bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2020‒08‒23
27 papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit

  1. Cell Rep. 2020 Aug 18. pii: S2211-1247(20)31014-7. [Epub ahead of print]32(7): 108029
    Funk CC, Casella AM, Jung S, Richards MA, Rodriguez A, Shannon P, Donovan-Maiye R, Heavner B, Chard K, Xiao Y, Glusman G, Ertekin-Taner N, Golde TE, Toga A, Hood L, Van Horn JD, Kesselman C, Foster I, Madduri R, Price ND, Ament SA.
      Characterizing the tissue-specific binding sites of transcription factors (TFs) is essential to reconstruct gene regulatory networks and predict functions for non-coding genetic variation. DNase-seq footprinting enables the prediction of genome-wide binding sites for hundreds of TFs simultaneously. Despite the public availability of high-quality DNase-seq data from hundreds of samples, a comprehensive, up-to-date resource for the locations of genomic footprints is lacking. Here, we develop a scalable footprinting workflow using two state-of-the-art algorithms: Wellington and HINT. We apply our workflow to detect footprints in 192 ENCODE DNase-seq experiments and predict the genomic occupancy of 1,515 human TFs in 27 human tissues. We validate that these footprints overlap true-positive TF binding sites from ChIP-seq. We demonstrate that the locations, depth, and tissue specificity of footprints predict effects of genetic variants on gene expression and capture a substantial proportion of genetic risk for complex traits.
    Keywords:  DNase-seq; ENCODE; footprinting; gene regulation; motifs; psychiatric genetics; transcription factors
  2. Cell Rep. 2020 Aug 18. pii: S2211-1247(20)31033-0. [Epub ahead of print]32(7): 108048
    Maqbool MA, Pioger L, El Aabidine AZ, Karasu N, Molitor AM, Dao LTM, Charbonnier G, van Laethem F, Fenouil R, Koch F, Lacaud G, Gut I, Gut M, Amigorena S, Joffre O, Sexton T, Spicuglia S, Andrau JC.
      During thymic development and upon peripheral activation, T cells undergo extensive phenotypic and functional changes coordinated by lineage-specific developmental programs. To characterize the regulatory landscape controlling T cell identity, we perform a wide epigenomic and transcriptional analysis of mouse thymocytes and naive CD4 differentiated T helper cells. Our investigations reveal a dynamic putative enhancer landscape, and we could validate many of the enhancers using the high-throughput CapStarr sequencing (CapStarr-seq) approach. We find that genes using multiple promoters display increased enhancer usage, suggesting that apparent "enhancer redundancy" might relate to isoform selection. Furthermore, we can show that two Runx3 promoters display long-range interactions with specific enhancers. Finally, our analyses suggest a novel function for the PRC2 complex in the control of alternative promoter usage. Altogether, our study has allowed for the mapping of an exhaustive set of active enhancers and provides new insights into their function and that of PRC2 in controlling promoter choice during T cell differentiation.
    Keywords:  CapSTARR-seq; T cell enhancerome; enhancer and promoter usage; enhancer redundancy; long-distance enhancer-promoter interactions
  3. Nat Commun. 2020 Aug 18. 11(1): 4136
    Tanaka H, Takizawa Y, Takaku M, Kato D, Kumagawa Y, Grimm SA, Wade PA, Kurumizaka H.
      During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5'-GAT-3' sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.
  4. Proc Natl Acad Sci U S A. 2020 Aug 18. pii: 201922216. [Epub ahead of print]
    Park A, Oh S, Jung KL, Choi UY, Lee HR, Rosenfeld MG, Jung JU.
      Enhancers play indispensable roles in cell proliferation and survival through spatiotemporally regulating gene transcription. Active enhancers and superenhancers often produce noncoding enhancer RNAs (eRNAs) that precisely control RNA polymerase II activity. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic gamma-2 herpesvirus that causes Kaposi's sarcoma and primary effusion lymphoma (PEL). It is well characterized that KSHV utilizes host epigenetic machineries to control the switch between two lifecycles, latency and lytic replication. However, how KSHV impacts host epigenome at different stages of viral lifecycle is not well understood. Using global run-on sequencing (GRO-seq) and chromatin-immunoprecipitation sequencing (ChIP-seq), we profiled the dynamics of host transcriptional regulatory elements during latency and lytic replication of KSHV-infected PEL cells. This revealed that a number of critical host genes for KSHV latency, including MYC proto-oncogene, were under the control of superenhancers whose activities were globally repressed upon viral reactivation. The eRNA-expressing MYC superenhancers were located downstream of the MYC gene in KSHV-infected PELs and played a key role in MYC expression. RNAi-mediated depletion or dCas9-KRAB CRISPR inhibition of eRNA expression significantly reduced MYC mRNA level in PELs, as did the treatment of an epigenomic drug that globally blocks superenhancer function. Finally, while cellular IRF4 acted upon eRNA expression and superenhancer function for MYC expression during latency, KSHV viral IRF4 repressed cellular IRF4 expression, decreasing MYC expression and thereby, facilitating lytic replication. These results indicate that KSHV acts as an epigenomic driver that modifies host epigenomic status upon reactivation by effectively regulating host enhancer function.
    Keywords:  Kaposi’s sarcoma-associated virus; enhancer; primary effusion lymphoma; viral epigenetics; viral genomics
  5. Cell. 2020 Aug 18. pii: S0092-8674(20)30940-5. [Epub ahead of print]
    Su JH, Zheng P, Kinrot SS, Bintu B, Zhuang X.
      The 3D organization of chromatin regulates many genome functions. Our understanding of 3D genome organization requires tools to directly visualize chromatin conformation in its native context. Here we report an imaging technology for visualizing chromatin organization across multiple scales in single cells with high genomic throughput. First we demonstrate multiplexed imaging of hundreds of genomic loci by sequential hybridization, which allows high-resolution conformation tracing of whole chromosomes. Next we report a multiplexed error-robust fluorescence in situ hybridization (MERFISH)-based method for genome-scale chromatin tracing and demonstrate simultaneous imaging of more than 1,000 genomic loci and nascent transcripts of more than 1,000 genes together with landmark nuclear structures. Using this technology, we characterize chromatin domains, compartments, and trans-chromosomal interactions and their relationship to transcription in single cells. We envision broad application of this high-throughput, multi-scale, and multi-modal imaging technology, which provides an integrated view of chromatin organization in its native structural and functional context.
    Keywords:  3D genome organization; MERFISH; chromosome compartments; chromosome conformation; genome-scale imaging; multiplexed FISH; nuclear lamina; nuclear speckles; topologically associated domains; trans-chromosomal interaction
  6. Nat Chem Biol. 2020 Aug 17.
    Kim KP, Choi J, Yoon J, Bruder JM, Shin B, Kim J, Arauzo-Bravo MJ, Han D, Wu G, Han DW, Kim J, Cramer P, Schöler HR.
      Identifying molecular and cellular processes that regulate reprogramming competence of transcription factors broadens our understanding of reprogramming mechanisms. In the present study, by a chemical screen targeting major epigenetic pathways in human reprogramming, we discovered that inhibiting specific epigenetic roadblocks including disruptor of telomeric silencing 1-like (DOT1L)-mediated H3K79/K27 methylation, but also other epigenetic pathways, catalyzed by lysine-specific histone demethylase 1A, DNA methyltransferases and histone deacetylases, allows induced pluripotent stem cell generation with almost all OCT factors. We found that simultaneous inhibition of these pathways not only dramatically enhances reprogramming competence of most OCT factors, but in fact enables dismantling of species-dependent reprogramming competence of OCT6, NR5A1, NR5A2, TET1 and GATA3. Harnessing these induced permissive epigenetic states, we performed an additional screen with 98 candidate genes. Thereby, we identified 25 transcriptional regulators (OTX2, SIX3, and so on) that can functionally replace OCT4 in inducing pluripotency. Our findings provide a conceptual framework for understanding how transcription factors elicit reprogramming in dependency of the donor cell epigenome that differs across species.
  7. Nat Commun. 2020 Aug 17. 11(1): 4118
    Djeghloul D, Patel B, Kramer H, Dimond A, Whilding C, Brown K, Kohler AC, Feytout A, Veland N, Elliott J, Bharat TAM, Tarafder AK, Löwe J, Ng BL, Guo Y, Guy J, Huseyin MK, Klose RJ, Merkenschlager M, Fisher AG.
      Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers such as PRC2 and DNA methyl-transferases, and proteins governing chromosome architecture. Deletion of PRC2, Dnmt1/3a/3b or Mecp2 in ESCs leads to an increase in the size of individual mitotic chromosomes, consistent with de-condensation. Similar results were obtained by the experimental cleavage of cohesin. Thus, we identify chromosome-bound factors in pluripotent stem cells during mitosis and reveal that PRC2, DNA methylation and Mecp2 are required to maintain chromosome compaction.
  8. Cancer Res. 2020 Aug 14. pii: canres.1259.2020. [Epub ahead of print]
    Du L, Fakih MG, Rosen ST, Chen Y.
      Elevated expression of EZH2, the enzymatic subunit of polycomb repressive complex 2 (PRC2), often occurs in cancer. EZH2 expression results in the silencing of genes that suppress tumor formation and metastasis through trimethylation of histone H3 at lysine 27 (H3K27me3) at said gene promoters. However, inhibitors of EZH2 enzymatic activity have not shown the expected efficacy against cancer in clinical trials, suggesting a need for other strategies to address EZH2 overexpression. Here we show that SUMOylation, a post-translational modification characterized by covalent attachment of small ubiquitin-like modifier (SUMO) proteins to a lysine (Lys) residue on target proteins, enhances EZH2 transcription. Either knockdown of the SUMO-activating enzyme SAE2 or pharmacological inhibition of SUMOylation resulted in decreased levels of EZH2 mRNA and protein as well as reduced H3K27me3 levels. SUMOylation regulated EZH2 expression by enhancing binding of the E2F1 transcriptional activator to the EZH2 promoter. Inhibition of SUMOylation not only resulted in reduced EZH2 mRNA and protein levels but also increased expression of genes silenced by EZH2, such as E-cadherin which suppresses epithelial-mesenchymal transition and metastasis. In more than 6,500 patient tumor samples across different cancer types, expression of UBA2 and EZH2 were positively correlated. Taken together, our findings suggest that inhibition of SUMOylation may serve as a potential strategy to address EZH2 overexpression and improve current cancer therapeutic approaches.
  9. Nucleic Acids Res. 2020 Aug 18. pii: gkaa671. [Epub ahead of print]
    Carullo NVN, Phillips Iii RA, Simon RC, Soto SAR, Hinds JE, Salisbury AJ, Revanna JS, Bunner KD, Ianov L, Sultan FA, Savell KE, Gersbach CA, Day JJ.
      Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer-gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR-based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer-gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CREB-binding protein (CBP). Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.
  10. Proc Natl Acad Sci U S A. 2020 Aug 19. pii: 202008672. [Epub ahead of print]
    Cai W, Huang J, Zhu Q, Li BE, Seruggia D, Zhou P, Nguyen M, Fujiwara Y, Xie H, Yang Z, Hong D, Ren P, Xu J, Pu WT, Yuan GC, Orkin SH.
      How overall principles of cell-type-specific gene regulation (the "logic") may change during ontogeny is largely unexplored. We compared transcriptomic, epigenomic, and three-dimensional (3D) genomic profiles in embryonic (EryP) and adult (EryD) erythroblasts. Despite reduced chromatin accessibility compared to EryP, distal chromatin of EryD is enriched in H3K27ac, Gata1, and Myb occupancy. EryP-/EryD-shared enhancers are highly correlated with red blood cell identity genes, whereas cell-type-specific regulation employs different cis elements in EryP and EryD cells. In contrast to EryP-specific genes, which exhibit promoter-centric regulation through Gata1, EryD-specific genes rely more on distal enhancers for regulation involving Myb-mediated enhancer activation. Gata1 HiChIP demonstrated an overall increased enhancer-promoter interactions at EryD-specific genes, whereas genome editing in selected loci confirmed distal enhancers are required for gene expression in EryD but not in EryP. Applying a metric for enhancer dependence of transcription, we observed a progressive reliance on cell-specific enhancers with increasing ontogenetic age among diverse tissues of mouse and human origin. Our findings highlight fundamental and conserved differences at distinct developmental stages, characterized by simpler promoter-centric regulation of cell-type-specific genes in embryonic cells and increased combinatorial enhancer-driven control in adult cells.
    Keywords:  GATA1; HiChIP; Myb; enhancer; erythropoiesis
  11. Cell Cycle. 2020 Aug 20. 1-22
    Adhikari A, Davie JK.
      The polycomb repressive complex 2 (PRC2) is an important developmental regulator responsible for the methylation of histone 3 lysine 27 (H3K27). Here, we show that the PRC2 complex regulates the cell cycle in skeletal muscle cells to control proliferation and mitotic exit. Depletions of the catalytic subunit of the PRC2 complex, EZH2, have shown that EZH2 is required for cell viability, suggesting that EZH2 promotes proliferation. We found that EZH2 directly represses both positive and negative cell cycle genes, thus enabling the PRC2 complex to tightly control the cell cycle. We show that modest inhibition or depletion of EZH2 leads to enhanced proliferation and an accumulation of cells in S phase. This effect is mediated by direct repression of cyclin D1 (Ccnd1) and cyclin E1 (Ccne1) by the PRC2 complex. Our results show that PRC2 has pleiotropic effects on proliferation as it serves to restrain cell growth, yet clearly has a function required for cell viability as well. Intriguingly, we also find that the retinoblastoma protein gene (Rb1) is a direct target of the PRC2 complex. However, modest depletion of EZH2 is not sufficient to maintain Rb1 expression, indicating that the PRC2 dependent upregulation of cyclin D1 is sufficient to inhibit Rb1 expression. Taken together, our results show that the PRC2 complex regulates skeletal muscle proliferation in a complex manner that involves the repression of Ccnd1 and Ccne1, thus restraining proliferation, and the repression of Rb1, which is required for mitotic exit and terminal differentiation.
    Keywords:  EZH2; PRC2; cell cycle; cyclin D1; cyclin E1; retinoblastoma protein (pRB, RB1); skeletal muscle
  12. Genes Dev. 2020 Aug 20.
    Basu A, Mestres I, Sahu SK, Tiwari N, Khongwir B, Baumgart J, Singh A, Calegari F, Tiwari VK.
      Cerebral cortical development in mammals involves a highly complex and organized set of events including the transition of neural stem and progenitor cells (NSCs) from proliferative to differentiative divisions to generate neurons. Despite progress, the spatiotemporal regulation of this proliferation-differentiation switch during neurogenesis and the upstream epigenetic triggers remain poorly known. Here we report a cortex-specific PHD finger protein, Phf21b, which is highly expressed in the neurogenic phase of cortical development and gets induced as NSCs begin to differentiate. Depletion of Phf21b in vivo inhibited neuronal differentiation as cortical progenitors lacking Phf21b were retained in the proliferative zones and underwent faster cell cycles. Mechanistically, Phf21b targets the regulatory regions of cell cycle promoting genes by virtue of its high affinity for monomethylated H3K4. Subsequently, Phf21b recruits the lysine-specific demethylase Lsd1 and histone deacetylase Hdac2, resulting in the simultaneous removal of monomethylation from H3K4 and acetylation from H3K27, respectively. Intriguingly, mutations in the Phf21b locus associate with depression and mental retardation in humans. Taken together, these findings establish how a precisely timed spatiotemporal expression of Phf21b creates an epigenetic program that triggers neural stem cell differentiation during cortical development.
    Keywords:  cortical development; epigenetics; gene regulation; genomics; neurogenesis
  13. Nat Protoc. 2020 Aug 17.
    Handa T, Harada A, Maehara K, Sato S, Nakao M, Goto N, Kurumizaka H, Ohkawa Y, Kimura H.
      Cell identity is determined by the selective activation or silencing of specific genes via transcription factor binding and epigenetic modifications on the genome. Chromatin immunoprecipitation (ChIP) has been the standard technique for mapping the sites of transcription factor binding and histone modification. Recently, alternative methods to ChIP have been developed for addressing the increasing demands for low-input epigenomic profiling. Chromatin integration labeling (ChIL) followed by sequencing (ChIL-seq) has been demonstrated to be particularly useful for epigenomic profiling of low-input samples or even single cells because the technique amplifies the target genomic sequence before cell lysis. After labeling the target protein or modification in situ with an oligonucleotide-conjugated antibody (ChIL probe), the nearby genome sequence is amplified by Tn5 transposase-mediated transposition followed by T7 RNA polymerase-mediated transcription. ChIL-seq enables the detection of the antibody target localization under a fluorescence microscope and at the genomic level. Here we describe the detailed protocol of ChIL-seq with assessment methods for the key steps, including ChIL probe reaction, transposition, in situ transcription and sequencing library preparation. The protocol usually takes 3 d to prepare the sequencing library, including overnight incubations for the ChIL probe reaction and in situ transcription. The ChIL probe can be separately prepared and stored for several months, and its preparation and evaluation protocols are also documented in detail. An optional analysis for multiple targets (multitarget ChIL-seq) is also described. We anticipate that the protocol presented here will make the ChIL technique more widely accessible for analyzing precious samples and facilitate further applications.
  14. Nat Commun. 2020 Aug 19. 11(1): 4153
    Vatapalli R, Sagar V, Rodriguez Y, Zhao JC, Unno K, Pamarthy S, Lysy B, Anker J, Han H, Yoo YA, Truica M, Chalmers ZR, Giles F, Yu J, Chakravarti D, Carneiro B, Abdulkadir SA.
      The histone methyltransferase DOT1L methylates lysine 79 (K79) on histone H3 and is involved in Mixed Lineage Leukemia (MLL) fusion leukemogenesis; however, its role in prostate cancer (PCa) is undefined. Here we show that DOT1L is overexpressed in PCa and is associated with poor outcome. Genetic and chemical inhibition of DOT1L selectively impaired the viability of androgen receptor (AR)-positive PCa cells and organoids, including castration-resistant and enzalutamide-resistant cells. The sensitivity of AR-positive cells is due to a distal K79 methylation-marked enhancer in the MYC gene bound by AR and DOT1L not present in AR-negative cells. DOT1L inhibition leads to reduced MYC expression and upregulation of MYC-regulated E3 ubiquitin ligases HECTD4 and MYCBP2, which promote AR and MYC degradation. This leads to further repression of MYC in a negative feed forward manner. Thus DOT1L selectively regulates the tumorigenicity of AR-positive prostate cancer cells and is a promising therapeutic target for PCa.
  15. Elife. 2020 Aug 17. pii: e59351. [Epub ahead of print]9
    Waymack R, Fletcher A, Enciso G, Wunderlich Z.
      Shadow enhancers, groups of seemingly redundant enhancers, are found in a wide range of organisms and are critical for robust developmental patterning. However, their mechanism of action is unknown. We hypothesized that shadow enhancers drive consistent expression levels by buffering upstream noise through a separation of transcription factor (TF) inputs at the individual enhancers. By measuring transcriptional dynamics of several Kruppel shadow enhancer configurations in live Drosophila embryos, we showed individual member enhancers act largely independently. We found that TF fluctuations are an appreciable source of noise that the shadow enhancer pair can better buffer than duplicated enhancers. The shadow enhancer pair is uniquely able to maintain low levels of expression noise across a wide range of temperatures. A stochastic model demonstrated the separation of TF inputs is sufficient to explain these findings. Our results suggest the widespread use of shadow enhancers is partially due to their noise suppressing ability.
    Keywords:  D. melanogaster; chromosomes; computational biology; gene expression; systems biology
  16. Nat Commun. 2020 Aug 17. 11(1): 4133
    Wang J, Wang J, Yang L, Zhao C, Wu LN, Xu L, Zhang F, Weng Q, Wegner M, Lu QR.
      Chromatin organization is critical for cell growth, differentiation, and disease development, however, its functions in peripheral myelination and myelin repair remain elusive. In this report, we demonstrate that the CCCTC-binding factor (CTCF), a crucial chromatin organizer, is essential for Schwann cell myelination and myelin regeneration after nerve injury. Inhibition of CTCF or its deletion blocks Schwann cell differentiation at the pro-myelinating stage, whereas overexpression of CTCF promotes the myelination program. We find that CTCF establishes chromatin interaction loops between enhancer and promoter regulatory elements and promotes expression of a key pro-myelinogenic factor EGR2. In addition, CTCF interacts with SUZ12, a component of polycomb-repressive-complex 2 (PRC2), to repress the transcriptional program associated with negative regulation of Schwann cell maturation. Together, our findings reveal a dual role of CTCF-dependent chromatin organization in promoting myelinogenic programs and recruiting chromatin-repressive complexes to block Schwann cell differentiation inhibitors to control peripheral myelination and repair.
  17. Cell Death Dis. 2020 Aug 21. 11(8): 673
    Wiese M, Hamdan FH, Kubiak K, Diederichs C, Gielen GH, Nussbaumer G, Carcaboso AM, Hulleman E, Johnsen SA, Kramm CM.
      Diffuse intrinsic pontine gliomas (DIPG) are the most aggressive brain tumors in children with 5-year survival rates of only 2%. About 85% of all DIPG are characterized by a lysine-to-methionine substitution in histone 3, which leads to global H3K27 hypomethylation accompanied by H3K27 hyperacetylation. Hyperacetylation in DIPG favors the action of the Bromodomain and Extra-Terminal (BET) protein BRD4, and leads to the reprogramming of the enhancer landscape contributing to the activation of DIPG super enhancer-driven oncogenes. The activity of the acetyltransferase CREB-binding protein (CBP) is enhanced by BRD4 and associated with acetylation of nucleosomes at super enhancers (SE). In addition, CBP contributes to transcriptional activation through its function as a scaffold and protein bridge. Monotherapy with either a CBP (ICG-001) or BET inhibitor (JQ1) led to the reduction of tumor-related characteristics. Interestingly, combined treatment induced strong cytotoxic effects in H3.3K27M-mutated DIPG cell lines. RNA sequencing and chromatin immunoprecipitation revealed that these effects were caused by the inactivation of DIPG SE-controlled tumor-related genes. However, single treatment with ICG-001 or JQ1, respectively, led to activation of a subgroup of detrimental super enhancers. Combinatorial treatment reversed the inadvertent activation of these super enhancers and rescued the effect of ICG-001 and JQ1 single treatment on enhancer-driven oncogenes in H3K27M-mutated DIPG, but not in H3 wild-type pedHGG cells. In conclusion, combinatorial treatment with CBP and BET inhibitors is highly efficient in H3K27M-mutant DIPG due to reversal of inadvertent activation of detrimental SE programs in comparison with monotherapy.
  18. Nucleic Acids Res. 2020 Aug 20. 48(14): 7712-7727
    Tellier M, Zaborowska J, Caizzi L, Mohammad E, Velychko T, Schwalb B, Ferrer-Vicens I, Blears D, Nojima T, Cramer P, Murphy S.
      Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.
  19. Genome Biol. 2020 Aug 20. 21(1): 210
    Mattioli K, Oliveros W, Gerhardinger C, Andergassen D, Maass PG, Rinn JL, Melé M.
      BACKGROUND: Gene expression differences between species are driven by both cis and trans effects. Whereas cis effects are caused by genetic variants located on the same DNA molecule as the target gene, trans effects are due to genetic variants that affect diffusible elements. Previous studies have mostly assessed the impact of cis and trans effects at the gene level. However, how cis and trans effects differentially impact regulatory elements such as enhancers and promoters remains poorly understood. Here, we use massively parallel reporter assays to directly measure the transcriptional outputs of thousands of individual regulatory elements in embryonic stem cells and measure cis and trans effects between human and mouse.RESULTS: Our approach reveals that cis effects are widespread across transcribed regulatory elements, and the strongest cis effects are associated with the disruption of motifs recognized by strong transcriptional activators. Conversely, we find that trans effects are rare but stronger in enhancers than promoters and are associated with a subset of transcription factors that are differentially expressed between human and mouse. While we find that cis-trans compensation is common within promoters, we do not see evidence of widespread cis-trans compensation at enhancers. Cis-trans compensation is inversely correlated with enhancer redundancy, suggesting that such compensation may often occur across multiple enhancers.
    CONCLUSIONS: Our results highlight differences in the mode of evolution between promoters and enhancers in complex mammalian genomes and indicate that studying the evolution of individual regulatory elements is pivotal to understand the tempo and mode of gene expression evolution.
    Keywords:  Cis and trans effects; Gene expression evolution; Massively parallel reporter assays; Regulatory element evolution
  20. Elife. 2020 08 18. pii: e58123. [Epub ahead of print]9
    Zimmerli D, Borrelli C, Jauregi-Miguel A, Söderholm S, Brütsch S, Doumpas N, Reichmuth J, Murphy-Seiler F, Aguet M, Basler K, Moor AE, Cantù C.
      BCL9 and PYGO are β-catenin cofactors that enhance the transcription of Wnt target genes. They have been proposed as therapeutic targets to diminish Wnt signaling output in intestinal malignancies. Here we find that, in colorectal cancer cells and in developing mouse forelimbs, BCL9 proteins sustain the action of β-catenin in a largely PYGO-independent manner. Our genetic analyses implied that BCL9 necessitates other interaction partners in mediating its transcriptional output. We identified the transcription factor TBX3 as a candidate tissue-specific member of the β-catenin transcriptional complex. In developing forelimbs, both TBX3 and BCL9 occupy a large number of Wnt-responsive regulatory elements, genome-wide. Moreover, mutations in Bcl9 affect the expression of TBX3 targets in vivo, and modulation of TBX3 abundance impacts on Wnt target genes transcription in a β-catenin- and TCF/LEF-dependent manner. Finally, TBX3 overexpression exacerbates the metastatic potential of Wnt-dependent human colorectal cancer cells. Our work implicates TBX3 as context-dependent component of the Wnt/β-catenin-dependent transcriptional complex.
    Keywords:  colorectal cancer; development; developmental biology; gene regulation; limb development; mouse; transcription; wnt signalling; zebrafish
  21. Elife. 2020 Aug 17. pii: e55124. [Epub ahead of print]9
    Stefanovic S, Laforest B, Desvignes JP, Lescroart F, Argiro L, Maurel-Zaffran C, Salgado D, Plaindoux E, De Bono C, Pazur K, Théveniau-Ruissy M, Béroud C, Puceat M, Gavalas A, Kelly RG, Zaffran S.
      Perturbation of addition of second heart field (SHF) cardiac progenitor cells to the poles of the heart tube results in congenital heart defects (CHD). The transcriptional programs and upstream regulatory events operating in different subpopulations of the SHF remain unclear. Here, we profile the transcriptome and chromatin accessibility of anterior and posterior SHF sub-populations at genome-wide levels and demonstrate that Hoxb1 negatively regulates differentiation in the posterior SHF. Spatial mis-expression of Hoxb1 in the anterior SHF results in hypoplastic right ventricle. Activation of Hoxb1 in embryonic stem cells arrests cardiac differentiation, whereas Hoxb1-deficient mouse embryos display premature cardiac differentiation. Moreover, ectopic differentiation in the posterior SHF of embryos lacking both Hoxb1 and its paralog Hoxa1 results in atrioventricular septal defects. Our results show that Hoxb1 plays a key role in patterning cardiac progenitor cells that contribute to both cardiac poles and provide new insights into the pathogenesis of CHD.
    Keywords:  developmental biology; mouse
  22. Cancer Res. 2020 Aug 19. pii: canres.1379.2020. [Epub ahead of print]
    Conway ME, McDaniel JM, Graham JM, Guillen KP, Oliver PG, Parker SL, Yue P, Turkson J, Buchsbaum DJ, Welm BE, Myers RM, Varley KE.
      Breast cancers are divided into subtypes with different prognoses and treatment responses based on global differences in gene expression. Luminal breast cancer gene expression and proliferation are driven by Estrogen Receptor alpha and targeting this transcription factor is the most effective therapy for this subtype. By contrast, it remains unclear which transcription factors drive the gene expression signature that defines basal-like triple-negative breast cancer and there are no targeted therapies approved to treat this aggressive subtype. In this study, we utilized integrated genomic analysis of DNA methylation, chromatin accessibility, transcription factor binding, and gene expression in large collections of breast cancer cell lines and patient tumors to identify transcription factors responsible for the basal-like gene expression program. Glucocorticoid receptor (GR) and signal transducer and activator of transcription 3 (STAT3) bind to the same genomic regulatory regions, which were specifically open and unmethylated in basal-like breast cancer. These transcription factors cooperated to regulate expression of hundreds of genes in the basal-like gene expression signature which were associated with poor prognosis. Combination treatment with small molecule inhibitors of both transcription factors resulted in synergistic decreases in cell growth in cell lines and patient-derived organoid models. This study demonstrates that GR and STAT3 cooperate to regulate the basal-like breast cancer gene expression program and provides the basis for improved therapy for basal-like triple-negative breast cancer through rational combination of STAT3 and GR inhibitors.
  23. Proc Natl Acad Sci U S A. 2020 Aug 19. pii: 202000516. [Epub ahead of print]
    Xiao Y, Thakkar KN, Zhao H, Broughton J, Li Y, Seoane JA, Diep AN, Metzner TJ, von Eyben R, Dill DL, Brooks JD, Curtis C, Leppert JT, Ye J, Peehl DM, Giaccia AJ, Sinha S, Rankin EB.
      Loss of the von Hippel-Lindau (VHL) tumor suppressor is a hallmark feature of renal clear cell carcinoma. VHL inactivation results in the constitutive activation of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 and their downstream targets, including the proangiogenic factors VEGF and PDGF. However, antiangiogenic agents and HIF-2 inhibitors have limited efficacy in cancer therapy due to the development of resistance. Here we employed an innovative computational platform, Mining of Synthetic Lethals (MiSL), to identify synthetic lethal interactions with the loss of VHL through analysis of primary tumor genomic and transcriptomic data. Using this approach, we identified a synthetic lethal interaction between VHL and the m6A RNA demethylase FTO in renal cell carcinoma. MiSL identified FTO as a synthetic lethal partner of VHL because deletions of FTO are mutually exclusive with VHL loss in pan cancer datasets. Moreover, FTO expression is increased in VHL-deficient ccRCC tumors compared to normal adjacent tissue. Genetic inactivation of FTO using multiple orthogonal approaches revealed that FTO inhibition selectively reduces the growth and survival of VHL-deficient cells in vitro and in vivo. Notably, FTO inhibition reduced the survival of both HIF wild type and HIF-deficient tumors, identifying FTO as an HIF-independent vulnerability of VHL-deficient cancers. Integrated analysis of transcriptome-wide m6A-seq and mRNA-seq analysis identified the glutamine transporter SLC1A5 as an FTO target that promotes metabolic reprogramming and survival of VHL-deficient ccRCC cells. These findings identify FTO as a potential HIF-independent therapeutic target for the treatment of VHL-deficient renal cell carcinoma.
    Keywords:  FTO; SLC1A5; kidney cancer; synthetic lethality; von Hippel–Lindau
  24. Nat Neurosci. 2020 Aug 17.
    Marshall LL, Killinger BA, Ensink E, Li P, Li KX, Cui W, Lubben N, Weiland M, Wang X, Gordevicius J, Coetzee GA, Ma J, Jovinge S, Labrie V.
      Parkinson's disease (PD) pathogenesis may involve the epigenetic control of enhancers that modify neuronal functions. Here, we comprehensively examine DNA methylation at enhancers, genome-wide, in neurons of patients with PD and of control individuals. We find a widespread increase in cytosine modifications at enhancers in PD neurons, which is partly explained by elevated hydroxymethylation levels. In particular, patients with PD exhibit an epigenetic and transcriptional upregulation of TET2, a master-regulator of cytosine modification status. TET2 depletion in a neuronal cell model results in cytosine modification changes that are reciprocal to those observed in PD neurons. Moreover, Tet2 inactivation in mice fully prevents nigral dopaminergic neuronal loss induced by previous inflammation. Tet2 loss also attenuates transcriptional immune responses to an inflammatory trigger. Thus, widespread epigenetic dysregulation of enhancers in PD neurons may, in part, be mediated by increased TET2 expression. Decreased Tet2 activity is neuroprotective, in vivo, and may be a new therapeutic target for PD.
  25. Genome Res. 2020 Aug 17.
    Nip KM, Chiu R, Yang C, Chu J, Mohamadi H, Warren RL, Birol I.
      Despite the rapid advance in single-cell RNA sequencing (scRNA-seq) technologies within the last decade, single-cell transcriptome analysis workflows have primarily used gene expression data while isoform sequence analysis at the single-cell level still remains fairly limited. Detection and discovery of isoforms in single cells is difficult because of the inherent technical shortcomings of scRNA-seq data, and existing transcriptome assembly methods are mainly designed for bulk RNA samples. To address this challenge, we developed RNA-Bloom, an assembly algorithm that leverages the rich information content aggregated from multiple single-cell transcriptomes to reconstruct cell-specific isoforms. Assembly with RNA-Bloom can be either reference-guided or reference-free, thus enabling unbiased discovery of novel isoforms or foreign transcripts. We compared both assembly strategies of RNA-Bloom against five state-of-the-art reference-free and reference-based transcriptome assembly methods. In our benchmarks on a simulated 384-cell data set, reference-free RNA-Bloom reconstructed 37.9%-38.3% more isoforms than the best reference-free assembler, whereas reference-guided RNA-Bloom reconstructed 4.1%-11.6% more isoforms than reference-based assemblers. When applied to a real 3840-cell data set consisting of more than 4 billion reads, RNA-Bloom reconstructed 9.7%-25.0% more isoforms than the best competing reference-based and reference-free approaches evaluated. We expect RNA-Bloom to boost the utility of scRNA-seq data beyond gene expression analysis, expanding what is informatically accessible now.
  26. Bioinformatics. 2020 Aug 19. pii: btaa735. [Epub ahead of print]
    Gupta R, Schrooders Y, Verheijen M, Roth A, Kleinjans J, Caiment F.
      SUMMARY: Typical RNA-Seq analyses are performed either at the gene level by summing all reads from the same locus, assuming that all transcripts from a gene make a protein or at the transcript level, assuming that each transcript displays unique function. However, these assumptions are flawed, as a gene can code for different types of transcripts and different transcripts are capable of synthesizing similar, different, or no protein. As a consequence, functional changes are not well illustrated by either gene or transcript analyses. We propose to improve RNA-Seq analyses by grouping the transcripts based on their similar functions. We developed FuSe to predict functional similarities using the primary and secondary structure of proteins. To estimate the likelihood of proteins with similar functions, FuSe computes two confidence scores: knowledge (KS) and discovery (DS) for protein pairs. Overlapping protein pairs exhibiting high confidence are grouped to form 'similar function protein groups' and expression is calculated for each functional group. The impact of using FuSe is demonstrated on in vitro cells exposed to paracetamol, which highlight genes responsible for cell adhesion and glycogen regulation which were earlier shown to be not differentially expressed with traditional analysis methods.AVAILABILITY: The source code is available at Data for APAP exposure are available in the BioStudies database ( under accession numbers S-HECA143, S-HECA(158), and S-HECA139.
    SUPPLEMENTARY INFORMATION: Data are available at Oxford Bioinformatics online.
  27. Nucleic Acids Res. 2020 Aug 21. pii: gkaa692. [Epub ahead of print]
    Tang Y, Chen K, Song B, Ma J, Wu X, Xu Q, Wei Z, Su J, Liu G, Rong R, Lu Z, de Magalhães JP, Rigden DJ, Meng J.
      N 6-Methyladenosine (m6A) is the most prevalent RNA modification on mRNAs and lncRNAs. It plays a pivotal role during various biological processes and disease pathogenesis. We present here a comprehensive knowledgebase, m6A-Atlas, for unraveling the m6A epitranscriptome. Compared to existing databases, m6A-Atlas features a high-confidence collection of 442 162 reliable m6A sites identified from seven base-resolution technologies and the quantitative (rather than binary) epitranscriptome profiles estimated from 1363 high-throughput sequencing samples. It also offers novel features, such as; the conservation of m6A sites among seven vertebrate species (including human, mouse and chimp), the m6A epitranscriptomes of 10 virus species (including HIV, KSHV and DENV), the putative biological functions of individual m6A sites predicted from epitranscriptome data, and the potential pathogenesis of m6A sites inferred from disease-associated genetic mutations that can directly destroy m6A directing sequence motifs. A user-friendly graphical user interface was constructed to support the query, visualization and sharing of the m6A epitranscriptomes annotated with sites specifying their interaction with post-transcriptional machinery (RBP-binding, microRNA interaction and splicing sites) and interactively display the landscape of multiple RNA modifications. These resources provide fresh opportunities for unraveling the m6A epitranscriptomes. m6A-Atlas is freely accessible at: