bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2020‒09‒13
thirty-two papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit


  1. Genome Biol. 2020 Sep 10. 21(1): 243
    Peng T, Zhai Y, Atlasi Y, Ter Huurne M, Marks H, Stunnenberg HG, Megchelenbrink W.
      BACKGROUND: Enhancers are distal regulators of gene expression that shape cell identity and control cell fate transitions. In mouse embryonic stem cells (mESCs), the pluripotency network is maintained by the function of a complex network of enhancers, that are drastically altered upon differentiation. Genome-wide chromatin accessibility and histone modification assays are commonly used as a proxy for identifying putative enhancers and for describing their activity levels and dynamics.RESULTS: Here, we applied STARR-seq, a genome-wide plasmid-based assay, as a read-out for the enhancer landscape in "ground-state" (2i+LIF; 2iL) and "metastable" (serum+LIF; SL) mESCs. This analysis reveals that active STARR-seq loci show modest overlap with enhancer locations derived from peak calling of ChIP-seq libraries for common enhancer marks. We unveil ZIC3-bound loci with significant STARR-seq activity in SL-ESCs. Knock-out of Zic3 removes STARR-seq activity only in SL-ESCs and increases their propensity to differentiate towards the endodermal fate. STARR-seq also reveals enhancers that are not accessible, masked by a repressive chromatin signature. We describe a class of dormant, p53 bound enhancers that gain H3K27ac under specific conditions, such as after treatment with Nocodazol, or transiently during reprogramming from fibroblasts to pluripotency.
    CONCLUSIONS: In conclusion, loci identified as active by STARR-seq often overlap with those identified by chromatin accessibility and active epigenetic marking, yet a significant fraction is epigenetically repressed or display condition-specific enhancer activity.
    DOI:  https://doi.org/10.1186/s13059-020-02156-3
  2. Cancer Res. 2020 Sep 09. pii: canres.0672.2020. [Epub ahead of print]
    Patil S, Steuber B, Kopp W, Kari V, Urbach L, Wang X, Küffer S, Bohnenberger H, Spyropoulou D, Zhang Z, Versemann L, Bösherz MS, Brunner M, Gaedcke J, Ströbel P, Zhang JS, Neesse A, Ellenrieder V, Singh SK, Johnsen SA, Hessmann E.
      Recent studies have thoroughly described genome-wide expression patterns defining molecular subtypes of pancreatic ductal adenocarcinoma (PDAC) with different prognostic and predictive implications. Although the reversible nature of key regulatory transcription circuits defining the two extreme PDAC subtype lineages "classical" and "basal-like" suggests that subtype states are not permanently encoded but underlie a certain degree of plasticity, pharmacologically actionable drivers of PDAC subtype identity remain elusive. Here we characterized the mechanistic and functional implications of the histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2) in controlling PDAC plasticity, dedifferentiation, and molecular subtype identity. Utilization of transgenic PDAC models and human PDAC samples linked EZH2 activity to PDAC dedifferentiation and tumor progression. Combined RNA- and ChIP-seq studies identified EZH2 as a pivotal suppressor of differentiation programs in PDAC and revealed EZH2-dependent transcriptional repression of the classical subtype defining transcription factor Gata6 as a mechanistic basis for EZH2-dependent PDAC progression. Importantly, genetic or pharmacological depletion of EZH2 sufficiently increased GATA6 expression, thus inducing a gene signature shift in favor of a less aggressive and more therapy-susceptible, classical PDAC subtype state. Consistently, abrogation of GATA6 expression in EZH2-deficient PDAC cells counteracted the acquisition of classical gene signatures and rescued their invasive capacities, suggesting that GATA6 derepression is critical to overcome PDAC progression in the context of EZH2 inhibition. Together our findings link the EZH2-GATA6 axis to PDAC subtype identity and uncover EZH2 inhibition as an appealing strategy to induce subtype-switching in favor of a less aggressive PDAC phenotype.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-0672
  3. Sci Adv. 2020 Jun;pii: eaay8299. [Epub ahead of print]6(24):
    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: http://rytenlab.com/browser/app/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.
    DOI:  https://doi.org/10.1126/sciadv.aay8299
  4. Elife. 2020 Sep 07. pii: e58029. [Epub ahead of print]9
    Mukherjee S, Chaturvedi P, Rankin SA, Fish MB, Wlizla M, Paraiso KD, MacDonald M, Chen X, Weirauch MT, Blitz IL, Cho KW, Zorn AM.
      Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN in Xenopus gastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and β-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and β-catenin synergistically activate transcription apparently independent of Tcfs, whereas on other enhancers, Sox17 represses β-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/β-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and β-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts.
    Keywords:  developmental biology; xenopus
    DOI:  https://doi.org/10.7554/eLife.58029
  5. J Exp Clin Cancer Res. 2020 Sep 07. 39(1): 181
    Ben Q, An W, Sun Y, Qian A, Liu J, Zou D, Yuan Y.
      BACKGROUND: Nicotine, an active ingredient in tobacco, can promote epithelial-to-mesenchymal transition (EMT) processes that enhance the aggressiveness of a number of human cancers. In the present study, we investigated whether cigarette smoke/nicotine drives EMT in pancreatic ductal adenocarcinoma (PDAC).METHODS: Quantitative real-time PCR, western blot, immunohistochemistry, and immunofluorescence assays were used to evaluate Yes-associated protein 1 (YAP1) expression associated with cigarette smoking in human PDAC tissue samples and with nicotine exposure in PDAC cell lines. Bioinformatics, loss- and gain- of- function experiments, luciferase reporter assays, chromatin immunoprecipitation (ChIP), and murine tumor xenograft models were performed to examine the function of YAP1 in PDAC and to identify potential mechanisms of action.
    RESULTS: Exposure to smoking or nicotine promoted EMT and tumor growth in PDAC cells and in xenograft tumors. Functional studies revealed that YAP1 might drive nicotine-stimulated EMT and oncogenic activity in vitro and in vivo. In human PDAC tissues, upregulation of YAP1 was associated with "ever smoking" status and poor overall survival. In term of mechanism, hypoxia inducible factor (HIF)1A promoted YAP1 nuclear localization and YAP1 transactivation by directly binding to the hypoxia responsive elements of the YAP1 promoter upon nicotine treatment. Nicotine stimulated HIF1A and YAP1 expression by activating cholinergic receptor nicotinic alpha7 (CHRNA7). In addition, YAP1 increased and sustained the protein stability of HIF1A.
    CONCLUSIONS: These data demonstrate that YAP1 enhances nicotine-stimulated EMT and tumor progression of PDAC through a HIF1A/YAP1 positive feedback loop. Developing inhibitors that specifically target YAP1 may provide a novel therapeutic approach to suppress PDAC growth, especially in PDAC patients who have a history of smoking.
    Keywords:  Epithelial-to-mesenchymal transition; HIF1A; Nicotine; Pancreatic ductal adenocarcinoma; YAP1
    DOI:  https://doi.org/10.1186/s13046-020-01689-6
  6. Nat Struct Mol Biol. 2020 Sep 07.
    Maezawa S, Sakashita A, Yukawa M, Chen X, Takahashi K, Alavattam KG, Nakata I, Weirauch MT, Barski A, Namekawa SH.
      Owing to bursts in the expression of thousands of germline-specific genes, the testis has the most diverse and complex transcriptome of all organs. By analyzing the male germline of mice, we demonstrate that the genome-wide reorganization of super-enhancers (SEs) drives bursts in germline gene expression after the mitosis-to-meiosis transition. SE reorganization is regulated by two molecular events: the establishment of meiosis-specific SEs via A-MYB (MYBL1), a key transcription factor for germline genes, and the resolution of SEs in mitotically proliferating cells via SCML2, a germline-specific Polycomb protein required for spermatogenesis-specific gene expression. Before entry into meiosis, meiotic SEs are preprogrammed in mitotic spermatogonia to ensure the unidirectional differentiation of spermatogenesis. We identify key regulatory factors for both mitotic and meiotic enhancers, revealing a molecular logic for the concurrent activation of mitotic enhancers and suppression of meiotic enhancers in the somatic and/or mitotic proliferation phases.
    DOI:  https://doi.org/10.1038/s41594-020-0488-3
  7. Science. 2020 Sep 11. pii: eaba3066. [Epub ahead of print]369(6509):
    Oliva M, Muñoz-Aguirre M, Kim-Hellmuth S, Wucher V, Gewirtz ADH, Cotter DJ, Parsana P, Kasela S, Balliu B, Viñuela A, Castel SE, Mohammadi P, Aguet F, Zou Y, Khramtsova EA, Skol AD, Garrido-Martín D, Reverter F, Brown A, Evans P, Gamazon ER, Payne A, Bonazzola R, Barbeira AN, Hamel AR, Martinez-Perez A, Soria JM, , Pierce BL, Stephens M, Eskin E, Dermitzakis ET, Segrè AV, Im HK, Engelhardt BE, Ardlie KG, Montgomery SB, Battle AJ, Lappalainen T, Guigó R, Stranger BE.
      Many complex human phenotypes exhibit sex-differentiated characteristics. However, the molecular mechanisms underlying these differences remain largely unknown. We generated a catalog of sex differences in gene expression and in the genetic regulation of gene expression across 44 human tissue sources surveyed by the Genotype-Tissue Expression project (GTEx, v8 release). We demonstrate that sex influences gene expression levels and cellular composition of tissue samples across the human body. A total of 37% of all genes exhibit sex-biased expression in at least one tissue. We identify cis expression quantitative trait loci (eQTLs) with sex-differentiated effects and characterize their cellular origin. By integrating sex-biased eQTLs with genome-wide association study data, we identify 58 gene-trait associations that are driven by genetic regulation of gene expression in a single sex. These findings provide an extensive characterization of sex differences in the human transcriptome and its genetic regulation.
    DOI:  https://doi.org/10.1126/science.aba3066
  8. Cell Rep. 2020 Sep 08. pii: S2211-1247(20)31095-0. [Epub ahead of print]32(10): 108106
    Topal S, Van C, Xue Y, Carey MF, Peterson CL.
      The proper coordination of transcription with DNA replication and repair is central for genomic stability. We investigate how the INO80C chromatin remodeling enzyme might coordinate these genomic processes. We find that INO80C co-localizes with the origin recognition complex (ORC) at yeast replication origins and is bound to replication initiation sites in mouse embryonic stem cells (mESCs). In yeast, INO80C recruitment requires origin sequences but does not require ORC, suggesting that recruitment is independent of pre-replication complex assembly. In both yeast and ESCs, INO80C co-localizes at origins with Mot1 and NC2 transcription factors, and genetic studies suggest that they function together to promote genome stability. Interestingly, nascent transcript sequencing demonstrates that INO80C and Mot1 prevent pervasive transcription through origin sequences, and absence of these factors leads to formation of new DNA double-strand breaks. We propose that INO80C and Mot1/NC2 function through distinct pathways to limit origin transcription, maintaining genomic stability.
    Keywords:  DSB; INO80; Mot1; NC2; NET-seq; ORC; chromatin; ncRNA; replication
    DOI:  https://doi.org/10.1016/j.celrep.2020.108106
  9. Development. 2020 Sep 11. pii: dev186015. [Epub ahead of print]147(17):
    Matern MS, Milon B, Lipford EL, McMurray M, Ogawa Y, Tkaczuk A, Song Y, Elkon R, Hertzano R.
      Despite the known importance of the transcription factors ATOH1, POU4F3 and GFI1 in hair cell development and regeneration, their downstream transcriptional cascades in the inner ear remain largely unknown. Here, we have used Gfi1cre;RiboTag mice to evaluate changes to the hair cell translatome in the absence of GFI1. We identify a systematic downregulation of hair cell differentiation genes, concomitant with robust upregulation of neuronal genes in the GFI1-deficient hair cells. This includes increased expression of neuronal-associated transcription factors (e.g. Pou4f1) as well as transcription factors that serve dual roles in hair cell and neuronal development (e.g. Neurod1, Atoh1 and Insm1). We further show that the upregulated genes are consistent with the NEUROD1 regulon and are normally expressed in hair cells prior to GFI1 onset. Additionally, minimal overlap of differentially expressed genes in auditory and vestibular hair cells suggests that GFI1 serves different roles in these systems. From these data, we propose a dual mechanism for GFI1 in promoting hair cell development, consisting of repression of neuronal-associated genes as well as activation of hair cell-specific genes required for normal functional maturation.
    Keywords:  GFI1; Hair cells; Inner ear
    DOI:  https://doi.org/10.1242/dev.186015
  10. Sci Adv. 2020 Sep;pii: eabc0367. [Epub ahead of print]6(37):
    Zwiggelaar RT, Lindholm HT, Fosslie M, Terndrup Pedersen M, Ohta Y, Díez-Sánchez A, Martín-Alonso M, Ostrop J, Matano M, Parmar N, Kvaløy E, Spanjers RR, Nazmi K, Rye M, Drabløs F, Arrowsmith C, Arne Dahl J, Jensen KB, Sato T, Oudhoff MJ.
      Intestinal epithelial homeostasis is maintained by adult intestinal stem cells, which, alongside Paneth cells, appear after birth in the neonatal period. We aimed to identify regulators of neonatal intestinal epithelial development by testing a small library of epigenetic modifier inhibitors in Paneth cell-skewed organoid cultures. We found that lysine-specific demethylase 1A (Kdm1a/Lsd1) is absolutely required for Paneth cell differentiation. Lsd1-deficient crypts, devoid of Paneth cells, are still able to form organoids without a requirement of exogenous or endogenous Wnt. Mechanistically, we find that LSD1 enzymatically represses genes that are normally expressed only in fetal and neonatal epithelium. This gene profile is similar to what is seen in repairing epithelium, and we find that Lsd1-deficient epithelium has superior regenerative capacities after irradiation injury. In summary, we found an important regulator of neonatal intestinal development and identified a druggable target to reprogram intestinal epithelium toward a reparative state.
    DOI:  https://doi.org/10.1126/sciadv.abc0367
  11. BMC Genomics. 2020 Sep 07. 21(1): 610
    Ahmed M, Min DS, Kim DR.
      BACKGROUND: Transcription factor binding to the regulatory region of a gene induces or represses its gene expression. Transcription factors share their binding sites with other factors, co-factors and/or DNA-binding proteins. These proteins form complexes which bind to the DNA as one-units. The binding of two factors to a shared site does not always lead to a functional interaction.RESULTS: We propose a method to predict the combined functions of two factors using comparable binding and expression data (target). We based this method on binding and expression target analysis (BETA), which we re-implemented in R and extended for this purpose. target ranks the factor's targets by importance and predicts the dominant type of interaction between two transcription factors. We applied the method to simulated and real datasets of transcription factor-binding sites and gene expression under perturbation of factors. We found that Yin Yang 1 transcription factor (YY1) and YY2 have antagonistic and independent regulatory targets in HeLa cells, but they may cooperate on a few shared targets.
    CONCLUSION: We developed an R package and a web application to integrate binding (ChIP-seq) and expression (microarrays or RNA-seq) data to determine the cooperative or competitive combined function of two transcription factors.
    Keywords:  BETA; Competitive-binding; Cooperative-binding; DNA-binding; R-package; Transcription-factor; YY1
    DOI:  https://doi.org/10.1186/s12864-020-06977-1
  12. Nat Commun. 2020 Sep 11. 11(1): 4544
    Fox S, Myers JA, Davidson C, Getman M, Kingsley PD, Frankiewicz N, Bulger M.
      Stratification of enhancers by signal strength in ChIP-seq assays has resulted in the establishment of super-enhancers as a widespread and useful tool for identifying cell type-specific, highly expressed genes and associated pathways. We examine a distinct method of stratification that focuses on peak breadth, termed hyperacetylated chromatin domains (HCDs), which classifies broad regions exhibiting histone modifications associated with gene activation. We find that this analysis serves to identify genes that are both more highly expressed and more closely aligned to cell identity than super-enhancer analysis does using multiple data sets. Moreover, genetic manipulations of selected gene loci suggest that some enhancers located within HCDs work at least in part via a distinct mechanism involving the modulation of histone modifications across domains and that this activity can be imported into a heterologous gene locus. In addition, such genetic dissection reveals that the super-enhancer concept can obscure important functions of constituent elements.
    DOI:  https://doi.org/10.1038/s41467-020-18303-0
  13. Cell Stem Cell. 2020 Aug 22. pii: S1934-5909(20)30360-X. [Epub ahead of print]
    Dost AFM, Moye AL, Vedaie M, Tran LM, Fung E, Heinze D, Villacorta-Martin C, Huang J, Hekman R, Kwan JH, Blum BC, Louie SM, Rowbotham SP, Sainz de Aja J, Piper ME, Bhetariya PJ, Bronson RT, Emili A, Mostoslavsky G, Fishbein GA, Wallace WD, Krysan K, Dubinett SM, Yanagawa J, Kotton DN, Kim CF.
      Mutant KRAS is a common driver in epithelial cancers. Nevertheless, molecular changes occurring early after activation of oncogenic KRAS in epithelial cells remain poorly understood. We compared transcriptional changes at single-cell resolution after KRAS activation in four sample sets. In addition to patient samples and genetically engineered mouse models, we developed organoid systems from primary mouse and human induced pluripotent stem cell-derived lung epithelial cells to model early-stage lung adenocarcinoma. In all four settings, alveolar epithelial progenitor (AT2) cells expressing oncogenic KRAS had reduced expression of mature lineage identity genes. These findings demonstrate the utility of our in vitro organoid approaches for uncovering the early consequences of oncogenic KRAS expression. This resource provides an extensive collection of datasets and describes organoid tools to study the transcriptional and proteomic changes that distinguish normal epithelial progenitor cells from early-stage lung cancer, facilitating the search for targets for KRAS-driven tumors.
    Keywords:  KRAS; alveolar; developmental programs; early-stage lung cancer; iPSC; loss of differentiation; organoid; single-cell RNA sequencing; stage IA lung adenocarcinoma; tumor progression
    DOI:  https://doi.org/10.1016/j.stem.2020.07.022
  14. Cell Syst. 2020 Sep 07. pii: S2405-4712(20)30288-X. [Epub ahead of print]
    Szczesnik T, Chu L, Ho JWK, Sherwood RI.
      Predicting where transcription factors bind in the genome from their in vitro DNA-binding affinity is confounded by the large number of possible interactions with nearby transcription factors. To characterize the in vivo binding logic for the Wnt effector Tcf7l2, we developed a high-throughput screening platform in which thousands of synthesized DNA phrases are inserted into a specific genomic locus, followed by measurement of Tcf7l2 binding by DamID. Using this platform at two genomic loci in mouse embryonic stem cells, we show that while the binding of Tcf7l2 closely follows the in vitro motif-binding strength and is influenced by local chromatin accessibility, it is also strongly affected by the surrounding 99 bp of sequence. Through controlled sequence perturbation, we show that Oct4 and Klf4 motifs promote Tcf7l2 binding, particularly in the adjacent ∼50 bp and oscillating with a 10.8-bp phasing relative to these cofactor motifs, which matches the turn of a DNA helix.
    Keywords:  CRISPR-Cas9; DamID; Gaussian process; Tcf7l2; transcription factor
    DOI:  https://doi.org/10.1016/j.cels.2020.08.004
  15. iScience. 2020 Aug 20. pii: S2589-0042(20)30667-2. [Epub ahead of print]23(9): 101475
    Liu X, Fang Z, Wen J, Tang F, Liao B, Jing N, Lai D, Jin Y.
      Region-specific neural progenitor cells (NPCs) can be generated from human embryonic stem cells (hESCs) by modulating signaling pathways. However, how intrinsic transcriptional factors contribute to the neural regionalization is not well characterized. Here, we generate region-specific NPCs from hESCs and find that SOX1 is highly expressed in NPCs with the rostral hindbrain identity. Moreover, we find that OTX2 inhibits SOX1 expression, displaying exclusive expression between the two factors. Furthermore, SOX1 knockout (KO) leads to the upregulation of midbrain genes and downregulation of rostral hindbrain genes, indicating that SOX1 is required for specification of rostral hindbrain NPCs. Our SOX1 chromatin immunoprecipitation sequencing analysis reveals that SOX1 binds to the distal region of GBX2 to activate its expression. Overexpression of GBX2 largely abrogates SOX1-KO-induced aberrant gene expression. Taken together, this study uncovers previously unappreciated role of SOX1 in early neural regionalization and provides new information for the precise control of the OTX2/GBX2 interface.
    Keywords:  Developmental Neuroscience; Molecular Genetics
    DOI:  https://doi.org/10.1016/j.isci.2020.101475
  16. Genome Biol. 2020 Sep 07. 21(1): 240
    Lawson JT, Smith JP, Bekiranov S, Garrett-Bakelman FE, Sheffield NC.
      A key challenge in epigenetics is to determine the biological significance of epigenetic variation among individuals. We present Coordinate Covariation Analysis (COCOA), a computational framework that uses covariation of epigenetic signals across individuals and a database of region sets to annotate epigenetic heterogeneity. COCOA is the first such tool for DNA methylation data and can also analyze any epigenetic signal with genomic coordinates. We demonstrate COCOA's utility by analyzing DNA methylation, ATAC-seq, and multi-omic data in supervised and unsupervised analyses, showing that COCOA provides new understanding of inter-sample epigenetic variation. COCOA is available on Bioconductor ( http://bioconductor.org/packages/COCOA ).
    Keywords:  Cancer; Chromatin accessibility; DNA methylation; Data integration; Dimensionality reduction; EZH2; Epigenetics; Multi-omics; Principal component analysis
    DOI:  https://doi.org/10.1186/s13059-020-02139-4
  17. Sci Adv. 2020 Sep;pii: eabb0333. [Epub ahead of print]6(36):
    Kassem S, Ferrari P, Hughes AL, Soudet J, Rando OJ, Strubin M.
      Transcription in eukaryotes correlates with major chromatin changes, including the replacement of old nucleosomal histones by new histones at the promoters of genes. The role of these histone exchange events in transcription remains unclear. In particular, the causal relationship between histone exchange and activator binding, preinitiation complex (PIC) assembly, and/or subsequent transcription remains unclear. Here, we provide evidence that histone exchange at gene promoters is not simply a consequence of PIC assembly or transcription but instead is mediated by activators. We further show that not all activators up-regulate gene expression by inducing histone turnover. Thus, histone exchange does not simply correlate with transcriptional activity, but instead reflects the mode of action of the activator. Last, we show that histone turnover is not only associated with activator function but also plays a role in transcriptional repression at the histone loci.
    DOI:  https://doi.org/10.1126/sciadv.abb0333
  18. Cell Syst. 2020 Sep 04. pii: S2405-4712(20)30293-3. [Epub ahead of print]
    Chen SY, Osimiri LC, Chevalier M, Bugaj LJ, Nguyen TH, Greenstein RA, Ng AH, Stewart-Ornstein J, Neves LT, El-Samad H.
      Gene expression is thought to be affected not only by the concentration of transcription factors (TFs) but also the dynamics of their nuclear translocation. Testing this hypothesis requires direct control of TF dynamics. Here, we engineer CLASP, an optogenetic tool for rapid and tunable translocation of a TF of interest. Using CLASP fused to Crz1, we observe that, for the same integrated concentration of nuclear TF over time, changing input dynamics changes target gene expression: pulsatile inputs yield higher expression than continuous inputs, or vice versa, depending on the target gene. Computational modeling reveals that a dose-response saturating at low TF input can yield higher gene expression for pulsatile versus continuous input, and that multi-state promoter activation can yield the opposite behavior. Our integrated tool development and modeling approach characterize promoter responses to Crz1 nuclear translocation dynamics, extracting quantitative features that may help explain the differential expression of target genes.
    Keywords:  dynamic decoding; nucleo-cytoplasmic pulsing; optogenetics; promoter interpretation; transcription factors; translocation dynamics
    DOI:  https://doi.org/10.1016/j.cels.2020.08.009
  19. Sci Adv. 2020 Sep;pii: eaba1190. [Epub ahead of print]6(37):
    Xing QR, El Farran CA, Gautam P, Chuah YS, Warrier T, Toh CXD, Kang NY, Sugii S, Chang YT, Xu J, Collins JJ, Daley GQ, Li H, Zhang LF, Loh YH.
      Cellular reprogramming suffers from low efficiency especially for the human cells. To deconstruct the heterogeneity and unravel the mechanisms for successful reprogramming, we adopted single-cell RNA sequencing (scRNA-Seq) and single-cell assay for transposase-accessible chromatin (scATAC-Seq) to profile reprogramming cells across various time points. Our analysis revealed that reprogramming cells proceed in an asynchronous trajectory and diversify into heterogeneous subpopulations. We identified fluorescent probes and surface markers to enrich for the early reprogrammed human cells. Furthermore, combinatory usage of the surface markers enabled the fine segregation of the early-intermediate cells with diverse reprogramming propensities. scATAC-Seq analysis further uncovered the genomic partitions and transcription factors responsible for the regulatory phasing of reprogramming process. Binary choice between a FOSL1 and a TEAD4-centric regulatory network determines the outcome of a successful reprogramming. Together, our study illuminates the multitude of diverse routes transversed by individual reprogramming cells and presents an integrative roadmap for identifying the mechanistic part list of the reprogramming machinery.
    DOI:  https://doi.org/10.1126/sciadv.aba1190
  20. Cell Rep. 2020 Sep 08. pii: S2211-1247(20)31109-8. [Epub ahead of print]32(10): 108120
    Liu J, Gao M, Xu S, Chen Y, Wu K, Liu H, Wang J, Yang X, Wang J, Liu W, Bao X, Chen J.
      N6-methyladenosine (m6A), the most abundant reversible modification on eukaryote messenger RNA, is recognized by a series of readers, including the YT521-B homology domain family (YTHDF) proteins, which are coupled to perform physiological functions. Here, we report that YTHDF2 and YTHDF3, but not YTHDF1, are required for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). Mechanistically, we found that YTHDF3 recruits the PAN2-PAN3 deadenylase complex and conduces to reprogramming by promoting mRNA clearance of somatic genes, including Tead2 and Tgfb1, which parallels the activity of the YTHDF2-CCR4-NOT deadenylase complex. Ythdf2/3 deficiency represses mesenchymal-to-epithelial transition (MET) and chromatin silencing at loci containing the TEAD motif, contributing to decreased reprogramming efficiency. Moreover, RNA interference of Tgfb1 or the Hippo signaling effectors Yap1, Taz, and Tead2 rescues Ythdf2/3-defective reprogramming. Overall, YTHDF2/3 couples RNA deadenylation and regulation with the clearance of somatic genes and provides insights into iPSC reprogramming at the posttranscriptional level.
    Keywords:  Hippo signaling pathway; RNA degradation; YTHDFs; reprogramming
    DOI:  https://doi.org/10.1016/j.celrep.2020.108120
  21. Elife. 2020 Sep 09. pii: e61090. [Epub ahead of print]9
    Sarthy JF, Meers MP, Janssens DH, Henikoff JG, Feldman H, Paddison PJ, Lockwood CM, Vitanza NA, Olson JM, Ahmad K, Henikoff S.
      Lysine 27-to-methionine (K27M) mutations in the H3.1 or H3.3 histone genes are characteristic of pediatric diffuse midline gliomas (DMGs). These oncohistone mutations dominantly inhibit histone H3K27 trimethylation and silencing, but it is unknown how oncohistone type affects gliomagenesis. We show that the genomic distributions of H3.1 and H3.3 oncohistones in human patient-derived DMG cells are consistent with the DNA replication-coupled deposition of histone H3.1 and the predominant replication-independent deposition of histone H3.3. Although H3K27 trimethylation is reduced for both oncohistone types, H3.3K27M-bearing cells retain some domains, and only H3.1K27M-bearing cells lack H3K27 trimethylation. Neither oncohistone interferes with PRC2 binding. Using Drosophila as a model, we demonstrate that inhibition of H3K27 trimethylation occurs only when H3K27M oncohistones are deposited into chromatin and only when expressed in cycling cells. We propose that oncohistones inhibit the H3K27 methyltransferase as chromatin patterns are being duplicated in proliferating cells, predisposing them to tumorigenesis.
    Keywords:  D. melanogaster; cancer biology; chromosomes; gene expression; human
    DOI:  https://doi.org/10.7554/eLife.61090
  22. Genome Biol. 2020 Sep 11. 21(1): 235
    He Y, Chhetri SB, Arvanitis M, Srinivasan K, Aguet F, Ardlie KG, Barbeira AN, Bonazzola R, Im HK, , Brown CD, Battle A.
      Genetic regulation of gene expression, revealed by expression quantitative trait loci (eQTLs), exhibits complex patterns of tissue-specific effects. Characterization of these patterns may allow us to better understand mechanisms of gene regulation and disease etiology. We develop a constrained matrix factorization model, sn-spMF, to learn patterns of tissue-sharing and apply it to 49 human tissues from the Genotype-Tissue Expression (GTEx) project. The learned factors reflect tissues with known biological similarity and identify transcription factors that may mediate tissue-specific effects. sn-spMF, available at https://github.com/heyuan7676/ts_eQTLs , can be applied to learn biologically interpretable patterns of eQTL tissue-specificity and generate testable mechanistic hypotheses.
    Keywords:  Matrix factorization; Tissue-specific eQTLs; Transcription factors; Ubiquitous eQTLs
    DOI:  https://doi.org/10.1186/s13059-020-02129-6
  23. Nat Struct Mol Biol. 2020 Sep 07.
    Torné J, Ray-Gallet D, Boyarchuk E, Garnier M, Le Baccon P, Coulon A, Orsi GA, Almouzni G.
      Nucleosomes represent a challenge in regard to transcription. Histone eviction enables RNA polymerase II (RNAPII) progression through DNA, but compromises chromatin integrity. Here, we used the SNAP-tag system to distinguish new and old histones and monitor chromatin reassembly coupled to transcription in human cells. We uncovered a transcription-dependent loss of old histone variants H3.1 and H3.3. At transcriptionally active domains, H3.3 enrichment reflected both old H3.3 retention and new deposition. Mechanistically, we found that the histone regulator A (HIRA) chaperone is critical to processing both new and old H3.3 via different pathways. De novo H3.3 deposition is totally dependent on HIRA trimerization as well as on its partner ubinuclein 1 (UBN1), while antisilencing function 1 (ASF1) interaction with HIRA can be bypassed. By contrast, recycling of H3.3 requires HIRA but proceeds independently of UBN1 or HIRA trimerization and shows absolute dependency on ASF1-HIRA interaction. We propose a model whereby HIRA coordinates these distinct pathways during transcription to fine-tune chromatin states.
    DOI:  https://doi.org/10.1038/s41594-020-0492-7
  24. Nat Plants. 2020 Sep 07.
    Karaaslan ES, Wang N, Faiß N, Liang Y, Montgomery SA, Laubinger S, Berendzen KW, Berger F, Breuninger H, Liu C.
      Information in the genome is not only encoded within sequence or epigenetic modifications, but is also found in how it folds in three-dimensional space. The formation of self-interacting genomic regions, named topologically associated domains (TADs), is known as a key feature of genome organization beyond the nucleosomal level. However, our understanding of the formation and function of TADs in plants is extremely limited. Here we show that the genome of Marchantia polymorpha, a member of a basal land plant lineage, exhibits TADs with epigenetic features similar to those of higher plants. By analysing various epigenetic marks across Marchantia TADs, we find that these regions generally represent interstitial heterochromatin and their borders are enriched with Marchantia transcription factor TCP1. We also identify a type of TAD that we name 'TCP1-rich TAD', in which genomic regions are highly accessible and are densely bound by TCP1 proteins. Transcription of TCP1 target genes differs on the basis gene location, and those in TCP1-rich TADs clearly show a lower expression level. In tcp1 mutant lines, neither TCP1-bound TAD borders nor TCP1-rich TADs display drastically altered chromatin organization patterns, suggesting that, in Marchantia, TCP1 is dispensable for TAD formation. However, we find that in tcp1 mutants, genes residing in TCP1-rich TADs have a greater extent of expression fold change as opposed to genes that do not belong to these TADs. Our results suggest that, besides standing as spatial chromatin-packing modules, plant TADs function as nuclear microcompartments associated with transcription factor activities.
    DOI:  https://doi.org/10.1038/s41477-020-00766-0
  25. Oncogene. 2020 Sep 11.
    Wen S, He Y, Wang L, Zhang J, Quan C, Niu Y, Huang H.
      Next generation antiandrogens such as enzalutamide (Enz) are effective initially for the treatment of castration-resistant prostate cancer (CRPC). However, the disease often relapses and the underlying mechanisms remain elusive. By performing H3-lysine-27 acetylation (H3K27ac) ChIP-seq in Enz-resistant CRPC cells, we identified a group of super enhancers (SEs) that are abnormally activated in Enz-resistant CRPC cells and associated with enhanced transcription of a subset of tumor promoting genes such as CHPT1, which catalyzes phosphatidylcholine (PtdCho) synthesis and regulates choline metabolism. Increased CHPT1 conferred CRPC resistance to Enz in vitro and in mice. While androgen receptor (AR) primarily binds to a putative CHPT1 enhancer and mediates androgen-dependent expression of CHPT1 gene in Enz-sensitive prostate cancer cells, AR binds to a different enhancer within the CHPT1 SE locus and facilities androgen-independent expression of CHPT1 in Enz-resistant cells. We further identified a long-non coding RNA transcribed at CHPT1 enhancer (also known as enhancer RNA) that binds to the H3K27ac reader BRD4 and participates in regulating CHPT1 SE activity and CHPT1 gene expression. Our findings demonstrate that aberrantly activated SE upregulates CHPT1 expression and confers Enz resistance in CRPC, suggesting that SE-mediated expression of downstream effectors such as CHPT1 can be viable targets to overcome Enz resistance in PCa.
    DOI:  https://doi.org/10.1038/s41388-020-01456-z
  26. Cell Mol Gastroenterol Hepatol. 2020 Sep 04. pii: S2352-345X(20)30137-5. [Epub ahead of print]
    Yagishita Y, McCallum ML, Kensler TW, Wakabayashi N.
      BACKGROUND & AIMS: Notch signaling coordinates cell differentiation processes in the intestinal epithelium. The transcription factor Nrf2 orchestrates defense mechanisms by regulating cellular redox homeostasis, which as shown previously in murine liver, can be amplified through signaling crosstalk with the Notch pathway. However, interplay between these two signaling pathways in the gut is unknown.METHODS: Mice modified genetically to amplify Nrf2 in the intestinal epithelium (Keap1f/f::VilCre) were generated as well as pharmacological activation of Nrf2 and subjected to phenotypic and cell lineage analyses. Cell lines were used for reporter gene assays together with Nrf2 overexpression to study transcriptional regulation of the Notch downstream effector.
    RESULTS: Constitutive activation of Nrf2 signaling caused increased intestinal length along with expanded cell number and thickness of enterocytes without any alterations of secretory lineage, outcomes abrogated by concomitant disruption of Nrf2. The Nrf2 and Notch pathways in epithelium showed inverse spatial profiles, where Nrf2 activity in crypts was lower than villi. In progenitor cells of Keap1f/f::VilCre mice, Notch downstream effector Math1, which regulates a differentiation balance of cell lineage through lateral inhibition, showed suppressed expression. In vitro results demonstrated Nrf2 negatively regulated Math1, where six antioxidant response elements located in the regulatory regions contributed to this repression.
    CONCLUSIONS: Activation of Nrf2 perturbed the dialog of the Notch cascade though negative regulation of Math1 in progenitor cells, leading to enhanced enterogenesis. The crosstalk between the Nrf2 and Notch pathways could be critical for fine-tuning intestinal homeostasis and point to new approaches for the pharmacological management of absorptive deficiencies.
    Keywords:  intestinal homeostasis; progenitor cells; signaling crosstalk
    DOI:  https://doi.org/10.1016/j.jcmgh.2020.08.013
  27. Am J Transl Res. 2020 ;12(8): 4478-4487
    Xie D, Yu S, Li L, Quan M, Gao Y.
      Both autotaxin (ATX) and Forkhead Box M1 (FOXM1) have been commonly recognized as oncogenes in multiple types of human malignancies. However, the expression and biological functions of ATX in pancreatic ductal adenocarcinoma (PDAC), and its correlation with FOXM1 are poorly understood. The present study aimed to investigate their correlation and biological consequences in PDAC development. By dual luciferase reporter and chromatin immunoprecipitation assays, we found that ATX was a downstream transcriptional target gene of FOXM1. Further cellular functional experiments indicated that ATX was required for FOXM1-mediated PDAC cell proliferation and migration. Data from molecular biological experiments showed that ATX could enhance FOXM1 expression in turn by inhibiting the Hippo signaling pathway, suggesting that ATX and FOXM1 formed a positive feedback loop to facilitate PDAC progression. Using immunohistochemistry (IHC) method, both ATX and FOXM1 expression were found to be frequently up-regulated in PDAC tumor tissues when compared with adjacent normal tissues, and elevated ATX and FOXM1 expression were positively correlated with each other. In conclusion, the present work identified a positive feedback loop between ATX and FOXM1 which promotes PDAC cell proliferation and migration.
    Keywords:  ATX; FOXM1; migration; pancreatic cancer; proliferation
  28. Nat Protoc. 2020 Sep 10.
    Kaya-Okur HS, Janssens DH, Henikoff JG, Ahmad K, Henikoff S.
      We recently introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei. These antibodies are then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds adapters ('tagmentation') for paired-end DNA sequencing. Here, we introduce a streamlined CUT&Tag protocol that suppresses DNA accessibility artefacts to ensure high-fidelity mapping of the antibody-targeted protein and improves the signal-to-noise ratio over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube, from cells to amplified libraries, providing low-cost genome-wide chromatin maps. By simplifying library preparation CUT&Tag requires less than a day at the bench, from live cells to sequencing-ready barcoded libraries. As a result of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for as little as $25 per sample. This enables routine genome-wide profiling of chromatin proteins and modifications and requires no special skills or equipment.
    DOI:  https://doi.org/10.1038/s41596-020-0373-x
  29. Sci Adv. 2020 Sep;pii: eaaz7364. [Epub ahead of print]6(36):
    Kim KP, Wu Y, Yoon J, Adachi K, Wu G, Velychko S, MacCarthy CM, Shin B, Röpke A, Arauzo-Bravo MJ, Stehling M, Han DW, Gao Y, Kim J, Gao S, Schöler HR.
      OCT4 (also known as POU5F1) plays an essential role in reprogramming. It is the only member of the POU (Pit-Oct-Unc) family of transcription factors that can induce pluripotency despite sharing high structural similarities to all other members. Here, we discover that OCT6 (also known as POU3F1) can elicit reprogramming specifically in human cells. OCT6-based reprogramming does not alter the mesenchymal-epithelial transition but is attenuated through the delayed activation of the pluripotency network in comparison with OCT4-based reprogramming. Creating a series of reciprocal domain-swapped chimeras and mutants across all OCT factors, we clearly delineate essential elements of OCT4/OCT6-dependent reprogramming and, conversely, identify the features that prevent induction of pluripotency by other OCT factors. With this strategy, we further discover various chimeric proteins that are superior to OCT4 in reprogramming. Our findings clarify how reprogramming competences of OCT factors are conferred through their structural components.
    DOI:  https://doi.org/10.1126/sciadv.aaz7364
  30. Science. 2020 Sep 11. pii: eaaz8528. [Epub ahead of print]369(6509):
    Kim-Hellmuth S, Aguet F, Oliva M, Muñoz-Aguirre M, Kasela S, Wucher V, Castel SE, Hamel AR, Viñuela A, Roberts AL, Mangul S, Wen X, Wang G, Barbeira AN, Garrido-Martín D, Nadel BB, Zou Y, Bonazzola R, Quan J, Brown A, Martinez-Perez A, Soria JM, , Getz G, Dermitzakis ET, Small KS, Stephens M, Xi HS, Im HK, Guigó R, Segrè AV, Stranger BE, Ardlie KG, Lappalainen T.
      The Genotype-Tissue Expression (GTEx) project has identified expression and splicing quantitative trait loci in cis (QTLs) for the majority of genes across a wide range of human tissues. However, the functional characterization of these QTLs has been limited by the heterogeneous cellular composition of GTEx tissue samples. We mapped interactions between computational estimates of cell type abundance and genotype to identify cell type-interaction QTLs for seven cell types and show that cell type-interaction expression QTLs (eQTLs) provide finer resolution to tissue specificity than bulk tissue cis-eQTLs. Analyses of genetic associations with 87 complex traits show a contribution from cell type-interaction QTLs and enables the discovery of hundreds of previously unidentified colocalized loci that are masked in bulk tissue.
    DOI:  https://doi.org/10.1126/science.aaz8528
  31. Nature. 2020 Sep 10.
    Pathare GR, Decout A, Glück S, Cavadini S, Makasheva K, Hovius R, Kempf G, Weiss J, Kozicka Z, Guey B, Melenec P, Fierz B, Thomä NH, Ablasser A.
      The DNA sensor cGAS initiates innate immune responses following microbial infection, cellular stress, and cancer1. Upon activation by double-stranded DNA, cytosolic cGAS produces 2'3' cyclic GMP-AMP and triggers inflammatory cytokine and type I interferon (IFN) induction2-7. cGAS is also present inside the cell nucleus replete with genomic DNA8, where chromatin has been implicated in restricting its enzymatic activity9. However, the structural basis for cGAS inhibition by chromatin has remained unknown. Here we present the cryo-electron microscopy structure of human cGAS bound to nucleosomes at 3.1 Å resolution. cGAS makes extensive contacts with both the acidic patch of the histone H2A-H2B heterodimer and nucleosomal DNA. The structural and complementary biochemical analysis also finds cGAS engaged to a second nucleosome in trans. Mechanistically, nucleosome binding locks cGAS in a monomeric state, in which steric hindrance suppresses spurious activation by genomic DNA. We find that mutations to the cGAS-acidic patch interface are necessary and sufficient to abolish the inhibitory effect of nucleosomes in vitro and to unleash cGAS activity on genomic DNA in living cells. Our work uncovers the structural basis of cGAS interaction with chromatin and defines a compelling mechanism that permits self-nonself discrimination of genomic DNA by cGAS.
    DOI:  https://doi.org/10.1038/s41586-020-2750-6
  32. Neuro Oncol. 2020 Sep 08. pii: noaa157. [Epub ahead of print]
    Johnson RM, Phillips HS, Bais C, Brennan CW, Cloughesy TF, Daemen A, Herrlinger U, Jenkins RB, Lai A, Mancao C, Weller M, Wick W, Bourgon R, Garcia J.
      BACKGROUND: We aimed to develop a gene expression-based prognostic signature for isocitrate dehydrogenase (IDH) wild-type glioblastoma using clinical trial datasets representative of glioblastoma clinical trial populations.METHODS: Samples were collected from newly diagnosed patients with IDH wild-type glioblastoma in the ARTE, TAMIGA, EORTC 26101 (referred to as "ATE"), AVAglio, and GLARIUS trials, or treated at UCLA. Transcriptional profiling was achieved with the NanoString gene expression platform. To identify genes prognostic for overall survival (OS), we built an elastic net penalized Cox proportional hazards regression model using the discovery ATE dataset. For validation in independent datasets (AVAglio, GLARIUS, UCLA), we combined elastic net-selected genes into a robust z-score signature (ATE score) to overcome gene expression platform differences between discovery and validation cohorts.
    RESULTS: NanoString data were available from 512 patients in the ATE dataset. Elastic net identified a prognostic signature of 9 genes (CHEK1, GPR17, IGF2BP3, MGMT, MTHFD1L, PTRH2, SOX11, S100A9, and TFRC). Translating weighted elastic net scores to the ATE score conserved the prognostic value of the genes. The ATE score was prognostic for OS in the ATE dataset (P < 0.0001), as expected, and in the validation cohorts (AVAglio, P < 0.0001; GLARIUS, P = 0.02; UCLA, P = 0.004). The ATE score remained prognostic following adjustment for O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status and corticosteroid use at baseline. A positive correlation between ATE score and proneural/proliferative subtypes was observed in patients with MGMT non-methylated promoter status.
    CONCLUSIONS: The ATE score showed prognostic value and may enable clinical trial stratification for IDH wild-type glioblastoma.
    Keywords:  NanoString; gene expression; glioblastoma; overall survival; prognostic signature
    DOI:  https://doi.org/10.1093/neuonc/noaa157