bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2020‒04‒19
forty-six papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit


  1. Elife. 2020 Apr 16. pii: e47333. [Epub ahead of print]9
    Criqui M, Qamra A, Chu TW, Sharma M, Tsao J, Henry DA, Barsyte-Lovejoy D, Arrowsmith CH, Winegarden N, Lupien M, Harrington L.
      The precise relationship between epigenetic perturbations and telomere dysfunction is an extant question. Previously, we showed that telomere dysfunction leads to differentiation instability in murine embryonic stem cells (mESCs) via perturbations in DNA methylation at pluripotency-factor promoters. Here, we uncovered that telomerase reverse transcriptase null (Tert-/-) mESCs exhibit genome-wide perturbations in chromatin accessibility and gene expression during differentiation. These changes were accompanied by an increase of H3K27me3 globally, an altered chromatin landscape at the Pou5f1/Oct4 pluripotency gene promoter, and impaired Tert-/- mESC differentiation. Inhibition of the Polycomb Repressive Complex 2 (PRC2), an H3K27 tri-methyltransferase, exacerbated the impairment in differentiation and pluripotency gene repression in Tert-/- mESCs but not wild-type mESCs, whereas inhibition of H3K27me3 demethylation led to a partial rescue of the Tert-/- phenotype. This data reveals a new interdependent relationship between H3K27me3 and telomere integrity in stem cell lineage commitment that may have implications in aging and cancer.
    Keywords:  chromosomes; gene expression; mouse
    DOI:  https://doi.org/10.7554/eLife.47333
  2. Nucleic Acids Res. 2020 Apr 14. pii: gkaa213. [Epub ahead of print]
    Li W, Karwacki-Neisius V, Ma C, Tan L, Shi Y, Wu F, Shi YG.
      NONO is a DNA/RNA-binding protein, which plays a critical regulatory role during cell stage transitions of mouse embryonic stem cells (mESCs). However, its function in neuronal lineage commitment and the molecular mechanisms of its action in such processes are largely unknown. Here we report that NONO plays a key role during neuronal differentiation of mESCs. Nono deletion impedes neuronal lineage commitment largely due to a failure of up-regulation of specific genes critical for neuronal differentiation. Many of the NONO regulated genes are also DNA demethylase TET1 targeted genes. Importantly, re-introducing wild type NONO to the Nono KO cells, not only restores the normal expression of the majority of NONO/TET1 coregulated genes but also rescues the defective neuronal differentiation of Nono-deficient mESCs. Mechanistically, our data shows that NONO directly interacts with TET1 via its DNA binding domain and recruits TET1 to genomic loci to regulate 5-hydroxymethylcytosine levels. Nono deletion leads to a significant dissociation of TET1 from chromatin and dysregulation of DNA hydroxymethylation of neuronal genes. Taken together, our findings reveal a key role and an epigenetic mechanism of action of NONO in regulation of TET1-targeted neuronal genes, offering new functional and mechanistic understanding of NONO in stem cell functions, lineage commitment and specification.
    DOI:  https://doi.org/10.1093/nar/gkaa213
  3. Nat Commun. 2020 Apr 14. 11(1): 1813
    Chen M, Zhu Q, Li C, Kou X, Zhao Y, Li Y, Xu R, Yang L, Yang L, Gu L, Wang H, Liu X, Jiang C, Gao S.
      The oocyte cytoplasm can reprogram the somatic cell nucleus into a totipotent state, but with low efficiency. The spatiotemporal chromatin organization of somatic cell nuclear transfer (SCNT) embryos remains elusive. Here, we examine higher order chromatin structures of mouse SCNT embryos using a low-input Hi-C method. We find that donor cell chromatin transforms to the metaphase state rapidly after SCNT along with the dissolution of typical 3D chromatin structure. Intriguingly, the genome undergoes a mitotic metaphase-like to meiosis metaphase II-like transition following activation. Subsequently, weak chromatin compartments and topologically associating domains (TADs) emerge following metaphase exit. TADs are further removed until the 2-cell stage before being progressively reestablished. Obvious defects including stronger TAD boundaries, aberrant super-enhancer and promoter interactions are found in SCNT embryos. These defects are partially caused by inherited H3K9me3, and can be rescued by Kdm4d overexpression. These observations provide insight into chromatin architecture reorganization during SCNT embryo development.
    DOI:  https://doi.org/10.1038/s41467-020-15607-z
  4. Epigenetics. 2020 Apr 14. 1-22
    Luz-Madrigal A, Grajales-Esquivel E, Tangeman J, Kosse S, Liu L, Wang K, Fausey A, Liang C, Tsonis PA, Del Rio-Tsonis K.
      Cellular reprogramming resets the epigenetic landscape to drive shifts in transcriptional programmes and cell identity. The embryonic chick can regenerate a complete neural retina, after retinectomy, via retinal pigment epithelium (RPE) reprogramming in the presence of FGF2. In this study, we systematically analysed the reprogramming competent chick RPE prior to injury, and during different stages of reprogramming. In addition to changes in the expression of genes associated with epigenetic modifications during RPE reprogramming, we observed dynamic changes in histone marks associated with bivalent chromatin (H3K27me3/H3K4me3) and intermediates of the process of DNA demethylation including 5hmC and 5caC. Comprehensive analysis of the methylome by whole-genome bisulphite sequencing (WGBS) confirmed extensive rearrangements of DNA methylation patterns including differentially methylated regions (DMRs) found at promoters of genes associated with chromatin organization and fibroblast growth factor production. We also identified Tet methylcytosine dioxygenase 3 (TET3) as an important factor for DNA demethylation and retina regeneration, capable of reprogramming RPE in the absence of exogenous FGF2. In conclusion, we demonstrate that injury early in RPE reprogramming triggers genome-wide dynamic changes in chromatin, including bivalent chromatin and DNA methylation. In the presence of FGF2, these dynamic modifications are further sustained in the commitment to form a new retina. Our findings reveal active DNA demethylation as an important process that may be applied to remove the epigenetic barriers in order to regenerate retina in mammals.Abbreviations: bp: Base pair; DMR: Differentially methylated region; DMC: Differentially methylated cytosines; GFP: Green fluorescent protein; PCR: Polymerase chain reaction. TET: Ten-eleven translocation; RPE: retinal pigment epithelium.
    Keywords:  Retina regeneration; demethylation; reprogramming; retinal pigment epithelium; tet3
    DOI:  https://doi.org/10.1080/15592294.2020.1747742
  5. Nat Commun. 2020 Apr 14. 11(1): 1796
    Diehl AG, Ouyang N, Boyle AP.
      Chromatin looping is important for gene regulation, and studies of 3D chromatin structure across species and cell types have improved our understanding of the principles governing chromatin looping. However, 3D genome evolution and its relationship with natural selection remains largely unexplored. In mammals, the CTCF protein defines the boundaries of most chromatin loops, and variations in CTCF occupancy are associated with looping divergence. While many CTCF binding sites fall within transposable elements (TEs), their contribution to 3D chromatin structural evolution is unknown. Here we report the relative contributions of TE-driven CTCF binding site expansions to conserved and divergent chromatin looping in human and mouse. We demonstrate that TE-derived CTCF binding divergence may explain a large fraction of variable loops. These variable loops contribute significantly to corresponding gene expression variability across cells and species, possibly by refining sub-TAD-scale loop contacts responsible for cell-type-specific enhancer-promoter interactions.
    DOI:  https://doi.org/10.1038/s41467-020-15520-5
  6. iScience. 2020 Mar 30. pii: S2589-0042(20)30202-9. [Epub ahead of print]23(4): 101018
    Romano O, Petiti L, Felix T, Meneghini V, Portafax M, Antoniani C, Amendola M, Bicciato S, Peano C, Miccio A.
      Erythroid commitment and differentiation are regulated by the coordinated action of a host of transcription factors, including GATA2 and GATA1. Here, we explored GATA-mediated transcriptional regulation through the integrative analysis of gene expression, chromatin modifications, and GATA factors' binding in human multipotent hematopoietic stem/progenitor cells, early erythroid progenitors, and late precursors. A progressive loss of H3K27 acetylation and a diminished usage of active enhancers and super-enhancers were observed during erythroid commitment and differentiation. GATA factors mediate transcriptional changes through a stage-specific interplay with regulatory elements: GATA1 binds different sets of regulatory elements in erythroid progenitors and precursors and controls the transcription of distinct genes during commitment and differentiation. Importantly, our results highlight a pivotal role of promoters in determining the transcriptional program activated upon erythroid differentiation. Finally, we demonstrated that GATA1 binding to a stage-specific super-enhancer sustains the expression of the KIT receptor in human erythroid progenitors.
    Keywords:  Biological Sciences; Cell Biology; Molecular Biology; Molecular Mechanism of Gene Regulation
    DOI:  https://doi.org/10.1016/j.isci.2020.101018
  7. Sci Adv. 2020 04;6(15): eaax5150
    Milstone ZJ, Saheera S, Bourke LM, Shpilka T, Haynes CM, Trivedi CM.
      Cryptic transcription occurs widely across the eukaryotic genome; however, its regulation during vertebrate development is not understood. Here, we show that two class I histone deacetylases, Hdac1 and Hdac2, silence cryptic transcription to promote mitochondrial function in developing murine hearts. Mice lacking Hdac1 and Hdac2 in heart exhibit defective developmental switch from anaerobic to mitochondrial oxidative phosphorylation (OXPHOS), severe defects in mitochondrial mass, mitochondrial function, and complete embryonic lethality. Hdac1/Hdac2 promotes the transition to OXPHOS by enforcing transcriptional fidelity of metabolic gene programs. Mechanistically, Hdac1/Hdac2 deacetylates histone residues including H3K23, H3K14, and H4K16 to suppress cryptic transcriptional initiation within the coding regions of actively transcribed metabolic genes. Thus, Hdac1/2-mediated epigenetic silencing of cryptic transcription is essential for mitochondrial function during early vertebrate development.
    DOI:  https://doi.org/10.1126/sciadv.aax5150
  8. Cell Rep. 2020 Apr 14. pii: S2211-1247(20)30393-4. [Epub ahead of print]31(2): 107503
    Justice M, Carico ZM, Stefan HC, Dowen JM.
      Chromosome structure is a key regulator of gene expression. CTCF and cohesin play critical roles in structuring chromosomes by mediating physical interactions between distant genomic sites. The resulting DNA loops often contain genes and their cis-regulatory elements. Despite the importance of DNA loops in maintaining proper transcriptional regulation and cell identity, there is limited understanding of the molecular mechanisms that regulate their dynamics and function. We report a previously unrecognized role for WIZ (widely interspaced zinc finger-containing protein) in DNA loop architecture and regulation of gene expression. WIZ forms a complex with cohesin and CTCF that occupies enhancers, promoters, insulators, and anchors of DNA loops. Aberrant WIZ function alters cohesin occupancy and increases the number of DNA loop structures in the genome. WIZ is required for proper gene expression and transcriptional insulation. Our results uncover an unexpected role for WIZ in DNA loop architecture, transcriptional control, and maintenance of cell identity.
    Keywords:  CTCF; DNA loop; WIZ; cellular identity; cohesin; gene expression; genome organization; stem cell; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2020.03.067
  9. Elife. 2020 Apr 17. pii: e52962. [Epub ahead of print]9
    Amândio AR, Lopez-Delisle L, Bolt CC, Mascrez B, Duboule D.
      Developmental genes are often controlled by large regulatory landscapes matching topologically associating domains (TADs). In various contexts, the associated chromatin backbone is modified by specific enhancer-enhancer and enhancer-promoter interactions. We used a TAD flanking the mouse HoxD cluster to study how these regulatory architectures are formed and deconstructed once their function achieved. We describe this TAD as a functional unit, with several regulatory sequences acting together to elicit a transcriptional response. With one exception, deletion of these sequences didn't modify the transcriptional outcome, a result at odds with a conventional view of enhancer function. The deletion and inversion of a CTCF site located near these regulatory sequences did not affect transcription of the target gene. Slight modifications were nevertheless observed, in agreement with the loop extrusion model. We discuss these unexpected results considering both conventional and alternative explanations relying on the accumulation of poorly specific factors within the TAD backbone.
    Keywords:  developmental biology; mouse
    DOI:  https://doi.org/10.7554/eLife.52962
  10. Oncogene. 2020 Apr 14.
    Liu C, Liu L, Wang K, Li XF, Ge LY, Ma RZ, Fan YD, Li LC, Liu ZF, Qiu M, Hao YC, Shi ZF, Xia CY, Strååt K, Huang Y, Ma LL, Xu D.
      It has been well established that the von Hippel-Lindau/hypoxia-inducible factor α (VHL-HIFα) axis and epidermal growth factor receptor (EGFR) signaling pathway play a critical role in the pathogenesis and progression of renal cell carcinoma (RCC). However, few studies have addressed the relationship between the two oncogenic drivers in RCC. SET and MYND domain-containing protein 3 (SMYD3) is a histone methyltransferase involved in gene transcription and oncogenesis, but its expression and function in RCC remain unclear. In the present study, we found that SMYD3 expression was significantly elevated in RCC tumors and correlated with advanced tumor stage, histological and nuclear grade, and shorter survival. Depletion of SMYD3 inhibited RCC cell proliferation, colony numbers, and xenograft tumor formation, while promoted apoptosis. Mechanistically, SMYD3 cooperates with SP1 to transcriptionally promote EGFR expression, amplifying its downstream signaling activity. TCGA data analyses revealed a significantly increased SMYD3 expression in primary RCC tumors carrying the loss-of-function VHL mutations. We further showed that HIF-2α can directly bind to the SMYD3 promoter and subsequently induced SMYD3 transcription and expression. Taken together, we identify the VHL/HIF-2α/SMYD3 signaling cascade-mediated EGFR hyperactivity through which SMYD3 promotes RCC progression. Our study suggests that SMYD3 is a potential therapeutic target and prognostic factor in RCC.
    DOI:  https://doi.org/10.1038/s41388-020-1291-7
  11. Genome Biol. 2020 Mar 23. 21(1): 75
    Jia Z, Li J, Ge X, Wu Y, Guo Y, Wu Q.
      BACKGROUND: CTCF is a key insulator-binding protein, and mammalian genomes contain numerous CTCF sites, many of which are organized in tandem.RESULTS: Using CRISPR DNA-fragment editing, in conjunction with chromosome conformation capture, we find that CTCF sites, if located between enhancers and promoters in the protocadherin (Pcdh) and β-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CTCF sites or not. Moreover, computational simulation in silico and genetic deletions in vivo as well as dCas9 blocking in vitro revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem CTCF sites in the Pcdh and immunoglobulin heavy chain (Igh) clusters. Furthermore, CTCF insulators promote, counter-intuitively, long-range chromatin interactions with distal directional CTCF sites, consistent with the cohesin "loop extrusion" model. Finally, gene expression levels are negatively correlated with CTCF insulators located between enhancers and promoters on a genome-wide scale. Thus, single CTCF insulators ensure proper enhancer insulation and promoter activation while tandem CTCF topological insulators determine balanced spatial contacts and promoter choice.
    CONCLUSIONS: These findings have interesting implications on the role of topological chromatin insulators in 3D genome folding and developmental gene regulation.
    Keywords:  3D genome; Bayesian networks; CTCF; Chromatin polymer simulation; Cohesin; Gene regulation; Insulator; Loop extrusion; Promoter/enhancer selection; Topological spatial contacts
    DOI:  https://doi.org/10.1186/s13059-020-01984-7
  12. Nat Commun. 2020 Apr 14. 11(1): 1805
    Holmes ZE, Hamilton DJ, Hwang T, Parsonnet NV, Rinn JL, Wuttke DS, Batey RT.
      Certain transcription factors are proposed to form functional interactions with RNA to facilitate proper regulation of gene expression. Sox2, a transcription factor critical for maintenance of pluripotency and neurogenesis, has been found associated with several lncRNAs, although it is unknown whether these interactions are direct or via other proteins. Here we demonstrate that human Sox2 interacts directly with one of these lncRNAs with high affinity through its HMG DNA-binding domain in vitro. These interactions are primarily with double-stranded RNA in a non-sequence specific fashion, mediated by a similar but not identical interaction surface. We further determined that Sox2 directly binds RNA in mouse embryonic stem cells by UV-cross-linked immunoprecipitation of Sox2 and more than a thousand Sox2-RNA interactions in vivo were identified using fRIP-seq. Together, these data reveal that Sox2 employs a high-affinity/low-specificity paradigm for RNA binding in vitro and in vivo.
    DOI:  https://doi.org/10.1038/s41467-020-15571-8
  13. Nucleic Acids Res. 2020 Apr 16. pii: gkaa234. [Epub ahead of print]
    Vaid R, Wen J, Mannervik M.
      A correlation between histone acetylation and transcription has been noted for a long time, but little is known about what step(s) in the transcription cycle is influenced by acetylation. We have examined the immediate transcriptional response to histone deacetylase (HDAC) inhibition, and find that release of promoter-proximal paused RNA polymerase II (Pol II) into elongation is stimulated, whereas initiation is not. Although histone acetylation is elevated globally by HDAC inhibition, less than 100 genes respond within 10 min. These genes are highly paused, are strongly associated with the chromatin regulators NURF and Trithorax, display a greater increase in acetylation of the first nucleosomes than other genes, and become transcriptionally activated by HDAC inhibition. Among these rapidly up-regulated genes are HDAC1 (Rpd3) and subunits of HDAC-containing co-repressor complexes, demonstrating feedback regulation upon HDAC inhibition. Our results suggest that histone acetylation stimulates transcription of paused genes by release of Pol II into elongation, and that increased acetylation is not a consequence of their enhanced expression. We propose that HDACs are major regulators of Pol II pausing and that this partly explains the presence of HDACs at active genes.
    DOI:  https://doi.org/10.1093/nar/gkaa234
  14. Cell Rep. 2020 Apr 14. pii: S2211-1247(20)30399-5. [Epub ahead of print]31(2): 107509
    Dinh TA, Sritharan R, Smith FD, Francisco AB, Ma RK, Bunaciu RP, Kanke M, Danko CG, Massa AP, Scott JD, Sethupathy P.
      Fibrolamellar carcinoma (FLC) is a rare, therapeutically intractable liver cancer that disproportionately affects youth. Although FLC tumors exhibit a distinct gene expression profile, the chromatin regulatory landscape and the genes most critical for tumor cell survival remain unclear. Here, we use chromatin run-on sequencing to discover ∼7,000 enhancers and 141 enhancer hotspots activated in FLC relative to nonmalignant liver. Bioinformatic analyses reveal aberrant ERK/MEK signaling and candidate master transcriptional regulators. We also define the genes most strongly associated with hotspots of FLC enhancer activity, including CA12 and SLC16A14. Treatment of FLC cell models with inhibitors of CA12 or SLC16A14 independently reduce cell viability and/or significantly enhance the effect of the MEK inhibitor cobimetinib. These findings highlight molecular targets for drug development, as well as drug combination approaches.
    Keywords:  MAPK; cancer; drug resistance; enhancers; fibrolamellar carcinoma; genomics
    DOI:  https://doi.org/10.1016/j.celrep.2020.03.073
  15. Elife. 2020 Apr 16. pii: e53659. [Epub ahead of print]9
    Kuang Y, Golan O, Preusse K, Cain B, Christensen CJ, Salomone J, Campbell I, Okwubido-Williams FV, Hass MR, Yuan Z, Eafergan N, Moberg KH, Kovall RA, Kopan R, Sprinzak D, Gebelein B.
      Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, we have a limited mechanistic understanding of phenotype variability due to gene dosage. Here, we unexpectedly found that inserting an enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can induce a subset of Notch haploinsufficiency phenotypes in Drosophila with wild type Notch gene dose. Using Drosophila genetics, we show that this enhancer induces Notch phenotypes in a Cdk8-dependent, transcription-independent manner. We further combined mathematical modeling with quantitative trait and expression analysis to build a model that describes how changes in Notch signal production versus degradation differentially impact cellular outcomes that require long versus short signal duration. Altogether, these findings support a 'bind and discard' mechanism in which enhancers with specific binding sites promote rapid Cdk8-dependent Notch turnover, and thereby reduce Notch-dependent transcription at other loci and sensitize tissues to gene dose based upon signal duration.
    Keywords:  D. melanogaster; developmental biology
    DOI:  https://doi.org/10.7554/eLife.53659
  16. Mol Cell. 2020 Apr 04. pii: S1097-2765(20)30191-X. [Epub ahead of print]
    Gerber A, Ito K, Chu CS, Roeder RG.
      Increasing evidence suggests that tRNA levels are dynamically and specifically regulated in response to internal and external cues to modulate the cellular translational program. However, the molecular players and the mechanisms regulating the gene-specific expression of tRNAs are still unknown. Using an inducible auxin-degron system to rapidly deplete RPB1 (the largest subunit of RNA Pol II) in living cells, we identified Pol II as a direct gene-specific regulator of tRNA transcription. Our data suggest that Pol II transcription robustly interferes with Pol III function at specific tRNA genes. This activity was further found to be essential for MAF1-mediated repression of a large set of tRNA genes during serum starvation, indicating that repression of tRNA genes by Pol II is dynamically regulated. Hence, Pol II plays a direct and central role in the gene-specific regulation of tRNA expression.
    Keywords:  RNA Pol II; RNA Pol III; auxin degron; tRNA differential expression
    DOI:  https://doi.org/10.1016/j.molcel.2020.03.023
  17. Mol Cell. 2020 Apr 04. pii: S1097-2765(20)30190-8. [Epub ahead of print]
    Slobodin B, Bahat A, Sehrawat U, Becker-Herman S, Zuckerman B, Weiss AN, Han R, Elkon R, Agami R, Ulitsky I, Shachar I, Dikstein R.
      Gene expression is regulated by the rates of synthesis and degradation of mRNAs, but how these processes are coordinated is poorly understood. Here, we show that reduced transcription dynamics of specific genes leads to enhanced m6A deposition, preferential activity of the CCR4-Not complex, shortened poly(A) tails, and reduced stability of the respective mRNAs. These effects are also exerted by internal ribosome entry site (IRES) elements, which we found to be transcriptional pause sites. However, when transcription dynamics, and subsequently poly(A) tails, are globally altered, cells buffer mRNA levels by adjusting the expression of mRNA degradation machinery. Stress-provoked global impediment of transcription elongation leads to a dramatic inhibition of the mRNA degradation machinery and massive mRNA stabilization. Accordingly, globally enhanced transcription, such as following B cell activation or glucose stimulation, has the opposite effects. This study uncovers two molecular pathways that maintain balanced gene expression in mammalian cells by linking transcription to mRNA stability.
    Keywords:  CCR4-Not complex; IRES; RNA polymerase II; gene expression buffering; m(6)A; m6A methyltransferase complex; mRNA buffering; mRNA degradation; poly(A) tails; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2020.03.022
  18. J Am Chem Soc. 2020 Apr 14.
    Palei S, Buchmuller B, Wolffgramm J, Muñoz-Lopez Á, Jung S, Czodrowski P, Summerer D.
      Ten-eleven-translocation (TET) dioxygenases catalyze the oxidation of 5-methylcytosine (5mC), the central epigenetic regulator of mammalian DNA. This activity dynamically reshapes the epigenome and transcriptome by depositing oxidized 5mC derivatives and initiating active DNA demethylation. However, studying this dynamic is hampered by the inability to selectively activate individual TETs with temporal control in cells. We report activation of TETs in mammalian cells by incorporation of genetically encoded 4,5-dimethoxy-2-nitrobenzyl-l-serine as a transient active-site block, and its subsequent deprotection with light. Our approach enables precise insights into the impact of cancer-associated TET2 mutations on the kinetics of TET2 catalysis in vivo, and allows time-resolved monitoring of target gene activation and transcriptome reorganization. This sets a basis for dissecting the order and kinetics of chromatin-associated events triggered by TET catalysis, ranging from DNA demethylation to chromatin and transcription regulation.
    DOI:  https://doi.org/10.1021/jacs.0c01193
  19. Mol Oncol. 2020 Apr 14.
    Wu J, Chai H, Xu X, Yu J, Gu Y.
      Growing tumors alter their metabolic profiles to support the increased cell proliferation. SETD1A, a histone lysine methyltransferase which specifically methylates H3K4, plays important roles in both normal cell and cancer cell functions. However, the function of SETD1A in gastric cancer (GC) progression and its role in GC metabolic reprogramming are still largely unknown. In the current study, we discovered that the expression of SETD1A was higher in GC tumor specimens compared to surrounding non-tumor tissues. Upregulation of SETD1A increased GC cell proliferation, whereas downregulation of SETD1A inhibited GC cell proliferation. Furthermore, knockdown of SETD1A reduced glucose uptake and production of lactate, and suppressed glycolysis by decreasing the expression of glycolytic genes, including GLUT1, HK2, PFK2, PKM2, LDHA and MCT4. Mechanistically, SETD1A interacted with HIF1α to strengthen its transactivation, indicating that SETD1A promotes glycolysis through coactivation of HIF1α. SETD1A and HIF1α were recruited to the promoter of HK2 and PFK2, where SETD1A could methylate H3K4. However, knockdown of SETD1A decreased the methylation of H3K4 on HK2 and PFK2 promoter, and reduced HIF1α recruitment necessary to promote transcription of glycolytic genes. Inhibition of HIF1α decelerated SETD1A-enhanced GC cell growth. In additional, there was a linear correlation between SETD1A and several key glycolytic genes in human GC specimens obtained from TCGA dataset. Thus, our results demonstrated that SETD1A interacted with HIF1α to promote glycolysis and accelerate GC progression, implicating that SETD1A may be a potential molecular target for GC treatment.
    Keywords:  HIF1α; Histone methyltransferase SETD1A; gastric cancer; progression glycolysis
    DOI:  https://doi.org/10.1002/1878-0261.12689
  20. Cell Stem Cell. 2020 Apr 15. pii: S1934-5909(20)30097-7. [Epub ahead of print]
    Gunne-Braden A, Sullivan A, Gharibi B, Sheriff RSM, Maity A, Wang YF, Edwards A, Jiang M, Howell M, Goldstone R, Wollman R, East P, Santos SDM.
      During early development, extrinsic triggers prompt pluripotent cells to begin the process of differentiation. When and how human embryonic stem cells (hESCs) irreversibly commit to differentiation is a fundamental yet unanswered question. By combining single-cell imaging, genomic approaches, and mathematical modeling, we find that hESCs commit to exiting pluripotency unexpectedly early. We show that bone morphogenetic protein 4 (BMP4), an important differentiation trigger, induces a subset of early genes to mirror the sustained, bistable dynamics of upstream signaling. Induction of one of these genes, GATA3, drives differentiation in the absence of BMP4. Conversely, GATA3 knockout delays differentiation and prevents fast commitment to differentiation. We show that positive feedback at the level of the GATA3-BMP4 axis induces fast, irreversible commitment to differentiation. We propose that early commitment may be a feature of BMP-driven fate choices and that interlinked feedback is the molecular basis for an irreversible transition from pluripotency to differentiation.
    Keywords:  BMP4; GATA3; bistability; commitment; differentiation; fate decisions; hESC; positive feedback
    DOI:  https://doi.org/10.1016/j.stem.2020.03.005
  21. Cell. 2020 Apr 09. pii: S0092-8674(20)30345-7. [Epub ahead of print]
    Nava MM, Miroshnikova YA, Biggs LC, Whitefield DB, Metge F, Boucas J, Vihinen H, Jokitalo E, Li X, García Arcos JM, Hoffmann B, Merkel R, Niessen CM, Dahl KN, Wickström SA.
      Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.
    Keywords:  DNA damage; chromatin; heterochromatin; mechanoprotection; mechanotransduction; nuclear architecture; nuclear lamina; nuclear mechanics; stem cells
    DOI:  https://doi.org/10.1016/j.cell.2020.03.052
  22. Sci Adv. 2020 04;6(15): eaax2746
    Ravasio R, Ceccacci E, Nicosia L, Hosseini A, Rossi PL, Barozzi I, Fornasari L, Zuffo RD, Valente S, Fioravanti R, Mercurio C, Varasi M, Mattevi A, Mai A, Pavesi G, Bonaldi T, Minucci S.
      The histone demethylase LSD1 is deregulated in several tumors, including leukemias, providing the rationale for the clinical use of LSD1 inhibitors. In acute promyelocytic leukemia (APL), pharmacological doses of retinoic acid (RA) induce differentiation of APL cells, triggering degradation of the PML-RAR oncogene. APL cells are resistant to LSD1 inhibition or knockout, but targeting LSD1 sensitizes them to physiological doses of RA without altering of PML-RAR levels, and extends survival of leukemic mice upon RA treatment. The combination of RA with LSD1 inhibition (or knockout) is also effective in other non-APL, acute myeloid leukemia (AML) cells. Nonenzymatic activities of LSD1 are essential to block differentiation, while RA with targeting of LSD1 releases a differentiation gene expression program, not strictly dependent on changes in histone H3K4 methylation. Integration of proteomic/epigenomic/mutational studies showed that LSD1 inhibitors alter the recruitment of LSD1-containing complexes to chromatin, inhibiting the interaction between LSD1 and the transcription factor GFI1.
    DOI:  https://doi.org/10.1126/sciadv.aax2746
  23. Nat Commun. 2020 Apr 15. 11(1): 1851
    Stojic L, Lun ATL, Mascalchi P, Ernst C, Redmond AM, Mangei J, Barr AR, Bousgouni V, Bakal C, Marioni JC, Odom DT, Gergely F.
      Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types. We perform transcriptome analysis of linc00899-depleted cells and identify the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. We further show that linc00899 binds TPPP/p25 and suppresses its transcription. In cells depleted of linc00899, upregulation of TPPP/p25 alters microtubule dynamics and delays mitosis. Overall, our comprehensive screen uncovers several lncRNAs involved in genome stability and reveals a lncRNA that controls microtubule behaviour with functional implications beyond cell division.
    DOI:  https://doi.org/10.1038/s41467-020-14978-7
  24. Elife. 2020 Apr 16. pii: e57519. [Epub ahead of print]9
    Mondal B, Jin H, Kallappagoudar S, Sedkov Y, Martinez T, Sentmanat MF, Poet GJ, Li C, Fan Y, Pruett-Miller SM, Herz HM.
      The mitotic deacetylase complex (MiDAC) is a recently identified histone deacetylase (HDAC) complex. While other HDAC complexes have been implicated in neurogenesis, the physiological role of MiDAC remains unknown. Here, we show that MiDAC constitutes an important regulator of neural differentiation. We demonstrate that MiDAC functions as a modulator of a neurodevelopmental gene expression program and binds to important regulators of neurite outgrowth. MiDAC upregulates gene expression of pro-neural genes such as those encoding the secreted ligands SLIT3 and NETRIN1 (NTN1) by a mechanism suggestive of H4K20ac removal on promoters and enhancers. Conversely, MiDAC inhibits gene expression by reducing H3K27ac on promoter-proximal and -distal elements of negative regulators of neurogenesis. Furthermore, loss of MiDAC results in neurite outgrowth defects that can be rescued by supplementation with SLIT3 and/or NTN1. These findings indicate a crucial role for MiDAC in regulating the ligands of the SLIT3 and NTN1 signaling axes to ensure the proper integrity of neurite development.
    Keywords:  chromosomes; gene expression; mouse
    DOI:  https://doi.org/10.7554/eLife.57519
  25. Nat Commun. 2020 Apr 14. 11(1): 1827
    Bywater MJ, Burkhart DL, Straube J, Sabò A, Pendino V, Hudson JE, Quaife-Ryan GA, Porrello ER, Rae J, Parton RG, Kress TR, Amati B, Littlewood TD, Evan GI, Wilson CH.
      It is unclear why some tissues are refractory to the mitogenic effects of the oncogene Myc. Here we show that Myc activation induces rapid transcriptional responses followed by proliferation in some, but not all, organs. Despite such disparities in proliferative response, Myc is bound to DNA at open elements in responsive (liver) and non-responsive (heart) tissues, but fails to induce a robust transcriptional and proliferative response in the heart. Using heart as an exemplar of a non-responsive tissue, we show that Myc-driven transcription is re-engaged in mature cardiomyocytes by elevating levels of the positive transcription elongation factor (P-TEFb), instating a large proliferative response. Hence, P-TEFb activity is a key limiting determinant of whether the heart is permissive for Myc transcriptional activation. These data provide a greater understanding of how Myc transcriptional activity is determined and indicate modification of P-TEFb levels could be utilised to drive regeneration of adult cardiomyocytes for the treatment of heart myopathies.
    DOI:  https://doi.org/10.1038/s41467-020-15552-x
  26. Nat Commun. 2020 Apr 14. 11(1): 1822
    Zhang F, Ma D, Zhao W, Wang D, Liu T, Liu Y, Yang Y, Liu Y, Mu J, Li B, Zhang Y, Pan Y, Guo C, Du H, Li L, Fu X, Cao Z, Jin L.
      B cell dysfunction due to obesity can be associated with alterations in the levels of micro-RNAs (miRNAs). However, the role of miRNAs in these processes remains elusive. Here, we show that miR-802 is increased in the pancreatic islets of obese mouse models and demonstrate that inducible transgenic overexpression of miR-802 in mice causes impaired insulin transcription and secretion. We identify Foxo1 as a transcription factor of miR-802 promoting its transcription, and NeuroD1 and Fzd5 as targets of miR-802-dependent silencing. Repression of NeuroD1 in β cell and primary islets impairs insulin transcription and reduction of Fzd5 in β cell, which, in turn, impairs Ca2+ signaling, thereby repressing calcium influx and decreasing insulin secretion. We functionally create a novel network between obesity and β cell dysfunction via miR-802 regulation. Elucidation of the impact of obesity on microRNA expression can broaden our understanding of pathophysiological development of diabetes.
    DOI:  https://doi.org/10.1038/s41467-020-15529-w
  27. Nat Commun. 2020 Apr 14. 11(1): 1818
    Li C, Liu B, Kang B, Liu Z, Liu Y, Chen C, Ren X, Zhang Z.
      Fast, robust and technology-independent computational methods are needed for supervised cell type annotation of single-cell RNA sequencing data. We present SciBet, a supervised cell type identifier that accurately predicts cell identity for newly sequenced cells with order-of-magnitude speed advantage. We enable web client deployment of SciBet for rapid local computation without uploading local data to the server. Facing the exponential growth in the size of single cell RNA datasets, this user-friendly and cross-platform tool can be widely useful for single cell type identification.
    DOI:  https://doi.org/10.1038/s41467-020-15523-2
  28. Nat Commun. 2020 Apr 14. 11(1): 1833
    Yin M, Guo Y, Hu R, Cai WL, Li Y, Pei S, Sun H, Peng C, Li J, Ye R, Yang Q, Wang N, Tao Y, Chen X, Yan Q.
      Small molecule inhibitor of the bromodomain and extraterminal domain (BET) family proteins is a promising option for cancer treatment. However, current BET inhibitors are limited by their potency or oral bioavailability. Here we report the discovery and characterization of NHWD-870, a BET inhibitor that is more potent than three major clinical stage BET inhibitors BMS-986158, OTX-015, and GSK-525762. NHWD-870 causes tumor shrinkage or significantly suppresses tumor growth in nine xenograft or syngeneic models. In addition to its ability to downregulate c-MYC and directly inhibit tumor cell proliferation, NHWD-870 blocks the proliferation of tumor associated macrophages (TAMs) through multiple mechanisms, partly by reducing the expression and secretion of macrophage colony-stimulating factor CSF1 by tumor cells. NHWD-870 inhibits CSF1 expression through suppressing BRD4 and its target HIF1α. Taken together, these results reveal a mechanism by which BRD4 inhibition suppresses tumor growth, and support further development of NHWD-870 to treat solid tumors.
    DOI:  https://doi.org/10.1038/s41467-020-15290-0
  29. Cell Death Dis. 2020 Apr 17. 11(4): 239
    Tan YF, Wang M, Chen ZY, Wang L, Liu XH.
      BRD4 has long been implicated in many different pathological processes, in particular, the development of cancer and inflammation. Pyroptosis is a newly recognized type of inflammatory programmed cell death. However, the correlation between BRD4 and pyroptosis in renal cell carcinoma (RCC) remains elusive. The present study demonstrates that BRD4 expression levels are markedly upregulated, while pyroptosis-associated proteins are significantly reduced, in RCC tissues and cells. Inhibition of BRD4, via either genetic knockdown or use of bromodomain inhibitor JQ1, prevented cell proliferation and epithelial-mesenchymal transition (EMT) progression and induced caspase-1-dependent pyroptosis in RCC both in vitro and in vivo. In addition, BRD4 inhibition suppressed proliferation and EMT though pyroptosis in vitro and in vivo. Moreover, NLRP3, which mediates caspase-1-dependent pyroptosis, was increased upon BRD4 inhibition. Furthermore, the transcriptional activity of NLRP3 was enhanced by BRD4 inhibition, and this enhancement was blocked by activation of NF-κB phosphorylation, indicating that NF-κB is an upstream regulator of NLRP3. Collectively, these results show that BRD4 inhibition prevents cell proliferation and EMT, and exerts an antitumor effect in RCC by activating the NF-κB-NLRP3-caspase-1 pyroptosis signaling pathway. Thus, BRD4 is a potential target for RCC treatment, and JQ1 shows promise as a therapeutic agent for this disease.
    DOI:  https://doi.org/10.1038/s41419-020-2431-2
  30. Cell Metab. 2020 Apr 13. pii: S1550-4131(20)30187-X. [Epub ahead of print]
    Camunas-Soler J, Dai XQ, Hang Y, Bautista A, Lyon J, Suzuki K, Kim SK, Quake SR, MacDonald PE.
      Impaired function of pancreatic islet cells is a major cause of metabolic dysregulation and disease in humans. Despite this, it remains challenging to directly link physiological dysfunction in islet cells to precise changes in gene expression. Here we show that single-cell RNA sequencing combined with electrophysiological measurements of exocytosis and channel activity (patch-seq) can be used to link endocrine physiology and transcriptomes at the single-cell level. We collected 1,369 patch-seq cells from the pancreata of 34 human donors with and without diabetes. An analysis of function and gene expression networks identified a gene set associated with functional heterogeneity in β cells that can be used to predict electrophysiology. We also report transcriptional programs underlying dysfunction in type 2 diabetes and extend this approach to cryopreserved cells from donors with type 1 diabetes, generating a valuable resource for understanding islet cell heterogeneity in health and disease.
    Keywords:  T1D; T2D; alpha cell; beta cell; cryopreservation; diabetes; islet; pancreas; patch-seq; single-cell RNA-seq
    DOI:  https://doi.org/10.1016/j.cmet.2020.04.005
  31. Mol Genet Genomics. 2020 Apr 15.
    Yu X, Buck MJ.
      Pioneer transcription factors are a special group of transcription factors that can interact with nucleosomal DNA and initiate regulatory events. Their binding to regulatory regions is the first event in gene activation and can occur in silent or heterochromatin regions. Several research groups have endeavored to define pioneer factors and study their binding characteristics using various techniques. In this review, we describe the in vitro methods used to define and characterize pioneer factors, paying particular attention to differences in methodologies and how these differences can affect results.
    Keywords:  Binding assays; Nucleosomes; Pioneer factors; Transcription factors
    DOI:  https://doi.org/10.1007/s00438-020-01675-9
  32. Sci Transl Med. 2020 Apr 15. pii: eaax2332. [Epub ahead of print]12(539):
    Smeriglio P, Grandi FC, Davala S, Masarapu V, Indelli PF, Goodman SB, Bhutani N.
      Osteoarthritis (OA) is a degenerative disease of the joint, which results in pain, loss of mobility, and, eventually, joint replacement. Currently, no disease-modifying drugs exist, partly because of the multiple levels at which cartilage homeostasis is disrupted. Recent studies have highlighted the importance of epigenetic dysregulation in OA, sparking interest in the epigenetic modulation for this disease. In our previous work, we characterized a fivefold increase in cytosine hydroxymethylation (5hmC), an oxidized derivative of cytosine methylation (5mC) associated with gene activation, accumulating at OA-associated genes. To test the role of 5hmC in OA, here, we used a mouse model of surgically induced OA and found that OA onset was accompanied by a gain of ~40,000 differentially hydroxymethylated sites before the notable histological appearance of disease. We demonstrated that ten-eleven-translocation enzyme 1 (TET1) mediates the 5hmC deposition because 98% of sites enriched for 5hmC in OA were lost in Tet1-/- mice. Loss of TET1-mediated 5hmC protected the Tet1-/- mice from OA development, including degeneration of the cartilage surface and osteophyte formation, by directly preventing the activation of multiple OA pathways. Loss of TET1 in human OA chondrocytes reduced the expression of the matrix metalloproteinases MMP3 and MMP13 and multiple inflammatory cytokines. Intra-articular injections of a dioxygenases inhibitor, 2-hydroxyglutarate, on mice after surgical induction of OA stalled disease progression. Treatment of human OA chondrocytes with the same inhibitor also phenocopied TET1 loss. Collectively, these data demonstrate that TET1-mediated 5hmC deposition regulates multiple OA pathways and can be modulated for therapeutic intervention.
    DOI:  https://doi.org/10.1126/scitranslmed.aax2332
  33. Blood. 2020 Apr 16. pii: blood.2020005301. [Epub ahead of print]
    Huang P, Peslak SA, Lan X, Khandros E, Yano JA, Sharma M, Keller CA, Giardine BM, Qin K, Abdulmalik O, Hardison R, Shi J, Blobel GA.
      Reactivation of fetal hemoglobin remains a critical goal in the treatment of patients with sickle cell disease and β-thalassemia. Previously, we discovered that silencing of the fetal γ-globin gene requires the erythroid-specific eIF2α kinase HRI, suggesting that HRI might present a pharmacologic target for raising fetal hemoglobin levels. Here, via a CRISPR-Cas9 guided loss-of-function screen in human erythroblasts we identify transcription factor ATF4, a known HRI-regulated protein, as a novel γ-globin regulator. ATF4 directly stimulates transcription of BCL11A, a repressor of γ-globin transcription, by binding to its enhancer and fostering enhancer-promoter contacts. Notably, HRI-deficient mice display normal Bcl11a levels, suggesting species-selective regulation, which we explain here by demonstrating that the analogous ATF4 motif at the murine Bcl11a enhancer is largely dispensable. Our studies uncover a linear signaling pathway from HRI to ATF4 to BCL11A to γ-globin and illustrate potential limits of murine models of globin gene regulation.
    DOI:  https://doi.org/10.1182/blood.2020005301
  34. Nat Immunol. 2020 Apr 13.
    Shulman Z, Stern-Ginossar N.
      Protection from harmful pathogens depends on activation of the immune system, which relies on tight regulation of gene expression. Recently, the RNA modification N6-methyladenosine (m6A) has been found to play an essential role in such regulation. Here, we summarize newly discovered functions of m6A in controlling various aspects of immunity, including immune recognition, activation of innate and adaptive immune responses, and cell fate decisions. We then discuss some of the current challenges in the field and describe future directions for uncovering the immunological functions of m6A and its mechanisms of action.
    DOI:  https://doi.org/10.1038/s41590-020-0650-4
  35. Elife. 2020 Apr 14. pii: e54695. [Epub ahead of print]9
    Zhong L, Yao L, Tower RJ, Wei Y, Miao Z, Park J, Shrestha R, Wang L, Yu W, Holdreith N, Huang X, Zhang Y, Tong W, Gong Y, Ahn J, Susztak K, Dyment N, Li M, Long F, Chen C, Seale P, Qin L.
      Bone marrow mesenchymal lineage cells are a heterogeneous cell population involved in bone homeostasis and diseases such as osteoporosis. While it is long postulated that they originate from mesenchymal stem cells, the true identity of progenitors and their in vivo bifurcated differentiation routes into osteoblasts and adipocytes remain poorly understood. Here, by employing large scale single cell transcriptome analysis, we computationally defined mesenchymal progenitors at different stages and delineated their bi-lineage differentiation paths in young, adult and aging mice. One identified subpopulation is a unique cell type that expresses adipocyte markers but contains no lipid droplets. As non-proliferative precursors for adipocytes, they exist abundantly as pericytes and stromal cells that form a ubiquitous 3D network inside the marrow cavity. Functionally they play critical roles in maintaining marrow vasculature and suppressing bone formation. Therefore, we name them marrow adipogenic lineage precursors (MALPs) and conclude that they are a new component of marrow adipose tissue.
    Keywords:  cell biology; mouse
    DOI:  https://doi.org/10.7554/eLife.54695
  36. FASEB J. 2020 Apr 12.
    Zhang Y, Zou J, Tolbert E, Zhao TC, Bayliss G, Zhuang S.
      Histone deacetylases (HDACs) have been shown to alleviate renal fibrosis, however, the role of individual HDAC isoforms in this process is poorly understood. In this study, we examined the role of HDAC8 in the development of renal fibrosis and partial epithelial-mesenchymal transitions (EMT). In a murine model of renal fibrosis induced by unilateral ureteral obstruction (UUO), HDAC8 was primarily expressed in renal tubular epithelial cells and time-dependently upregulated. This occurred in parallel with the deacetylation of cortactin, a nonhistone substrate of HDAC8, and increased expression of three fibrotic markers: α-smooth muscle actin, collagen 1, and fibronectin. Administration of PCI34051, a highly selective inhibitor of HDAC8, restored acetylation of contactin and reduced expression of those proteins. PCI34051 treatment also reduced the number of renal tubular epithelial cells arrested at the G2/M phase of the cell cycle and suppressed phosphorylation of Smad3, STAT3, β-catenin, and expression of Snail after ureteral obstruction. In contrast, HDAC8 inhibition reversed UUO-induced downregulation of BMP7 and Klotho, two renoprotective proteins. In cultured murine proximal tubular cells, treatment with PCI34051 or specific HDAC8 siRNA was also effective in inhibiting transforming growth factor β1 (TGFβ1)-induced deacetylation of contactin, EMT, phosphorylation of Smad3, STAT3, and β-catenin, upregulation of Snail, and downregulation of BMP7 and Klotho. Collectively, these results suggest that HDAC8 activation is required for the EMT and renal fibrogenesis by activation of multiple profibrotic signaling and transcription factors, and suppression of antifibrotic proteins. Therefore, targeting HDAC8 may be novel therapeutic approach for treatment of renal fibrosis.
    Keywords:  epithelial-mesenchymal transition; histone deacetylase 8; renal fibrosis; transforming growth factor β1; unilateral ureteral obstruction; β-catenin
    DOI:  https://doi.org/10.1096/fj.201903254R
  37. Sci Adv. 2020 04;6(15): eaax3969
    Zhou C, Miao Y, Cui Z, ShiYang X, Zhang Y, Xiong B.
      During mitotic prophase, cohesins are removed from chromosome arms by Wapl to ensure faithful sister chromatid separation. However, during female meiosis I, the resolution of chiasmata requires the proteolytic cleavage of cohesin subunit Rec8 along chromosome arms by Separase to separate homologs, and thus the role of Wapl remained unknown. Here, we report that Wapl functions as a regulator of spindle assembly checkpoint (SAC) to prevent aneuploidy in meiosis I. Depletion of Wapl accelerates meiotic progression, inactivates SAC, and causes meiotic defects such as aberrant spindle/chromosome structure and incorrect kinetochore-microtubule (K-MT) attachment, consequently leading to aneuploid eggs. Notably, we identify Bub3 as a binding partner of Wapl by immunoprecipitation and mass spectrometry analysis. We further determine that Wapl controls the SAC activity by maintaining Bub3 protein level and document that exogenous Bub3 restores the normal meiosis in Wapl-depleted oocytes. Together, our findings uncover unique, noncanonical roles for Wapl in mediating control of the SAC in female meiosis I.
    DOI:  https://doi.org/10.1126/sciadv.aax3969
  38. Cancer Res. 2020 Apr 14. pii: canres.3230.2019. [Epub ahead of print]
    Patel A, Fraile Garcia L, Mannella V, Gammon L, Borg TM, Maffucci T, Scatolini M, Chiorino G, Vergani E, Rodolfo M, Maurichi A, Posch C, Matin RN, Harwood CA, Bergamaschi D.
      Targeting the MAPK pathway by combined inhibition of BRAF and MEK has increased overall survival in advanced BRAF-mutant melanoma in both therapeutic and adjuvant clinical settings. However, a significant proportion of tumors develop acquired resistance, leading to treatment failure. We have previously shown p63 to be an important inhibitor of p53-induced apoptosis in melanoma following genotoxic drug exposure. Here we investigated the role of p63 in acquired resistance to MAPK inhibition and show that p63 isoforms are upregulated in melanoma cell lines chronically exposed to BRAF and MEK inhibition, with consequent increased resistance to apoptosis. This p63 upregulation was the result of its reduced degradation by the E3 ubiquitin ligase FBXW7. FBXW7 was itself regulated by MDM2, and in therapy-resistant melanoma cell lines, nuclear accumulation of MDM2 caused downregulation of FBXW7 and consequent upregulation of p63. Consistent with this, both FBXW7 inactivating mutations and MDM2 upregulation were found in melanoma clinical samples. Treatment of MAPK inhibitor-resistant melanoma cells with MDM2 inhibitor Nutlin-3A restored FBXW7 expression and p63 degradation in a dose-dependent manner and sensitized these cells to apoptosis. Collectively, these data provide a compelling rationale for future investigation of nutlin-3A as an approach to abrogate acquired resistance of melanoma to MAPK inhibitor targeted therapy.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-19-3230
  39. PLoS Comput Biol. 2020 Apr 14. 16(4): e1007819
    Vsevolozhskaya OA, Shi M, Hu F, Zaykin DV.
      Historically, the majority of statistical association methods have been designed assuming availability of SNP-level information. However, modern genetic and sequencing data present new challenges to access and sharing of genotype-phenotype datasets, including cost of management, difficulties in consolidation of records across research groups, etc. These issues make methods based on SNP-level summary statistics particularly appealing. The most common form of combining statistics is a sum of SNP-level squared scores, possibly weighted, as in burden tests for rare variants. The overall significance of the resulting statistic is evaluated using its distribution under the null hypothesis. Here, we demonstrate that this basic approach can be substantially improved by decorrelating scores prior to their addition, resulting in remarkable power gains in situations that are most commonly encountered in practice; namely, under heterogeneity of effect sizes and diversity between pairwise LD. In these situations, the power of the traditional test, based on the added squared scores, quickly reaches a ceiling, as the number of variants increases. Thus, the traditional approach does not benefit from information potentially contained in any additional SNPs, while our decorrelation by orthogonal transformation (DOT) method yields steady gain in power. We present theoretical and computational analyses of both approaches, and reveal causes behind sometimes dramatic difference in their respective powers. We showcase DOT by analyzing breast cancer and cleft lip data, in which our method strengthened levels of previously reported associations and implied the possibility of multiple new alleles that jointly confer disease risk.
    DOI:  https://doi.org/10.1371/journal.pcbi.1007819
  40. RNA. 2020 Apr 13. pii: rna.074922.120. [Epub ahead of print]
    Zhao S, Ye Z, Stanton R.
      In recent years RNA-sequencing (RNA-seq) has emerged as a powerful technology for transcriptome profiling. For a given gene, the number of mapped reads is not only dependent on its expression level and gene length, but also the sequencing depth. To normalize these dependencies, RPKM (Reads Per Kilobase of transcript per Million reads mapped) and TPM (Transcripts Per Million) were used to measure gene or transcript expression levels. A common misconception is that RPKM and TPM values are already normalized, and thus should be comparable across samples or RNA-seq projects. However, RPKM and TPM represent the relative abundance of a transcript among a population of sequenced transcripts, and therefore depend on the composition of the RNA population in a sample. Quite often, it is reasonable to assume that total RNA concentration and distributions is very close across compared samples. Nevertheless, the sequenced RNA repertoires may differ significantly under different experimental conditions and/or across sequencing protocols; thus, the proportion of gene expression is not directly comparable in such cases. In this review, we illustrate typical scenarios in which RPKM and TPM are misused, unintentionally, and hope to raise scientists' awareness of this issue when comparing them across samples or different sequencing protocols.
    Keywords:  Normalization; RNA-seq; RPKM; TPM
    DOI:  https://doi.org/10.1261/rna.074922.120
  41. PLoS Genet. 2020 Apr 17. 16(4): e1008666
    Marinić M, Lynch VJ.
      The steroid hormone progesterone, acting through the progesterone receptor (PR), a ligand-activated DNA-binding transcription factor, plays an essential role in regulating nearly every aspect of female reproductive biology. While many reproductive traits regulated by PR are conserved in mammals, Catarrhine primates evolved several derived traits including spontaneous decidualization, menstruation, and a divergent (and unknown) parturition signal, suggesting that PR may also have evolved divergent functions in Catarrhines. There is conflicting evidence, however, whether the progesterone receptor gene (PGR) was positively selected in the human lineage. Here we show that PGR evolved rapidly in the human stem-lineage (as well as other Catarrhine primates), which likely reflects an episode of relaxed selection intensity rather than positive selection. Coincident with the episode of relaxed selection intensity, ancestral sequence resurrection and functional tests indicate that the major human PR isoforms (PR-A and PR-B) evolved divergent functions in the human stem-lineage. These results suggest that the regulation of progesterone signaling by PR-A and PR-B may also have diverged in the human lineage and that non-human animal models of progesterone signaling may not faithfully recapitulate human biology.
    DOI:  https://doi.org/10.1371/journal.pgen.1008666
  42. Nucleic Acids Res. 2020 Apr 16. pii: gkaa244. [Epub ahead of print]
    Warman EA, Singh SS, Gubieda AG, Grainger DC.
      RNA polymerases initiate transcription at DNA sequences called promoters. In bacteria, the best conserved promoter feature is the AT-rich -10 element; a sequence essential for DNA unwinding. Further elements, and gene regulatory proteins, are needed to recruit RNA polymerase to the -10 sequence. Hence, -10 elements cannot function in isolation. Many horizontally acquired genes also have a high AT-content. Consequently, sequences that resemble the -10 element occur frequently. As a result, foreign genes are predisposed to spurious transcription. However, it is not clear how RNA polymerase initially recognizes such sequences. Here, we identify a non-canonical promoter element that plays a key role. The sequence, itself a short AT-tract, resides 5 base pairs upstream of otherwise cryptic -10 elements. The AT-tract alters DNA conformation and enhances contacts between the DNA backbone and RNA polymerase.
    DOI:  https://doi.org/10.1093/nar/gkaa244
  43. Science. 2020 Apr 17. 368(6488): 303-306
    Weber C, Zhou Y, Lee JG, Looger LL, Qian G, Ge C, Capel B.
      In many reptiles, including the red-eared slider turtle Trachemys scripta elegans (T. scripta), sex is determined by ambient temperature during embryogenesis. We previously showed that the epigenetic regulator Kdm6b is elevated at the male-producing temperature and essential to activate the male pathway. In this work, we established a causal link between temperature and transcriptional regulation of Kdm6b We show that signal transducer and activator of transcription 3 (STAT3) is phosphorylated at the warmer, female-producing temperature, binds the Kdm6b locus, and represses Kdm6b transcription, blocking the male pathway. Influx of Ca2+, a mediator of STAT3 phosphorylation, is elevated at the female temperature and acts as a temperature-sensitive regulator of STAT3 activation.
    DOI:  https://doi.org/10.1126/science.aaz4165
  44. Front Oncol. 2020 ;10 368
    Li J, Ye M, Zhou C.
      The homeobox A cluster (HOXA) gene family, comprising 11 members, is involved in a wide spectrum of biological functions in human cancers. However, there is little research on the expression profile and prognostic values of HOXA genes in laryngeal squamous cell cancer (LSCC). Based on updated public resources and integrative bioinformatics analysis, we assessed the expression profile and prognostic values of the HOXA family members. Expression and methylation data on HOXA family members were obtained from The Cancer Genome Atlas (TCGA). The prognostic values of HOXA members and clinical features were identified. A gene set enrichment analysis (GSEA) was conducted to explore the mechanism underlying the involvement of HOXA members in LSCC. The associations between tumor immune infiltrating cells (TIICs) and the HOXA family members were evaluated using the Tumor Immune Estimation Resource (TIMER) database. HOXA2 and HOXA4 were downregulated and HOXA7 and HOXA9-13 were upregulated in LSCC. Upregulation of HOXA10, HOXA11, and HOXA13, along with two clinical characteristics (M stage and gender), were associated with a poor LSCC prognosis based on the results of univariate and multivariate Cox proportional hazards regression analyses. Although there were no significant correlations between TIICs and HOXA members, the GSEA results indicated that HOXA members participate in multiple biological processes underlying tumorigenesis. This study comprehensively analyzed the HOXA members, providing insights for further investigation of the HOXA family members as potential targets in LSCC.
    Keywords:  GSEA; HOXA family; LSCC; TCGA; prognosis
    DOI:  https://doi.org/10.3389/fonc.2020.00368
  45. Pancreas. 2020 Apr;49(4): 514-523
    Han X, Chen W, Chen P, Zhou W, Rong Y, Lv Y, Li JA, Ji Y, Chen W, Lou W, Xu X.
      OBJECTIVE: The genetic aberrations that underlie chromatin remodeling in sporadic nonfunctional pancreatic neuroendocrine tumors (NF-pNETs) remain largely unknown. Here, we investigated the dysregulation of the switch/sucrose nonfermentable (SWI/SNF) component ARID1A and its correlation with clinicopathological features and prognosis.METHODS: We sequenced the exomes of sporadic NF-pNETs. Quantitative real-time polymerase chain reaction and immunohistochemistry were used to determine messenger RNA level and protein expression.
    RESULTS: The sporadic NF-pNETs harbored 264 somatic mutations in 228 different genes, most commonly affecting the SWI/SNF components ARID1B (57.1%) and ARID1A (42.9%). The expression of ARID1A was remarkably downregulated in NF-pNETs and corresponding liver metastases compared with that in normal pancreatic islet tissue. Reduced expression of ARID1A was associated with malignant clinicopathological features (P < 0.05). The loss of ARID1A was related to a high Ki-67 index (P < 0.05). Patients with ARID1A-negative expression had a significantly worse overall survival rate than those with ARID1A-positive expression (P < 0.05). The ARID1A status was an independent predictor of overall survival, and a nomogram integrating ARID1A with clinicopathological features was proposed.
    CONCLUSIONS: The loss of SWI/SNF components ARID1A may be associated with malignant behaviors and an unfavorable prognosis. Aberrations of ARID1A may contribute to tumorigenesis and metastasis in sporadic NF-pNETs.
    DOI:  https://doi.org/10.1097/MPA.0000000000001535
  46. Nature. 2020 Apr;580(7803): 402-408
    Luck K, Kim DK, Lambourne L, Spirohn K, Begg BE, Bian W, Brignall R, Cafarelli T, Campos-Laborie FJ, Charloteaux B, Choi D, Coté AG, Daley M, Deimling S, Desbuleux A, Dricot A, Gebbia M, Hardy MF, Kishore N, Knapp JJ, Kovács IA, Lemmens I, Mee MW, Mellor JC, Pollis C, Pons C, Richardson AD, Schlabach S, Teeking B, Yadav A, Babor M, Balcha D, Basha O, Bowman-Colin C, Chin SF, Choi SG, Colabella C, Coppin G, D'Amata C, De Ridder D, De Rouck S, Duran-Frigola M, Ennajdaoui H, Goebels F, Goehring L, Gopal A, Haddad G, Hatchi E, Helmy M, Jacob Y, Kassa Y, Landini S, Li R, van Lieshout N, MacWilliams A, Markey D, Paulson JN, Rangarajan S, Rasla J, Rayhan A, Rolland T, San-Miguel A, Shen Y, Sheykhkarimli D, Sheynkman GM, Simonovsky E, Taşan M, Tejeda A, Tropepe V, Twizere JC, Wang Y, Weatheritt RJ, Weile J, Xia Y, Yang X, Yeger-Lotem E, Zhong Q, Aloy P, Bader GD, De Las Rivas J, Gaudet S, Hao T, Rak J, Tavernier J, Hill DE, Vidal M, Roth FP, Calderwood MA.
      Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships1,2. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.
    DOI:  https://doi.org/10.1038/s41586-020-2188-x