bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2021‒02‒14
thirty-one papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit

  1. Nat Genet. 2021 Feb 08.
    Iurlaro M, Stadler MB, Masoni F, Jagani Z, Galli GG, Schübeler D.
      Chromatin accessibility is a hallmark of regulatory regions, entails transcription factor (TF) binding and requires nucleosomal reorganization. However, it remains unclear how dynamic this process is. In the present study, we use small-molecule inhibition of the catalytic subunit of the mouse SWI/SNF remodeler complex to show that accessibility and reduced nucleosome presence at TF-binding sites rely on persistent activity of nucleosome remodelers. Within minutes of remodeler inhibition, accessibility and TF binding decrease. Although this is irrespective of TF function, we show that the activating TF OCT4 (POU5F1) exhibits a faster response than the repressive TF REST. Accessibility, nucleosome depletion and gene expression are rapidly restored on inhibitor removal, suggesting that accessible chromatin is regenerated continuously and in a largely cell-autonomous fashion. We postulate that TF binding to chromatin and remodeler-mediated nucleosomal removal do not represent a stable situation, but instead accessible chromatin reflects an average of a dynamic process under continued renewal.
  2. Cell Rep. 2021 Feb 09. pii: S2211-1247(21)00055-3. [Epub ahead of print]34(6): 108742
    Greulich F, Wierer M, Mechtidou A, Gonzalez-Garcia O, Uhlenhaut NH.
      Glucocorticoids (GCs) are effective anti-inflammatory drugs; yet, their mechanisms of action are poorly understood. GCs bind to the glucocorticoid receptor (GR), a ligand-gated transcription factor controlling gene expression in numerous cell types. Here, we characterize GR's protein interactome and find the SETD1A (SET domain containing 1A)/COMPASS (complex of proteins associated with Set1) histone H3 lysine 4 (H3K4) methyltransferase complex highly enriched in activated mouse macrophages. We show that SETD1A/COMPASS is recruited by GR to specific cis-regulatory elements, coinciding with H3K4 methylation dynamics at subsets of sites, upon treatment with lipopolysaccharide (LPS) and GCs. By chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq, we identify subsets of GR target loci that display SETD1A occupancy, H3K4 mono-, di-, or tri-methylation patterns, and transcriptional changes. However, our data on methylation status and COMPASS recruitment suggest that SETD1A has additional transcriptional functions. Setd1a loss-of-function studies reveal that SETD1A/COMPASS is required for GR-controlled transcription of subsets of macrophage target genes. We demonstrate that the SETD1A/COMPASS complex cooperates with GR to mediate anti-inflammatory effects.
    Keywords:  COMPASS complex; H3K4 methylation; SETD1A; dexamethasone; enhancers; genomics; glucocorticoid receptor; inflammation; macrophages; transcription
  3. Nat Commun. 2021 02 09. 12(1): 887
    Huseyin MK, Klose RJ.
      Polycomb repressive complex 1 (PRC1) is an essential chromatin-based repressor of gene transcription. How PRC1 engages with chromatin to identify its target genes and achieve gene repression remains poorly defined, representing a major hurdle to our understanding of Polycomb system function. Here, we use genome engineering and single particle tracking to dissect how PRC1 binds to chromatin in live mouse embryonic stem cells. We observe that PRC1 is highly dynamic, with only a small fraction stably interacting with chromatin. By integrating subunit-specific dynamics, chromatin binding, and abundance measurements, we discover that PRC1 exhibits low occupancy at target sites. Furthermore, we employ perturbation approaches to uncover how specific components of PRC1 define its kinetics and chromatin binding. Together, these discoveries provide a quantitative understanding of chromatin binding by PRC1 in live cells, suggesting that chromatin modification, as opposed to PRC1 complex occupancy, is central to gene repression.
  4. Mol Oncol. 2021 Feb 11.
    Severson TM, Zhu Y, De Marzo AM, Jones T, Simons JW, Nelson WG, Yegnasubramanian S, Freedman ML, Wessels L, Bergman AM, Haffner MC, Zwart W.
      The epigenomic regulation of transcriptional programs in metastatic prostate cancer is poorly understood. We studied the epigenomic landscape of prostate cancer drivers using transcriptional profiling and chromatin immunoprecipitation sequencing (ChIP-seq) in four clonal metastatic tumors derived from a single prostate cancer patient. Our epigenomic analyses focused on Androgen Receptor (AR), which is a key oncogenic driver in prostate cancer , the AR pioneer factor FOXA1, chromatin insulator CTCF, as well as for modified histones H3K27ac and H3K27me3. The vast majority of AR binding sites were shared among healthy prostate, primary prostate cancer and metastatic tumor samples, signifying core AR-driven transcriptional regulation within the prostate cell lineage. Genes associated with core AR-binding events were significantly enriched for essential genes in prostate cancer cell proliferation. Remarkably, the metastasis-specific active AR binding sites showed no differential transcriptional output, indicating a robust transcriptional program across metastatic samples. Combined, our data reveal a core transcriptional program in clonal metastatic prostate cancer, despite epigenomic differences in the AR cistrome.
    Keywords:  ChIP-seq; cistrome; epigenomics; metastasis; prostate cancer; transcriptomics
  5. Cell Death Discov. 2020 Apr 29. 6(1): 30
    He F, Wu H, Zhou L, Lin Q, Cheng Y, Sun YE.
      DNA methylation and demethylation at CpG di-nucleotide sites plays important roles in cell fate specification of neural stem cells (NSCs). We have previously reported that DNA methyltransferases, Dnmt1and Dnmt3a, serve to suppress precocious astrocyte differentiation from NSCs via methylation of astroglial lineage genes. However, whether active DNA demethylase also participates in astrogliogenesis remains undetermined. In this study, we discovered that a Ten-eleven translocation (Tet) protein, Tet2, which was critically involved in active DNA demethylation through oxidation of 5-Methylcytosine (5mC), drove astrocyte differentiation from NSCs by demethylation of astroglial lineage genes including Gfap. Moreover, we found that an NSC-specific bHLH transcription factor Olig2 was an upstream inhibitor for Tet2 expression through direct association with the Tet2 promoter, and indirectly inhibited astrocyte differentiation. Our research not only revealed a brand-new function of Tet2 to promote NSC differentiation into astrocytes, but also a novel mechanism for Olig2 to inhibit astrocyte formation.
  6. Nat Commun. 2021 02 09. 12(1): 896
    Li J, Mahata B, Escobar M, Goell J, Wang K, Khemka P, Hilton IB.
      Histone phosphorylation is a ubiquitous post-translational modification that allows eukaryotic cells to rapidly respond to environmental stimuli. Despite correlative evidence linking histone phosphorylation to changes in gene expression, establishing the causal role of this key epigenomic modification at diverse loci within native chromatin has been hampered by a lack of technologies enabling robust, locus-specific deposition of endogenous histone phosphorylation. To address this technological gap, here we build a programmable chromatin kinase, called dCas9-dMSK1, by directly fusing nuclease-null CRISPR/Cas9 to a hyperactive, truncated variant of the human MSK1 histone kinase. Targeting dCas9-dMSK1 to human promoters results in increased target histone phosphorylation and gene activation and demonstrates that hyperphosphorylation of histone H3 serine 28 (H3S28ph) in particular plays a causal role in the transactivation of human promoters. In addition, we uncover mediators of resistance to the BRAF V600E inhibitor PLX-4720 in human melanoma cells using genome-scale screening with dCas9-dMSK1. Collectively, our findings enable a facile way to reshape human chromatin using CRISPR/Cas9-based epigenome editing and further define the causal link between histone phosphorylation and human gene activation.
  7. Nat Genet. 2021 Feb 11.
    Belaghzal H, Borrman T, Stephens AD, Lafontaine DL, Venev SV, Weng Z, Marko JF, Dekker J.
      Nuclear compartmentalization of active and inactive chromatin is thought to occur through microphase separation mediated by interactions between loci of similar type. The nature and dynamics of these interactions are not known. We developed liquid chromatin Hi-C to map the stability of associations between loci. Before fixation and Hi-C, chromosomes are fragmented, which removes strong polymeric constraint, enabling detection of intrinsic locus-locus interaction stabilities. Compartmentalization is stable when fragments are larger than 10-25 kb. Fragmentation of chromatin into pieces smaller than 6 kb leads to gradual loss of genome organization. Lamin-associated domains are most stable, whereas interactions for speckle- and polycomb-associated loci are more dynamic. Cohesin-mediated loops dissolve after fragmentation. Liquid chromatin Hi-C provides a genome-wide view of chromosome interaction dynamics.
  8. EMBO J. 2021 Feb 08. e107015
    Sawicka A, Villamil G, Lidschreiber M, Darzacq X, Dugast-Darzacq C, Schwalb B, Cramer P.
      Eukaryotic RNA polymerase II (Pol II) contains a tail-like, intrinsically disordered carboxy-terminal domain (CTD) comprised of heptad-repeats, that functions in coordination of the transcription cycle and in coupling transcription to co-transcriptional processes. The CTD repeat number varies between species and generally increases with genome size, but the reasons for this are unclear. Here, we show that shortening the CTD in human cells to half of its length does not generally change pre-mRNA synthesis or processing in cells. However, CTD shortening decreases the duration of promoter-proximal Pol II pausing, alters transcription of putative enhancer elements, and delays transcription activation after stimulation of the MAP kinase pathway. We suggest that a long CTD is required for efficient enhancer-dependent recruitment of Pol II to target genes for their rapid activation.
    Keywords:  RNA polymerase II; enhancer; gene expression; transcription kinetics
  9. Proc Natl Acad Sci U S A. 2021 Feb 16. pii: e2015675118. [Epub ahead of print]118(7):
    Zhang P, Katzaroff AJ, Buttitta LA, Ma Y, Jiang H, Nickerson DW, Øvrebø JI, Edgar BA.
      Using a gain-of-function screen in Drosophila, we identified the Krüppel-like factor Cabut (Cbt) as a positive regulator of cell cycle gene expression and cell proliferation. Enforced cbt expression is sufficient to induce an extra cell division in the differentiating fly wing or eye, and also promotes intestinal stem cell divisions in the adult gut. Although inappropriate cell proliferation also results from forced expression of the E2f1 transcription factor or its target, Cyclin E, Cbt does not increase E2F1 or Cyclin E activity. Instead, Cbt regulates a large set of E2F1 target genes independently of E2F1, and our data suggest that Cbt acts via distinct binding sites in target gene promoters. Although Cbt was not required for cell proliferation during wing or eye development, Cbt is required for normal intestinal stem cell divisions in the midgut, which expresses E2F1 at relatively low levels. The E2F1-like functions of Cbt identify a distinct mechanism for cell cycle regulation that may be important in certain normal cell cycles, or in cells that cycle inappropriately, such as cancer cells.
    Keywords:  Cabut (Cbt); E2F1; cell cycle exit; intestinal stem cell (ISC); proliferation
  10. Oncogene. 2021 Feb;40(6): 1091-1105
    Swoboda A, Soukup R, Eckel O, Kinslechner K, Wingelhofer B, Schörghofer D, Sternberg C, Pham HTT, Vallianou M, Horvath J, Stoiber D, Kenner L, Larue L, Poli V, Beermann F, Yokota T, Kubicek S, Krausgruber T, Rendeiro AF, Bock C, Zenz R, Kovacic B, Aberger F, Hengstschläger M, Petzelbauer P, Mikula M, Moriggl R.
      Metastatic melanoma is hallmarked by its ability of phenotype switching to more slowly proliferating, but highly invasive cells. Here, we tested the impact of signal transducer and activator of transcription 3 (STAT3) on melanoma progression in association with melanocyte inducing transcription factor (MITF) expression levels. We established a mouse melanoma model for deleting Stat3 in melanocytes with specific expression of human hyperactive NRASQ61K in an Ink4a-deficient background, two frequent driver mutations in human melanoma. Mice devoid of Stat3 showed early disease onset with higher proliferation in primary tumors, but displayed significantly diminished lung, brain, and liver metastases. Whole-genome expression profiling of tumor-derived cells also showed a reduced invasion phenotype, which was further corroborated by 3D melanoma model analysis. Notably, loss or knockdown of STAT3 in mouse or human cells resulted in the upregulation of MITF and induction of cell proliferation. Mechanistically we show that STAT3-induced CAAT Box Enhancer Binding Protein (CEBP) expression was sufficient to suppress MITF transcription. Epigenetic analysis by ATAC-seq confirmed that CEBPa/b binding to the MITF enhancer region silenced the MITF locus. Finally, by classification of patient-derived melanoma samples, we show that STAT3 and MITF act antagonistically and hence contribute differentially to melanoma progression. We conclude that STAT3 is a driver of the metastatic process in melanoma and able to antagonize MITF via direct induction of CEBP family member transcription.
  11. Proc Natl Acad Sci U S A. 2021 Feb 16. pii: e2023127118. [Epub ahead of print]118(7):
    Xiao T, Li X, Felsenfeld G.
      The Myc-associated zinc finger protein (MAZ) is often found at genomic binding sites adjacent to CTCF, a protein which affects large-scale genome organization through its interaction with cohesin. We show here that, like CTCF, MAZ physically interacts with a cohesin subunit and can arrest cohesin sliding independently of CTCF. It also shares with CTCF the ability to independently pause the elongating form of RNA polymerase II, and consequently affects RNA alternative splicing. CTCF/MAZ double sites are more effective at sequestering cohesin than sites occupied only by CTCF. Furthermore, depletion of CTCF results in preferential loss of CTCF from sites not occupied by MAZ. In an assay for insulation activity like that used for CTCF, binding of MAZ to sites between an enhancer and promoter results in down-regulation of reporter gene expression, supporting a role for MAZ as an insulator protein. Hi-C analysis of the effect of MAZ depletion on genome organization shows that local interactions within topologically associated domains (TADs) are disrupted, as well as contacts that establish the boundaries of individual TADs. We conclude that MAZ augments the action of CTCF in organizing the genome, but also shares properties with CTCF that allow it to act independently.
    Keywords:  CTCF; MAZ; RNA Pol II pausing; cohesin arrest; insulation
  12. Sci Adv. 2021 Jan;pii: eabe3445. [Epub ahead of print]7(5):
    Wang Y, Wu J, Chen H, Yang Y, Xiao C, Yi X, Shi C, Zhong K, He H, Li Y, Wu Z, Zhou G, Rao Q, Wang X, Zhou X, Lomberk G, Liu B, Zhao J, Ge J, Zhou W, Chu X, Chen C, Zhou X, Wang L, Guan K, Qu L.
      Cancer stem cells (CSCs) are involved in tumorigenesis, recurrence, and therapy resistance. To identify critical regulators of sarcoma CSCs, we performed a reporter-based genome-wide CRISPR-Cas9 screen and uncovered Kruppel-like factor 11 (KLF11) as top candidate. In vitro and in vivo functional annotation defined a negative role of KLF11 in CSCs. Mechanistically, KLF11 and YAP/TEAD bound to adjacent DNA sites along with direct interaction. KLF11 recruited SIN3A/HDAC to suppress the transcriptional output of YAP/TEAD, which, in turn, promoted KLF11 transcription, forming a negative feedback loop. However, in CSCs, this negative feedback was lost because of epigenetic silence of KLF11, causing sustained YAP activation. Low KLF11 was associated with poor prognosis and chemotherapy response in patients with sarcoma. Pharmacological activation of KLF11 by thiazolidinedione effectively restored chemotherapy response. Collectively, our study identifies KLF11 as a negative regulator in sarcoma CSCs and potential therapeutic target.
  13. Cell Rep. 2021 Feb 09. pii: S2211-1247(21)00048-6. [Epub ahead of print]34(6): 108735
    Zhu Q, Sang F, Withey S, Tang W, Dietmann S, Klisch D, Ramos-Ibeas P, Zhang H, Requena CE, Hajkova P, Loose M, Surani MA, Alberio R.
      Investigations of the human germline and programming are challenging because of limited access to embryonic material. However, the pig as a model may provide insights into transcriptional network and epigenetic reprogramming applicable to both species. Here we show that, during the pre- and early migratory stages, pig primordial germ cells (PGCs) initiate large-scale epigenomic reprogramming, including DNA demethylation involving TET-mediated hydroxylation and, potentially, base excision repair (BER). There is also macroH2A1 depletion and increased H3K27me3 as well as X chromosome reactivation (XCR) in females. Concomitantly, there is dampening of glycolytic metabolism genes and re-expression of some pluripotency genes like those in preimplantation embryos. We identified evolutionarily young transposable elements and gene coding regions resistant to DNA demethylation in acutely hypomethylated gonadal PGCs, with potential for transgenerational epigenetic inheritance. Detailed insights into the pig germline will likely contribute significantly to advances in human germline biology, including in vitro gametogenesis.
    Keywords:  DNA demethylation; X-chromosome reactivation; epigenetic resetting; escapees; germ cells; pig; single-cell RNA-seq; transgenerational inheritance
  14. Nucleic Acids Res. 2021 Feb 12. pii: gkab065. [Epub ahead of print]
    Hou G, Zhao X, Li L, Yang Q, Liu X, Huang C, Lu R, Chen R, Wang Y, Jiang B, Yu J.
      N 6-Methyladenosine (m6A) is the most abundant modification within diverse RNAs including mRNAs and lncRNAs and is regulated by a reversible process with important biological functions. Human YTH domain family 2 (YTHDF2) selectively recognized m6A-RNAs to regulate degradation. However, the possible regulation of YTHDF2 by protein post-translational modification remains unknown. Here, we show that YTHDF2 is SUMOylated in vivo and in vitro at the major site of K571, which can be induced by hypoxia while reduced by oxidative stress and SUMOylation inhibitors. SUMOylation of YTHDF2 has little impact on its ubiquitination and localization, but significantly increases its binding affinity of m6A-modified mRNAs and subsequently results in deregulated gene expressions which accounts for cancer progression. Moreover, Disease-free survival analysis of patients with lung adenocarcinoma derived from TCGA dataset reveals that higher expression of YTHDF2 together with higher expression of SUMO1 predicts poor prognosis. Our works uncover a new regulatory mechanism for YTHDF2 recognition of m6A-RNAs and highlight the importance of YTHDF2 SUMOylation in post-transcriptional gene expression regulation and cancer progression.
  15. Hum Mol Genet. 2021 Feb 09. pii: ddab042. [Epub ahead of print]
    Xu K, Zhang W, Wang C, Hu L, Wang R, Wang C, Tang L, Zhou G, Zou B, Xie H, Tang J, Guan X.
      The potentially different genetics and epigenetics in the primary tumors and metastases affect the efficacy of treatment in breast cancer patients. Nevertheless, the cellular and molecular mechanisms of breast cancer lymph node metastasis still remain elusive. Here, we employed single-cell RNA sequencing (scRNA-seq) to acquire the transcriptomic profiles of individual cells from primary tumours, negative, and positive lymph nodes. We also performed a single-cell assay for transposase-accessible chromatin (ATAC) sequencing (scATAC-seq) of the positive and negative lymph node samples to get the chromatin accessibility profile. We identified a novel cell subpopulation with an abnormally high expression level of CXCL14 in the positive lymph node of breast cancer patients. Cell trajectory analysis also revealed that CXCL14 was increased expressed in the late pseudo-time. Moreover. Based on a tissue microarray of 55 patients and the Oncomine database, We validated that CXCL14 expression was significantly higher in breast cancer patients with lymph node metastasis. Furthermore, scATAC-seq identified several transcription factors (TFs) that may be potential regulation factors for the lymph node metastasis of breast cancer. Thus, our findings will improve our current understanding of the mechanism for lymph node metastasis, and are potentially valuable in providing novel prognosis markers for lymphatic metastasis of breast cancer.
  16. Mol Cell. 2021 Feb 03. pii: S1097-2765(21)00013-7. [Epub ahead of print]
    Garcia DA, Johnson TA, Presman DM, Fettweis G, Wagh K, Rinaldi L, Stavreva DA, Paakinaho V, Jensen RAM, Mandrup S, Upadhyaya A, Hager GL.
      Transcription factors (TFs) regulate gene expression by binding to specific consensus motifs within the local chromatin context. The mechanisms by which TFs navigate the nuclear environment as they search for binding sites remain unclear. Here, we used single-molecule tracking and machine-learning-based classification to directly measure the nuclear mobility of the glucocorticoid receptor (GR) in live cells. We revealed two distinct and dynamic low-mobility populations. One accounts for specific binding to chromatin, while the other represents a confinement state that requires an intrinsically disordered region (IDR), implicated in liquid-liquid condensate subdomains. Further analysis showed that the dwell times of both subpopulations follow a power-law distribution, consistent with a broad distribution of affinities on the GR cistrome and interactome. Together, our data link IDRs with a confinement state that is functionally distinct from specific chromatin binding and modulates the transcriptional output by increasing the local concentration of TFs at specific sites.
    Keywords:  GRdim; GRmon; PPAR; bi-exponential; chromatin binding; confinement; glucocorticoid receptor; intrinsically disordered regions; power-law; single-molecule; transcription dynamics
  17. Sci Adv. 2021 Feb;pii: eabe5905. [Epub ahead of print]7(7):
    Ryu JK, Bouchoux C, Liu HW, Kim E, Minamino M, de Groot R, Katan AJ, Bonato A, Marenduzzo D, Michieletto D, Uhlmann F, Dekker C.
      Structural maintenance of chromosome (SMC) protein complexes are able to extrude DNA loops. While loop extrusion constitutes a fundamental building block of chromosomes, other factors may be equally important. Here, we show that yeast cohesin exhibits pronounced clustering on DNA, with all the hallmarks of biomolecular condensation. DNA-cohesin clusters exhibit liquid-like behavior, showing fusion of clusters, rapid fluorescence recovery after photobleaching and exchange of cohesin with the environment. Strikingly, the in vitro clustering is DNA length dependent, as cohesin forms clusters only on DNA exceeding 3 kilo-base pairs. We discuss how bridging-induced phase separation, a previously unobserved type of biological condensation, can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in yeast cells in vivo, a fraction of cohesin associates with chromatin in a manner consistent with bridging-induced phase separation. Biomolecular condensation by SMC proteins constitutes a new basic principle by which SMC complexes direct genome organization.
  18. J Genet Genomics. 2020 Dec 01. pii: S1673-8527(20)30188-0. [Epub ahead of print]
    Zheng W, Yang Z, Ge X, Feng Y, Wang Y, Liu C, Luan Y, Cai K, Vakal S, You F, Guo W, Wang W, Feng Z, Li F.
      Chromatin interactions functionally affect genome architecture and gene regulation, but to date, only fresh samples must be used in Hi-C (High-through chromosome conformation capture) to keep natural chromatin conformation intact. This requirement has impeded the advancement of 3D genome research by limiting sample collection and storage options for researchers and severely limiting the number of samples that can be processed in a short time. Here, we developed a freeze substitution Hi-C (FS-Hi-C) technique that overcomes the need for fresh samples. FS-Hi-C can be used with samples stored in liquid nitrogen (LN2): the water in a vitreous form in the sample cells is replaced with ethanol via automated freeze substitution. After confirming that the FS step preserves the natural chromosome conformation during sample thawing, we tested the performance of FS-Hi-C with Drosophila melanogaster and Gossypium hirsutum. Beyond allowing the use of frozen samples and confirming that FS-Hi-C delivers robust data for generating contact heat maps and delineating A/B compartments and topologically associating domains, we found that FS-Hi-C outperforms the in situ Hi-C in terms of library quality, reproducibility, and valid interactions. Thus, FS-Hi-C will probably extend the application of 3D genome structure analysis to the vast number of experimental contexts in biological and medical research for which Hi-C methods have been unfeasible to date.
    Keywords:  Chromosome conformation; Drosophila melanogaster; FS-Hi-C; Frozen sample; Gossypium hirsutum
  19. Nucleic Acids Res. 2021 Feb 12. pii: gkab086. [Epub ahead of print]
    Li J, Dai S, Chen X, Liang X, Qu L, Jiang L, Guo M, Zhou Z, Wei H, Zhang H, Chen Z, Chen L, Chen Y.
      Forkhead transcription factors bind a canonical consensus DNA motif, RYAAAYA (R = A/G, Y = C/T), as a monomer. However, the molecular mechanisms by which forkhead transcription factors bind DNA as a dimer are not well understood. In this study, we show that FOXO1 recognizes a palindromic DNA element DIV2, and mediates transcriptional regulation. The crystal structure of FOXO1/DIV2 reveals that the FOXO1 DNA binding domain (DBD) binds the DIV2 site as a homodimer. The wing1 region of FOXO1 mediates the dimerization, which enhances FOXO1 DNA binding affinity and complex stability. Further biochemical assays show that FOXO3, FOXM1 and FOXI1 also bind the DIV2 site as homodimer, while FOXC2 can only bind this site as a monomer. Our structural, biochemical and bioinformatics analyses not only provide a novel mechanism by which FOXO1 binds DNA as a homodimer, but also shed light on the target selection of forkhead transcription factors.
  20. Sci Rep. 2021 Feb 09. 11(1): 3381
    Kashima K, Kawai T, Nishimura R, Shiwa Y, Urayama KY, Kamura H, Takeda K, Aoto S, Ito A, Matsubara K, Nagamatsu T, Fujii T, Omori I, Shimizu M, Hyodo H, Kugu K, Matsumoto K, Shimizu A, Oka A, Mizuguchi M, Nakabayashi K, Hata K, Takahashi N.
      Preterm birth is known to be associated with chronic disease risk in adulthood whereby epigenetic memory may play a mechanistic role in disease susceptibility. Gestational age (GA) is the most important prognostic factor for preterm infants, and numerous DNA methylation alterations associated with GA have been revealed by epigenome-wide association studies. However, in human preterm infants, whether the methylation changes relate to transcription in the fetal state and persist after birth remains to be elucidated. Here, we identified 461 transcripts associated with GA (range 23-41 weeks) and 2093 candidate CpG sites for GA-involved epigenetic memory through analysis of methylome (110 cord blood and 47 postnatal blood) and transcriptional data (55 cord blood). Moreover, we discovered the trends of chromatin state, such as polycomb-binding, among these candidate sites. Fifty-four memory candidate sites showed correlation between methylation and transcription, and the representative corresponding gene was UCN, which encodes urocortin.
  21. Nat Commun. 2021 Feb 12. 12(1): 1011
    Kaushal A, Mohana G, Dorier J, Özdemir I, Omer A, Cousin P, Semenova A, Taschner M, Dergai O, Marzetta F, Iseli C, Eliaz Y, Weisz D, Shamim MS, Guex N, Lieberman Aiden E, Gambetta MC.
      Vertebrate genomes are partitioned into contact domains defined by enhanced internal contact frequency and formed by two principal mechanisms: compartmentalization of transcriptionally active and inactive domains, and stalling of chromosomal loop-extruding cohesin by CTCF bound at domain boundaries. While Drosophila has widespread contact domains and CTCF, it is currently unclear whether CTCF-dependent domains exist in flies. We genetically ablate CTCF in Drosophila and examine impacts on genome folding and transcriptional regulation in the central nervous system. We find that CTCF is required to form a small fraction of all domain boundaries, while critically controlling expression patterns of certain genes and supporting nervous system function. We also find that CTCF recruits the pervasive boundary-associated factor Cp190 to CTCF-occupied boundaries and co-regulates a subset of genes near boundaries together with Cp190. These results highlight a profound difference in CTCF-requirement for genome folding in flies and vertebrates, in which a large fraction of boundaries are CTCF-dependent and suggest that CTCF has played mutable roles in genome architecture and direct gene expression control during metazoan evolution.
  22. EMBO J. 2021 Feb 08. e104913
    Bright AR, van Genesen S, Li Q, Grasso A, Frölich S, van der Sande M, van Heeringen SJ, Veenstra GJC.
      During vertebrate gastrulation, mesoderm is induced in pluripotent cells, concomitant with dorsal-ventral patterning and establishing of the dorsal axis. We applied single-cell chromatin accessibility and transcriptome analyses to explore the emergence of cellular heterogeneity during gastrulation in Xenopus tropicalis. Transcriptionally inactive lineage-restricted genes exhibit relatively open chromatin in animal caps, whereas chromatin accessibility in dorsal marginal zone cells more closely reflects transcriptional activity. We characterized single-cell trajectories and identified head and trunk organizer cell clusters in early gastrulae. By integrating chromatin accessibility and transcriptome data, we inferred the activity of transcription factors in single-cell clusters and tested the activity of organizer-expressed transcription factors in animal caps, alone or in combination. The expression profile induced by a combination of Foxb1 and Eomes most closely resembles that observed in the head organizer. Genes induced by Eomes, Otx2, or the Irx3-Otx2 combination are enriched for maternally regulated H3K4me3 modifications, whereas Lhx8-induced genes are marked more frequently by zygotically controlled H3K4me3. Taken together, our results show that transcription factors cooperate in a combinatorial fashion in generally open chromatin to orchestrate zygotic gene expression.
    Keywords:  cell trajectories; chromatin accessibility; mesendoderm; organizer; transcription factors
  23. Mol Cell. 2021 Jan 28. pii: S1097-2765(21)00016-2. [Epub ahead of print]
    Rawat P, Boehning M, Hummel B, Aprile-Garcia F, Pandit AS, Eisenhardt N, Khavaran A, Niskanen E, Vos SM, Palvimo JJ, Pichler A, Cramer P, Sawarkar R.
      In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.
    Keywords:  CDK9; RNA polymerase II; SUMO; heat shock; negative elongation factor (NELF); pausing; phase separation; proteostasis; transcriptional condensates; transcriptional stress response
  24. Nat Commun. 2021 02 09. 12(1): 898
    Stewart-Ornstein J, Iwamoto Y, Miller MA, Prytyskach MA, Ferretti S, Holzer P, Kallen J, Furet P, Jambhekar A, Forrester WC, Weissleder R, Lahav G.
      Radiation sensitivity varies greatly between tissues. The transcription factor p53 mediates the response to radiation; however, the abundance of p53 protein does not correlate well with the extent of radiosensitivity across tissues. Given recent studies showing that the temporal dynamics of p53 influence the fate of cultured cells in response to irradiation, we set out to determine the dynamic behavior of p53 and its impact on radiation sensitivity in vivo. We find that radiosensitive tissues show prolonged p53 signaling after radiation, while more resistant tissues show transient p53 activation. Sustaining p53 using a small molecule (NMI801) that inhibits Mdm2, a negative regulator of p53, reduced viability in cell culture and suppressed tumor growth. Our work proposes a mechanism for the control of radiation sensitivity and suggests tools to alter the dynamics of p53 to enhance tumor clearance. Similar approaches can be used to enhance killing of cancer cells or reduce toxicity in normal tissues following genotoxic therapies.
  25. Commun Biol. 2021 Feb 12. 4(1): 191
    Hirano R, Arimura Y, Kujirai T, Shibata M, Okuda A, Morishima K, Inoue R, Sugiyama M, Kurumizaka H.
      H2A.B is an evolutionarily distant histone H2A variant that accumulates on DNA repair sites, DNA replication sites, and actively transcribing regions in genomes. In cells, H2A.B exchanges rapidly in chromatin, but the mechanism has remained enigmatic. In the present study, we found that the H2A.B-H2B dimer incorporated within the nucleosome exchanges with the canonical H2A-H2B dimer without assistance from additional factors, such as histone chaperones and nucleosome remodelers. High-speed atomic force microscopy revealed that the H2A.B nucleosome, but not the canonical H2A nucleosome, transiently forms an intermediate "open conformation", in which two H2A.B-H2B dimers may be detached from the H3-H4 tetramer and bind to the DNA regions near the entry/exit sites. Mutational analyses revealed that the H2A.B C-terminal region is responsible for the adoption of the open conformation and the H2A.B-H2B exchange in the nucleosome. These findings provide mechanistic insights into the histone exchange of the H2A.B nucleosome.
  26. Nat Immunol. 2021 Feb 11.
    Yao C, Lou G, Sun HW, Zhu Z, Sun Y, Chen Z, Chauss D, Moseman EA, Cheng J, D'Antonio MA, Shi W, Shi J, Kometani K, Kurosaki T, Wherry EJ, Afzali B, Gattinoni L, Zhu Y, McGavern DB, O'Shea JJ, Schwartzberg PL, Wu T.
      During chronic infection and cancer, a self-renewing CD8+ T cell subset maintains long-term immunity and is critical to the effectiveness of immunotherapy. These stem-like CD8+ T cells diverge from other CD8+ subsets early after chronic viral infection. However, pathways guarding stem-like CD8+ T cells against terminal exhaustion remain unclear. Here, we show that the gene encoding transcriptional repressor BACH2 is transcriptionally and epigenetically active in stem-like CD8+ T cells but not terminally exhausted cells early after infection. BACH2 overexpression enforced stem-like cell fate, whereas BACH2 deficiency impaired stem-like CD8+ T cell differentiation. Single-cell transcriptomic and epigenomic approaches revealed that BACH2 established the transcriptional and epigenetic programs of stem-like CD8+ T cells. In addition, BACH2 suppressed the molecular program driving terminal exhaustion through transcriptional repression and epigenetic silencing. Thus, our study reveals a new pathway that enforces commitment to stem-like CD8+ lineage and prevents an alternative terminally exhausted cell fate.
  27. Elife. 2021 Feb 11. pii: e61070. [Epub ahead of print]10
    Mukherjee K, Xue L, Planutis A, Gnanapragasam MN, Chess A, Bieker JJ.
      Erythroblastic islands are a specialized niche that contain a central macrophage surrounded by erythroid cells at various stages of maturation. However, identifying the precise genetic and transcriptional control mechanisms in the island macrophage remains difficult due to macrophage heterogeneity. Using unbiased global sequencing and directed genetic approaches focused on early mammalian development, we find that fetal liver macrophage exhibit a unique expression signature that differentiates them from erythroid and adult macrophage cells. The importance of EKLF/KLF1 in this identity is shown by expression analyses in EKLF-/- and in EKLF-marked macrophage cells. Single cell sequence analysis simplifies heterogeneity and identifies clusters of genes important for EKLF-dependent macrophage function and novel cell surface biomarkers. Remarkably, this singular set of macrophage island cells appears transiently during embryogenesis. Together these studies provide a detailed perspective on the importance of EKLF in establishment of the dynamic gene expression network within erythroblastic islands in the developing embryo and provide the means for their efficient isolation.
    Keywords:  developmental biology; mouse
  28. NAR Genom Bioinform. 2020 Dec;2(4): lqaa091
    Sharma R, Pandey N, Mongia A, Mishra S, Majumdar A, Kumar V.
      The advent of single-cell open-chromatin profiling technology has facilitated the analysis of heterogeneity of activity of regulatory regions at single-cell resolution. However, stochasticity and availability of low amount of relevant DNA, cause high drop-out rate and noise in single-cell open-chromatin profiles. We introduce here a robust method called as forest of imputation trees (FITs) to recover original signals from highly sparse and noisy single-cell open-chromatin profiles. FITs makes multiple imputation trees to avoid bias during the restoration of read-count matrices. It resolves the challenging issue of recovering open chromatin signals without blurring out information at genomic sites with cell-type-specific activity. Besides visualization and classification, FITs-based imputation also improved accuracy in the detection of enhancers, calculating pathway enrichment score and prediction of chromatin-interactions. FITs is generalized for wider applicability, especially for highly sparse read-count matrices. The superiority of FITs in recovering signals of minority cells also makes it highly useful for single-cell open-chromatin profile from in vivo samples. The software is freely available at
  29. Cancer Res. 2021 Feb 11. pii: canres.2489.2020. [Epub ahead of print]
    Laterra J, Ma T, Hu C, Lal B, Zhou W, Ma Y, Ying M, Prinos P, Quiñones-Hinojosa A, Lim M, Li Y.
      A subset of stem-like tumor-propagating cells in GBM (GSC) underlies tumor propagation, therapeutic resistance and tumor recurrence. Immune evasion is critical for GSCs to carry out these functions. However, the molecular mechanisms employed by GSCs to escape anti-tumor immunity remain largely unknown. The reprogramming transcription factors Oct4 and Sox2 function as core multipotency factors and play an essential role in the formation and maintenance of GSCs, but the roles of these transcription factors in GSC immune escape have not been well explored. Here we examine how Oct4/Sox2 co-expression contributes to the immunosuppressive phenotype of GSCs. Combined transcription profiling and functional studies of Oct4/Sox2 co-expressing GSCs and differentiated GBM cells demonstrated that Oct4 and Sox2 cooperatively induce an immunosuppressive transcriptome consisting of multiple immunosuppressive checkpoints (i.e., PD-L1, CD70, A2aR, TDO) and dysregulation of cytokines and chemokines that are associated with an immunosuppressive tumor microenvironment. Mechanistically, induction and function of BRD/H3k27Ac-dependent immunosuppressive genes played a role in the immunosuppressive phenotype of GSCs. Pan-BET bromodomain inhibitors (e.g., JQ1) and shBRD4 constructs significantly inhibited the immunosuppressive transcriptome and immunosuppressive biological responses induced by Oct4/Sox2. Our findings identify targetable mechanisms by which tumor-propagating GSCs contribute to the immunosuppressive microenvironment in GBM.
  30. Cancer Res. 2021 Feb 11. pii: canres.2801.2020. [Epub ahead of print]
    Quinn HM, Vogel R, Popp O, Mertins P, Lan L, Messerschmidt C, Landshammer A, Lisek K, Chateau-Joubert S, Marangoni E, Koren E, Fuchs Y, Birchmeier W.
      Targeting cancer stem cells (CSC) can serve as an effective approach toward limiting resistance to therapies. While basal-like (triple-negative) breast cancers encompass cells with CSC features, rational therapies remain poorly established. We show here that the receptor tyrosine kinase Met promotes YAP activity in basal-like breast cancer and find enhanced YAP activity within the CSC population. Interfering with YAP activity delayed basal-like cancer formation, prevented luminal to basal trans-differentiation, and reduced CSC. YAP knockout mammary glands revealed a decrease in β-catenin target genes, suggesting that YAP is required for nuclear β-catenin activity. Mechanistically, nuclear YAP interacted with β-catenin and TEAD4 at gene regulatory elements. Proteomic patient data revealed an upregulation of the YAP signature in basal-like breast cancers. Our findings demonstrate that in basal-like breast cancers, β-catenin activity is dependent on YAP signalling and controls the CSC program. These findings suggest that targeting the YAP/TEAD4/β-catenin complex offers a potential therapeutic strategy for eradicating CSCs in basal-like breast cancers.
  31. NAR Genom Bioinform. 2020 Jun;2(2): lqaa031
    Kuang S, Wang L.
      CCCTC-binding factor (CTCF) is a key regulator of 3D genome organization and gene expression. Recent studies suggest that RNA transcripts, mostly long non-coding RNAs (lncRNAs), can serve as locus-specific factors to bind and recruit CTCF to the chromatin. However, it remains unclear whether specific sequence patterns are shared by the CTCF-binding RNA sites, and no RNA motif has been reported so far for CTCF binding. In this study, we have developed DeepLncCTCF, a new deep learning model based on a convolutional neural network and a bidirectional long short-term memory network, to discover the RNA recognition patterns of CTCF and identify candidate lncRNAs binding to CTCF. When evaluated on two different datasets, human U2OS dataset and mouse ESC dataset, DeepLncCTCF was shown to be able to accurately predict CTCF-binding RNA sites from nucleotide sequence. By examining the sequence features learned by DeepLncCTCF, we discovered a novel RNA motif with the consensus sequence, AGAUNGGA, for potential CTCF binding in humans. Furthermore, the applicability of DeepLncCTCF was demonstrated by identifying nearly 5000 candidate lncRNAs that might bind to CTCF in the nucleus. Our results provide useful information for understanding the molecular mechanisms of CTCF function in 3D genome organization.