.
      Tumor cells are characterized by unlimited proliferation and perturbed differentiation. Using single-cell RNA sequencing, we demonstrate that tumor cells in medulloblastoma (MB) retain their capacity to differentiate in a similar way as their normal originating cells, cerebellar granule neuron precursors. Once they differentiate, MB cells permanently lose their proliferative capacity and tumorigenic potential. Differentiated MB cells highly express NeuroD1, a helix-loop-helix transcription factor, and forced expression of NeuroD1 promotes the differentiation of MB cells. The expression of NeuroD1 in bulk MB cells is repressed by trimethylation of histone 3 lysine-27 (H3K27me3). Inhibition of the histone lysine methyltransferase EZH2 prevents H3K27 trimethylation, resulting in increased NeuroD1 expression and enhanced differentiation in MB cells, which consequently reduces tumor growth. These studies reveal the mechanisms underlying MB cell differentiation and provide rationales to treat MB (potentially other malignancies) by stimulating tumor cell differentiation.
    Keywords:  EZH2 inhibitors; H3k27me3; NeuroD1; Tag1; differentiation therapy; epigenetic compound screening; granule neuron precursors; hedgehog signaling; medulloblastoma; tumor cell differentiation
  13. J Cell Sci. 2020 Jun 23. pii: jcs.243444. [Epub ahead of print]
    Hayashi-Takanaka Y, Kina Y, Nakamura F, Becking LE, Nakao Y, Nagase T, Nozaki N, Kimura H.
      Post-translational modifications on histones can be stable epigenetic marks and transient signals that can occur in response to internal and external stimuli. Levels of histone modifications fluctuate during the cell cycle and vary among different cell types. Here we describe a simple system to monitor the levels of multiple histone modifications in single cells by multicolor immunofluorescence using directly labeled modification-specific antibodies. We analyzed histone H3 and H4 modifications during the cell cycle. Levels of active marks, such as acetylation and H3K4 methylation, were increased during the S phase, in association with chromatin duplication. By contrast, levels of some repressive modifications gradually increased during the G2 and the next G1 phases. We applied this method to validate the target modifications of various histone demethylases in cells using a transient overexpression system. We also screened chemical compounds in marine organism extracts that affect histone modifications and identified psammaplin A, which was previously reported to inhibit histone deacetylases. Thus, the method presented here is a powerful and convenient tool for analyzing the changes in histone modifications.
    Keywords:  Chemical biology; Chromatin; Epigenetics; Histone modification; Monoclonal antibody
  14. Science. 2020 Jun 26. 368(6498): 1449-1454
    Stergachis AB, Debo BM, Haugen E, Churchman LS, Stamatoyannopoulos JA.
      Gene regulation is chiefly determined at the level of individual linear chromatin molecules, yet our current understanding of cis-regulatory architectures derives from fragmented sampling of large numbers of disparate molecules. We developed an approach for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using nonspecific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (Fiber-seq). Fiber-seq exposed widespread plasticity in the linear organization of individual chromatin fibers and illuminated principles guiding regulatory DNA actuation, the coordinated actuation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. Our approach and results open new vistas on the primary architecture of gene regulation.
  15. Nat Immunol. 2020 Jun 22.
    Tanaka S, Ise W, Inoue T, Ito A, Ono C, Shima Y, Sakakibara S, Nakayama M, Fujii K, Miura I, Sharif J, Koseki H, Koni PA, Raman I, Li QZ, Kubo M, Fujiki K, Nakato R, Shirahige K, Araki H, Miura F, Ito T, Kawakami E, Baba Y, Kurosaki T.
      A contribution of epigenetic modifications to B cell tolerance has been proposed but not directly tested. Here we report that deficiency of ten-eleven translocation (Tet) DNA demethylase family members Tet2 and Tet3 in B cells led to hyperactivation of B and T cells, autoantibody production and lupus-like disease in mice. Mechanistically, in the absence of Tet2 and Tet3, downregulation of CD86, which normally occurs following chronic exposure of self-reactive B cells to self-antigen, did not take place. The importance of dysregulated CD86 expression in Tet2- and Tet3-deficient B cells was further demonstrated by the restriction, albeit not complete, on aberrant T and B cell activation following anti-CD86 blockade. Tet2- and Tet3-deficient B cells had decreased accumulation of histone deacetylase 1 (HDAC1) and HDAC2 at the Cd86 locus. Thus, our findings suggest that Tet2- and Tet3-mediated chromatin modification participates in repression of CD86 on chronically stimulated self-reactive B cells, which contributes, at least in part, to preventing autoimmunity.
  16. Int J Cancer. 2020 Jun 23.
    Wu Y, Yang Y, Gu H, Tao B, Zhang E, Wei J, Wang Z, Liu A, Sun R, Chen M, Fan Y, Mao R.
      Enhancer can transcribe RNAs, however, most of them were neglected in traditional RNA-seq analysis workflow. Here, we developed a Pipeline for Enhancer Transcription (PET, for quantifying enhancer RNAs (eRNAs) from RNA-seq. By applying this pipeline on lung cancer samples and cell lines, we show that the transcribed enhancers are enriched with histone marks and transcription factor motifs (JUNB, Hand1-Tcf3, and GATA4). By training a machine learning model, we demonstrate that enhancers can predict prognosis better than their nearby genes. Integrating the Hi-C, ChIP-seq, and RNA-seq data, we observe that transcribed enhancers associate with cancer hallmarks or oncogenes, among which LcsMYC-1 (Lung cancer-specific MYC eRNA-1) potentially supports MYC expression. Surprisingly, a significant proportion of transcribed enhancers contain small protein-coding open reading frames (sORFs) and can be translated into microproteins. Our study provides a computational method for eRNA quantification and deepens our understandings of the DNA, RNA, and protein nature of enhancers. This article is protected by copyright. All rights reserved.
    Keywords:  Enhancer RNA; eRNA pipeline; sORF; transcription factor
  17. Cell Rep. 2020 Jun 23. pii: S2211-1247(20)30790-7. [Epub ahead of print]31(12): 107809
    Holden JK, Crawford JJ, Noland CL, Schmidt S, Zbieg JR, Lacap JA, Zang R, Miller GM, Zhang Y, Beroza P, Reja R, Lee W, Tom JYK, Fong R, Steffek M, Clausen S, Hagenbeek TJ, Hu T, Zhou Z, Shen HC, Cunningham CN.
      The transcriptional enhanced associate domain (TEAD) family of transcription factors serves as the receptors for the downstream effectors of the Hippo pathway, YAP and TAZ, to upregulate the expression of multiple genes involved in cellular proliferation and survival. Recent work identified TEAD S-palmitoylation as critical for protein stability and activity as the lipid tail extends into a hydrophobic core of the protein. Here, we report the identification and characterization of a potent small molecule that binds the TEAD lipid pocket (LP) and disrupts TEAD S-palmitoylation. Using a variety of biochemical, structural, and cellular methods, we uncover that TEAD S-palmitoylation functions as a TEAD homeostatic protein level checkpoint and that dysregulation of this lipidation affects TEAD transcriptional activity in a dominant-negative manner. Furthermore, we demonstrate that targeting the TEAD LP is a promising therapeutic strategy for modulating the Hippo pathway, showing tumor stasis in a mouse xenograft model.
    Keywords:  Hippo pathway; TEAD; YAP; cancer; lipidation; therapeutic; transcription factor
  18. Genome Med. 2020 Jun 24. 12(1): 54
    Erger F, Nörling D, Borchert D, Leenen E, Habbig S, Wiesener MS, Bartram MP, Wenzel A, Becker C, Toliat MR, Nürnberg P, Beck BB, Altmüller J.
      Cell-free DNA (cfDNA) analysis has become essential in cancer diagnostics and prenatal testing. We present cfNOMe, a two-in-one method of measuring cfDNA cytosine methylation and nucleosome occupancy in a single assay using non-disruptive enzymatic cytosine conversion and a custom bioinformatic pipeline. We show that enzymatic cytosine conversion better preserves cfDNA fragmentation information than does bisulfite conversion. Whereas previously separate experiments were required to study either epigenetic marking, cfNOMe delivers reliable results for both, enabling more comprehensive and inexpensive epigenetic cfDNA profiling. cfNOMe has the potential to advance biomarker discovery and diagnostic usage in diseases with systemic perturbations of cfDNA composition.
    Keywords:  Analysis software; Cell-free DNA; Methylome; Nucleosome footprints; cfNOMe
  19. Oncogene. 2020 Jun 25.
    Hruschka N, Kalisz M, Subijana M, Graña-Castro O, Del Cano-Ochoa F, Brunet LP, Chernukhin I, Sagrera A, De Reynies A, Kloesch B, Chin SF, Burgués O, Andreu D, Bermejo B, Cejalvo JM, Sutton J, Caldas C, Ramón-Maiques S, Carroll JS, Prat A, Real FX, Martinelli P.
      As the catalog of oncogenic driver mutations is expanding, it becomes clear that alterations in a given gene might have different functions and should not be lumped into one class. The transcription factor GATA3 is a paradigm of this. We investigated the functions of the most common GATA3 mutation (X308_Splice) and five additional mutations, which converge into a neoprotein that we called "neoGATA3," associated with excellent prognosis in patients. Analysis of available molecular data from >3000 breast cancer patients revealed a dysregulation of the ER-dependent transcriptional response in tumors carrying neoGATA3-generating mutations. Mechanistic studies in vitro showed that neoGATA3 interferes with the transcriptional programs controlled by estrogen and progesterone receptors, without fully abrogating them. ChIP-Seq analysis indicated that ER binding is reduced in neoGATA3-expressing cells, especially at distal regions, suggesting that neoGATA3 interferes with the fine tuning of ER-dependent gene expression. This has opposite outputs in distinct hormonal context, having pro- or anti-proliferative effects, depending on the estrogen/progesterone ratio. Our data call for functional analyses of putative cancer drivers to guide clinical application.
  20. Nat Commun. 2020 Jun 23. 11(1): 3171
    Wosika V, Pelet S.
      Precise regulation of gene expression in response to environmental changes is crucial for cell survival, adaptation and proliferation. In eukaryotic cells, extracellular signal integration is often carried out by Mitogen-Activated Protein Kinases (MAPK). Despite a robust MAPK signaling activity, downstream gene expression can display a great variability between single cells. Using a live mRNA reporter, here we monitor the dynamics of transcription in Saccharomyces cerevisiae upon hyper-osmotic shock. We find that the transient activity of the MAPK Hog1 opens a temporal window where stress-response genes can be activated. We show that the first minutes of Hog1 activity are essential to control the activation of a promoter. Chromatin repression on a locus slows down this transition and contributes to the variability in gene expression, while binding of transcription factors increases the level of transcription. However, soon after Hog1 activity peaks, negative regulators promote chromatin closure of the locus and transcription progressively stops.
  21. Nature. 2020 Jun 24.
    Wheat JC, Sella Y, Willcockson M, Skoultchi AI, Bergman A, Singer RH, Steidl U.
      Molecular noise is a natural phenomenon that is inherent to all biological systems1,2. How stochastic processes give rise to the robust outcomes that support tissue homeostasis remains unclear. Here we use single-molecule RNA fluorescent in situ hybridization (smFISH) on mouse stem cells derived from haematopoietic tissue to measure the transcription dynamics of three key genes that encode transcription factors: PU.1 (also known as Spi1), Gata1 and Gata2. We find that infrequent, stochastic bursts of transcription result in the co-expression of these antagonistic transcription factors in the majority of haematopoietic stem and progenitor cells. Moreover, by pairing smFISH with time-lapse microscopy and the analysis of pedigrees, we find that although individual stem-cell clones produce descendants that are in transcriptionally related states-akin to a transcriptional priming phenomenon-the underlying transition dynamics between states are best captured by stochastic and reversible models. As such, a stochastic process can produce cellular behaviours that may be incorrectly inferred to have arisen from deterministic dynamics. We propose a model whereby the intrinsic stochasticity of gene expression facilitates, rather than impedes, the concomitant maintenance of transcriptional plasticity and stem cell robustness.
  22. Sci Rep. 2020 Jun 25. 10(1): 10343
    Li H, Hu Z, Jiang H, Pu J, Selli I, Qiu J, Zhang B, Feng J.
      The TET family of 5-methylcytosine (5mC) dioxygenases plays critical roles in development by modifying DNA methylation. Using CRISPR, we inactivated the TET1 gene in H9 human embryonic stem cells (hESCs). Mutant H9 hESCs remained pluripotent, even though the level of hydroxymethylcytosine (5hmC) decreased to 30% of that in wild-type cells. Neural differentiation induced by dual SMAD inhibitors was not significantly affected by loss of TET1 activity. However, in a morphogen-free condition, TET1 deficiency significantly reduced the generation of NESTIN+SOX1+ neuroectoderm cells from 70% in wild-type cells to 20% in mutant cells. This was accompanied by a 20-fold reduction in the expression level of PAX6 and a significant decrease in the amount of 5hmC on the PAX6 promoter. Overexpression of the TET1 catalytic domain in TET1-deficient hESCs significantly increased 5hmC levels and elevated PAX6 expression during differentiation. Consistent with these in vitro data, PAX6 expression was significantly decreased in teratomas formed by TET1-deficient hESCs. However, TET1 deficiency did not prevent the formation of neural tube-like structures in teratomas. Our results suggest that TET1 deficiency impairs the intrinsic ability of hESCs to differentiate to neuroectoderm, presumably by decreasing the expression of PAX6, a key regulator in the development of human neuroectoderm.
  23. Sci Adv. 2020 Jun;6(24): eaay8299
    Zhang D, Guelfi S, Garcia-Ruiz S, Costa B, Reynolds RH, D'Sa K, Liu W, Courtin T, Peterson A, Jaffe AE, Hardy J, Botía JA, Collado-Torres L, Ryten M.
      Growing evidence suggests that human gene annotation remains incomplete; however, it is unclear how this affects different tissues and our understanding of different disorders. Here, we detect previously unannotated transcription from Genotype-Tissue Expression RNA sequencing data across 41 human tissues. We connect this unannotated transcription to known genes, confirming that human gene annotation remains incomplete, even among well-studied genes including 63% of the Online Mendelian Inheritance in Man-morbid catalog and 317 neurodegeneration-associated genes. We find the greatest abundance of unannotated transcription in brain and genes highly expressed in brain are more likely to be reannotated. We explore examples of reannotated disease genes, such as SNCA, for which we experimentally validate a previously unidentified, brain-specific, potentially protein-coding exon. We release all tissue-specific transcriptomes through vizER: We anticipate that this resource will facilitate more accurate genetic analysis, with the greatest impact on our understanding of Mendelian and complex neurogenetic disorders.
  24. J Vis Exp. 2020 Jun 05.
    Liao Z, Lee JH, Krakowiak J, Wang R, Li W.
      Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence has shown that enhancers not only provide DNA binding motifs for transcription factors (TFs) but also generate non-coding RNAs that are referred to as eRNAs. Studies have demonstrated that eRNA transcripts can play significant roles in gene regulation in both physiology and disease. Commonly used methods to investigate the function of eRNAs are constrained to "loss-of-function" approaches by knockdown of eRNAs, or by chemical inhibition of the enhancer transcription. There has not been a robust method to conduct "gain-of-function" studies of eRNAs to mimic specific disease conditions such as human cancer, where eRNAs are often overexpressed. Here, we introduce a method for precisely and robustly activating eRNAs for functional interrogation of their roles by applying the dCas9 mediated Synergistic Activation Mediators (SAM) system. We present the entire workflow of eRNA activation, from the selection of eRNAs, the design of gRNAs to the validation of eRNA activation by RT-qPCR. This method represents a unique approach to study the roles of a particular eRNA in gene regulation and disease development. In addition, this system can be employed for unbiased CRISPR screening to identify phenotype-driving eRNA targets in the context of a specific disease.
  25. Biochemistry. 2020 Jun 26.
    Dhall A, Shelton PMM, Delachat AM, Leonen CJA, Fierz B, Chatterjee C.
      The essential human enzyme lysine specific demethylase 1 (LSD1) silences genes by demethylating mono- and dimethylated lysine 4 in histone H3 (H3K4me1/2). Studies of the minimal requirements for LSD1 activity are complicated by the heterogeneity of histone modification states in cells. We overcame this challenge by generating homogeneous mononucleosome substrates containing semisynthetic H3K4me2. Biophysical and biochemical assays with full-length LSD1 revealed its ability to bind and demethylate nucleosomes. Consistent with a requirement for nucleosome binding prior to demethylation, a competing nucleosome-binding peptide from the high-mobility group protein effectively inhibited LSD1 activity. Thus, our studies provide the first glimpse of nucleosome demethylation by LSD1 in the absence of other scaffolding proteins.
  26. PLoS One. 2020 ;15(6): e0235343
    Raisner R, Bainer R, Haverty PM, Benedetti KL, Gascoigne KE.
      Triple Negative Breast Cancer (TNBC) is a heterogeneous disease lacking known molecular drivers and effective targeted therapies. Cytotoxic chemotherapy remains the mainstay of treatment for TNBCs, which have significantly poorer survival rates compared to other breast cancer subtypes. In addition to changes within the coding genome, aberrant enhancer activity is a well-established contributor to tumorigenesis. Here we use H3K27Ac chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map the active cis-regulatory landscape in TNBC. We identify distinct disease subtypes associated with specific enhancer activity, and over 2,500 unique superenhancers acquired by tumor cells but absent from normal breast tissue. To identify potential actionable disease drivers, we probed the dependency on genes that associate with tumor-specific enhancers by CRISPR screening. In this way we identify a number of tumor-specific dependencies, including a previously uncharacterized dependency on the TGFβ pseudo-receptor BAMBI.
  27. Dev Cell. 2020 Jun 19. pii: S1534-5807(20)30453-6. [Epub ahead of print]
    Hur W, Kemp JP, Tarzia M, Deneke VE, Marzluff WF, Duronio RJ, Di Talia S.
      Many membraneless organelles form through liquid-liquid phase separation, but how their size is controlled and whether size is linked to function remain poorly understood. The histone locus body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of histone mRNAs. Here, we show that Drosophila HLBs form through phase separation. During embryogenesis, the size of HLBs is controlled in a precise and dynamic manner that is dependent on the cell cycle and zygotic histone gene activation. Control of HLB growth is achieved by a mechanism integrating nascent mRNAs at the histone locus, which facilitates phase separation, and the nuclear concentration of the scaffold protein multi-sex combs (Mxc), which is controlled by the activity of cyclin-dependent kinases. Reduced Cdk2 activity results in smaller HLBs and the appearance of nascent, misprocessed histone mRNAs. Thus, our experiments identify a mechanism linking nuclear body growth and size with gene expression.
    Keywords:  cell cycle; cyclin-dependent kinase; histone locus body; histone mRNA processing; histone transcription; membraneless organelle; multi-sex combs; nuclear body; phase separation; size control
  28. Mol Cancer Res. 2020 Jun 22. pii: molcanres.0108.2020. [Epub ahead of print]
    Tian Y, Liu Q, Yu S, Chu Q, Chen Y, Wu K, Wang L.
      Constitutive NRF2 activation by disrupted KEAP1-NRF2 interaction has been reported in a variety of human cancers. However, studies focusing on NRF2-driven KEAP1 expression under human cancer contexts are still uncommon. We examined mRNA expression correlation between NRF2 and KEAP1 in multiple human cancers. We measured KEAP1 mRNA and protein alterations in response to the activation or silencing of NRF2. We queried ChIP-seq datasets to identify NRF2 binding to KEAP1 promoters in human cells. We used reporter assay and CRISPR editing to assess KEAP1 promoter activity and mRNA abundance change. To determine specimen implication of the feedback pattern, we used gene expression ratio to predict NRF2 signal disruption as well as patients' prognosis. Correlation analysis showed KEAP1 mRNA expression was in positive association with NRF2 in multiple squamous cell cancers. The positive correlations were consistent across all squamous cell lung cancer cohorts, but not in adenocarcinomas. In human lung cells, NRF2 interventions significantly altered KEAP1 mRNA and protein expressions. ChIP-qPCR and sequencing data demonstrated consistent NRF2 occupancy to KEAP1 promoter. Deleting NRF2 binding site significantly reduced baseline and inducible KEAP1 promoter activity and KEAP1 mRNA expression. By incorporating tumor tissue KEAP1 mRNA expressions in estimating NRF2 signaling disruptions, we found increased TXN/KEAP1 mRNA ratio in cases with NRF2 gain or KEAP1 loss and decreased NRF2/KEAP1 mRNA ratio in cases with NRF2-KEAP1 somatic mutations. In TCGA PanCancer datasets, we also identified that cases with loss-of-function mutations in NRF2 pathway recurrently appeared above the NRF2-KEAP1 mRNA expression regression lines. Moreover, compared with previous NRF2 signatures, the ratio-based strategy showed better predictive performance in survival analysis with multiple SQC cohort validations. Implications: NRF2-driven KEAP1 transcription is a crucial component of NRF2 signaling modulation. This hidden circuit will provide in-depth insight into novel cancer prevention and therapeutic strategies.
  29. Cell Rep. 2020 Jun 23. pii: S2211-1247(20)30769-5. [Epub ahead of print]31(12): 107789
    Odell SC, Taki F, Klein SL, Chen RJ, Levine OB, Skelly MJ, Nabila A, Brindley E, Gal Toth J, Dündar F, Sheridan CK, Fetcho RN, Alonso A, Liston C, Landau DA, Pleil KE, Toth M.
      Sensory inputs activate sparse neuronal ensembles in the dentate gyrus of the hippocampus, but how eligibility of individual neurons to recruitment is determined remains elusive. We identify thousands of largely bistable (CpG methylated or unmethylated) regions within neuronal gene bodies, established during mouse dentate gyrus development. Reducing DNA methylation and the proportion of the methylated epialleles at bistable regions compromises novel context-induced neuronal activation. Conversely, increasing methylation and the frequency of the methylated epialleles at bistable regions enhances intrinsic excitability. Single-nucleus profiling reveals enrichment of specific epialleles related to a subset of primarily exonic, bistable regions in activated neurons. Genes displaying both differential methylation and expression in activated neurons define a network of proteins regulating neuronal excitability and structural plasticity. We propose a model in which bistable regions create neuron heterogeneity and constellations of exonic methylation, which may contribute to cell-specific gene expression, excitability, and eligibility to a coding ensemble.
    Keywords:  DNA methylation; dentate gyrus; encoding; ensemble; epialleles; epigenetics; neuron recruitment; neuronal excitability; neuronal heterogeneity
  30. FASEB J. 2020 Jun 22.
    Ruiz CF, Montal ED, Haley JA, Bott AJ, Haley JD.
      Cancer cells require extensive metabolic reprograming in order to provide the bioenergetics and macromolecular precursors needed to sustain a malignant phenotype. Mutant KRAS is a driver oncogene that is well-known for its ability to regulate the ERK and PI3K signaling pathways. However, it is now appreciated that KRAS can promote the tumor growth via upregulation of anabolic metabolism. We recently reported that oncogenic KRAS promotes a gene expression program of de novo lipogenesis in non-small cell lung cancer (NSCLC). To define the mechanism(s) responsible, we focused on the lipogenic transcription factor SREBP1. We observed that KRAS increases SREBP1 expression and genetic knockdown of SREBP1 significantly inhibited the cell proliferation of mutant KRAS-expressing cells. Unexpectedly, lipogenesis was not significantly altered in cells subject to SREBP1 knockdown. Carbon tracing metabolic studies showed a significant decrease in oxidative phosphorylation and RNA-seq data revealed a significant decrease in mitochondrial encoded subunits of the electron transport chain (ETC). Taken together, these data support a novel role, distinct from lipogenesis, of SREBP1 on mitochondrial function in mutant KRAS NSCLC.
    Keywords:  cancer metabolism; de novo lipogenesis; electron transport chain; oxidative phosphorylation
  31. Nat Commun. 2020 Jun 26. 11(1): 3224
    Le NT, Harukawa Y, Miura S, Boer D, Kawabe A, Saze H.
      In plants, epigenetic regulation is critical for silencing transposons and maintaining proper gene expression. However, its impact on the genome-wide transcription initiation landscape remains elusive. By conducting a genome-wide analysis of transcription start sites (TSSs) using cap analysis of gene expression (CAGE) sequencing, we show that thousands of TSSs are exclusively activated in various epigenetic mutants of Arabidopsis thaliana and referred to as cryptic TSSs. Many have not been identified in previous studies, of which up to 65% are contributed by transposons. They possess similar genetic features to regular TSSs and their activation is strongly associated with the ectopic recruitment of RNAPII machinery. The activation of cryptic TSSs significantly alters transcription of nearby TSSs, including those of genes important for development and stress responses. Our study, therefore, sheds light on the role of epigenetic regulation in maintaining proper gene functions in plants by suppressing transcription from cryptic TSSs.
  32. PLoS Comput Biol. 2020 Jun 25. 16(6): e1008006
    Lee D, Zhang J, Liu J, Gerstein M.
      Alternative RNA splicing provides an important means to expand metazoan transcriptome diversity. Contrary to what was accepted previously, splicing is now thought to predominantly take place during transcription. Motivated by emerging data showing the physical proximity of the spliceosome to Pol II, we surveyed the effect of epigenetic context on co-transcriptional splicing. In particular, we observed that splicing factors were not necessarily enriched at exon junctions and that most epigenetic signatures had a distinctly asymmetric profile around known splice sites. Given this, we tried to build an interpretable model that mimics the physical layout of splicing regulation where the chromatin context progressively changes as the Pol II moves along the guide DNA. We used a recurrent-neural-network architecture to predict the inclusion of a spliced exon based on adjacent epigenetic signals, and we showed that distinct spatio-temporal features of these signals were key determinants of model outcome, in addition to the actual nucleotide sequence of the guide DNA strand. After the model had been trained and tested (with >80% precision-recall curve metric), we explored the derived weights of the latent factors, finding they highlight the importance of the asymmetric time-direction of chromatin context during transcription.