bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2020‒05‒03
forty-two papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit


  1. Nature. 2020 Apr;580(7805): 669-672
    Dodonova SO, Zhu F, Dienemann C, Taipale J, Cramer P.
      'Pioneer' transcription factors are required for stem-cell pluripotency, cell differentiation and cell reprogramming1,2. Pioneer factors can bind nucleosomal DNA to enable gene expression from regions of the genome with closed chromatin. SOX2 is a prominent pioneer factor that is essential for pluripotency and self-renewal of embryonic stem cells3. Here we report cryo-electron microscopy structures of the DNA-binding domains of SOX2 and its close homologue SOX11 bound to nucleosomes. The structures show that SOX factors can bind and locally distort DNA at superhelical location 2. The factors also facilitate detachment of terminal nucleosomal DNA from the histone octamer, which increases DNA accessibility. SOX-factor binding to the nucleosome can also lead to a repositioning of the N-terminal tail of histone H4 that includes residue lysine 16. We speculate that this repositioning is incompatible with higher-order nucleosome stacking, which involves contacts of the H4 tail with a neighbouring nucleosome. Our results indicate that pioneer transcription factors can use binding energy to initiate chromatin opening, and thereby facilitate nucleosome remodelling and subsequent transcription.
    DOI:  https://doi.org/10.1038/s41586-020-2195-y
  2. J Clin Invest. 2020 Apr 28. pii: 134260. [Epub ahead of print]
    Ramanand SG, Chen Y, Yuan J, Daescu K, Lambros M, Houlahan KE, Carreira S, Yuan W, Baek G, Sharp A, Paschalis A, Kanchwala M, Gao Y, Aslam A, Safdar N, Zhan X, Raj GV, Xing C, Boutros PC, de Bono J, Zhang MQ, Mani RS.
      Transcriptional dysregulation is a hallmark of prostate cancer (PCa). We mapped the RNA Polymerase II (RNA Pol II) associated chromatin interactions in normal prostate cells and PCa cells. We discovered thousands of enhancer-promoter, enhancer-enhancer, as well as promoter-promoter chromatin interactions. These transcriptional hubs operate within the framework set by structural proteins-CTCF and cohesins, and are regulated by the cooperative action of master transcription factors, such as the Androgen Receptor (AR) and FOXA1. By combining analyses from metastatic castration resistant PCa (mCRPC) specimens, we show that AR locus amplification contributes to the transcriptional up-regulation of AR gene by increasing the total number of chromatin interaction modules comprising of the AR gene and its distal enhancer. We deconvoluted the transcription control modules of several PCa genes, notably, the biomarker KLK3, lineage-restricted genes (KRT8, KRT18, HOXB13, FOXA1, ZBTB16), the drug target EZH2, and the oncogene MYC. By integrating clinical PCa data, we defined a novel germline-somatic interplay between the PCa risk allele rs684232 and the somatically acquired TMPRSS2-ERG gene fusion in the transcriptional regulation of multiple target genes-VPS53, FAM57A and GEMIN4. Our studies implicate changes in genome organization as a critical determinant of aberrant transcriptional regulation in PCa.
    Keywords:  Epigenetics; Genetics; Oncology; Prostate cancer; Transcription
    DOI:  https://doi.org/10.1172/JCI134260
  3. Mol Cell. 2020 Apr 23. pii: S1097-2765(20)30192-1. [Epub ahead of print]
    Nanavaty V, Abrash EW, Hong C, Park S, Fink EE, Li Z, Sweet TJ, Bhasin JM, Singuri S, Lee BH, Hwang TH, Ting AH.
      Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer and have been studied as independent processes. We discovered a DNA methylation-regulated APA mechanism when we compared genome-wide DNA methylation and polyadenylation site usage between DNA methylation-competent and DNA methylation-deficient cells. Here, we show that removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which, in turn, form chromatin loops that promote proximal polyadenylation site usage. In this DNA demethylated context, either deletion of the CTCF binding site or depletion of RAD21 cohesin complex protein can recover distal polyadenylation site usage. Using data from The Cancer Genome Atlas, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression in vivo. This DNA methylation-regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment.
    Keywords:  CTCF; alternative cleavage and polyadenylation; cohesin; gene-body DNA methylation; transcriptome diversity
    DOI:  https://doi.org/10.1016/j.molcel.2020.03.024
  4. Genome Biol. 2020 Apr 28. 21(1): 100
    Liu S, Zhu A, He C, Chen M.
      The REPIC (RNA EPItranscriptome Collection) database records about 10 million peaks called from publicly available m6A-seq and MeRIP-seq data using our unified pipeline. These data were collected from 672 samples of 49 studies, covering 61 cell lines or tissues in 11 organisms. REPIC allows users to query N6-methyladenosine (m6A) modification sites by specific cell lines or tissue types. In addition, it integrates m6A/MeRIP-seq data with 1418 histone ChIP-seq and 118 DNase-seq data tracks from the ENCODE project in a modern genome browser to present a comprehensive atlas of m6A methylation sites, histone modification sites, and chromatin accessibility regions. REPIC is accessible at https://repicmod.uchicago.edu/repic.
    Keywords:  Database; Genome browser; Tissue specificity; m6A modification
    DOI:  https://doi.org/10.1186/s13059-020-02012-4
  5. Cell Death Dis. 2020 Apr 27. 11(4): 287
    Xie W, Miehe M, Laufer S, Johnsen SA.
      Direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) requires a resetting of the epigenome in order to facilitate a cell fate transition. Previous studies have shown that epigenetic modifying enzymes play a central role in controlling induced pluripotency and the generation of iPSC. Here we show that RNF40, a histone H2B lysine 120 E3 ubiquitin-protein ligase, is specifically required for early reprogramming during induced pluripotency. Loss of RNF40-mediated H2B monoubiquitination (H2Bub1) impaired early gene activation in reprogramming. We further show that RNF40 contributes to tissue-specific gene suppression via indirect effects by controlling the expression of the polycomb repressive complex-2 histone methyltransferase component EZH2, as well as through more direct effects by promoting the resolution of H3K4me3/H3K27me3 bivalency on H2Bub1-occupied pluripotency genes. Thus, we identify RNF40 as a central epigenetic mediator of cell state transition with distinct functions in resetting somatic cell state to pluripotency.
    DOI:  https://doi.org/10.1038/s41419-020-2482-4
  6. Elife. 2020 Apr 29. pii: e53916. [Epub ahead of print]9
    Soluri IV, Zumerling LM, Payan Parra OA, Clark EG, Blythe SA.
      Because chromatin determines whether information encoded in DNA is accessible to transcription factors, dynamic chromatin states in development may constrain how gene regulatory networks impart embryonic pattern. To determine the interplay between chromatin states and regulatory network function, we performed ATAC-seq on Drosophila embryos during the establishment of the segmentation network, comparing wild-type and mutant embryos in which all graded maternal patterning inputs are eliminated. While during the period between zygotic genome activation and gastrulation many regions maintain stable accessibility, cis-regulatory modules (CRMs) within the network undergo extensive patterning-dependent changes in accessibility. A component of the network, Odd-paired (opa), is necessary for pioneering accessibility of late segmentation network CRMs. opa-driven changes in accessibility are accompanied by equivalent changes in gene expression. Interfering with the timing of opa activity impacts the proper patterning of expression. These results indicate that dynamic systems for chromatin regulation directly impact the reading of embryonic patterning information.
    Keywords:  D. melanogaster; chromatin; chromosomes; developmental biology; gene expression; patterning; transcription factors
    DOI:  https://doi.org/10.7554/eLife.53916
  7. Nucleic Acids Res. 2020 Apr 30. pii: gkaa284. [Epub ahead of print]
    Pan H, Jin M, Ghadiyaram A, Kaur P, Miller HE, Ta HM, Liu M, Fan Y, Mahn C, Gorthi A, You C, Piehler J, Riehn R, Bishop AJR, Tao YJ, Wang H.
      Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.
    DOI:  https://doi.org/10.1093/nar/gkaa284
  8. J Bioinform Comput Biol. 2020 Feb;18(1): 2040003
    Salehin N, Tam PPL, Osteil P.
      Assays for transposase-accessible chromatin sequencing (ATAC-seq) provides an innovative approach to study chromatin status in multiple cell types. Moreover, it is also possible to efficiently extract differentially accessible chromatin (DACs) regions by using state-of-the-art algorithms (e.g. DESeq2) to predict gene activity in specific samples. Furthermore, it has recently been shown that small dips in sequencing peaks can be attributed to the binding of transcription factors. These dips, also known as footprints, can be used to identify trans-regulating interactions leading to gene expression. Current protocols used to identify footprints (e.g. pyDNAse and HINT-ATAC) have shown limitations resulting in the discovery of many false positive footprints. We generated a novel approach to identify genuine footprints within any given ATAC-seq dataset. Herein, we developed a new pipeline embedding DACs together with bona fide footprints resulting in the generation of a Predictive gene regulatory Network (PreNet) simply from ATAC-seq data. We further demonstrated that PreNet can be used to unveil meaningful molecular regulatory pathways in a given cell type.
    Keywords:  ATAC-seq; footprints; gene regulatory network
    DOI:  https://doi.org/10.1142/S021972002040003X
  9. Blood. 2020 Apr 29. pii: blood.2020005204. [Epub ahead of print]
    Taveirne S, Wahlen S, Van Loocke W, Kiekens L, Persyn E, Van Ammel E, De Mulder K, Roels J, Tilleman L, Aumercier M, Matthys P, Van Nieuwerburgh F, Kerre TCC, Taghon T, Van Vlierberghe P, Vandekerckhove B, Leclercq G.
      Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and regulate other immune cells by cytokine secretion. Whereas murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells (hESC) and by expressing the dominant-negative ETS1 p27 isoform in cord blood (CB) hematopoietic progenitor cells (HPCs), we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation-sequencing (ChIP-seq) analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation, i.e. E4BP4, TXNIP, TBET, GATA3, HOBIT and BLIMP1. In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation.
    DOI:  https://doi.org/10.1182/blood.2020005204
  10. Nat Commun. 2020 Apr 29. 11(1): 2082
    Vinckier NK, Patel NA, Geusz RJ, Wang A, Wang J, Matta I, Harrington AR, Wortham M, Wetton N, Wang J, Jhala US, Rosenfeld MG, Benner CW, Shih HP, Sander M.
      Developmental progression depends on temporally defined changes in gene expression mediated by transient exposure of lineage intermediates to signals in the progenitor niche. To determine whether cell-intrinsic epigenetic mechanisms contribute to signal-induced transcriptional responses, here we manipulate the signalling environment and activity of the histone demethylase LSD1 during differentiation of hESC-gut tube intermediates into pancreatic endocrine cells. We identify a transient requirement for LSD1 in endocrine cell differentiation spanning a short time-window early in pancreas development, a phenotype we reproduced in mice. Examination of enhancer and transcriptome landscapes revealed that LSD1 silences transiently active retinoic acid (RA)-induced enhancers and their target genes. Furthermore, prolonged RA exposure phenocopies LSD1 inhibition, suggesting that LSD1 regulates endocrine cell differentiation by limiting the duration of RA signalling. Our findings identify LSD1-mediated enhancer silencing as a cell-intrinsic epigenetic feedback mechanism by which the duration of the transcriptional response to a developmental signal is limited.
    DOI:  https://doi.org/10.1038/s41467-020-16017-x
  11. Nat Commun. 2020 May 01. 11(1): 2181
    Bae HJ, Dubarry M, Jeon J, Soares LM, Dargemont C, Kim J, Geli V, Buratowski S.
      Methylation of histone H3 lysine 4 (H3K4) by Set1/COMPASS occurs co-transcriptionally, and is important for gene regulation. Set1/COMPASS associates with the RNA polymerase II C-terminal domain (CTD) to establish proper levels and distribution of H3K4 methylations. However, details of CTD association remain unclear. Here we report that the Set1 N-terminal region and the COMPASS subunit Swd2, which interact with each other, are both needed for efficient CTD binding in Saccharomyces cerevisiae. Moreover, a single point mutation in Swd2 that affects its interaction with Set1 also impairs COMPASS recruitment to chromatin and H3K4 methylation. A CTD interaction domain (CID) from the protein Nrd1 can partially substitute for the Set1 N-terminal region to restore CTD interactions and histone methylation. However, even when Set1/COMPASS is recruited via the Nrd1 CID, histone H2B ubiquitylation is still required for efficient H3K4 methylation, indicating that H2Bub acts after the initial recruitment of COMPASS to chromatin.
    DOI:  https://doi.org/10.1038/s41467-020-16082-2
  12. Elife. 2020 Apr 27. pii: e55667. [Epub ahead of print]9
    Ranjan A, Nguyen VQ, Liu S, Wisniewski J, Kim JM, Tang X, Mizuguchi G, Elalaoui E, Nickels TJ, Jou V, English BP, Zheng Q, Luk E, Lavis LD, Lionnet T, Wu C.
      The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.
    Keywords:  S. cerevisiae; chromosomes; gene expression
    DOI:  https://doi.org/10.7554/eLife.55667
  13. Genome Biol. 2020 Apr 29. 21(1): 104
    Concia L, Veluchamy A, Ramirez-Prado JS, Martin-Ramirez A, Huang Y, Perez M, Domenichini S, Rodriguez Granados NY, Kim S, Blein T, Duncan S, Pichot C, Manza-Mianza D, Juery C, Paux E, Moore G, Hirt H, Bergounioux C, Crespi M, Mahfouz MM, Bendahmane A, Liu C, Hall A, Raynaud C, Latrasse D, Benhamed M.
      BACKGROUND: Polyploidy is ubiquitous in eukaryotic plant and fungal lineages, and it leads to the co-existence of several copies of similar or related genomes in one nucleus. In plants, polyploidy is considered a major factor in successful domestication. However, polyploidy challenges chromosome folding architecture in the nucleus to establish functional structures.RESULTS: We examine the hexaploid wheat nuclear architecture by integrating RNA-seq, ChIP-seq, ATAC-seq, Hi-C, and Hi-ChIP data. Our results highlight the presence of three levels of large-scale spatial organization: the arrangement into genome territories, the diametrical separation between facultative and constitutive heterochromatin, and the organization of RNA polymerase II around transcription factories. We demonstrate the micro-compartmentalization of transcriptionally active genes determined by physical interactions between genes with specific euchromatic histone modifications. Both intra- and interchromosomal RNA polymerase-associated contacts involve multiple genes displaying similar expression levels.
    CONCLUSIONS: Our results provide new insights into the physical chromosome organization of a polyploid genome, as well as on the relationship between epigenetic marks and chromosome conformation to determine a 3D spatial organization of gene expression, a key factor governing gene transcription in polyploids.
    Keywords:  DNA loops; Genome territories; Hi-C; Hi-ChIP; Transcription factories
    DOI:  https://doi.org/10.1186/s13059-020-01998-1
  14. Biochemistry. 2020 Apr 30.
    Nikolova EN, Stanfield RL, Dyson HJ, Wright PE.
      Recognition of the epigenetic mark 5-methylcytosine (mC) at CpG sites in DNA has emerged as a novel function of many eukaryotic transcription factors (TFs). It remains unclear why the sequence specificity of these TFs differs for CpG-methylated motifs and consensus motifs. Here, we dissect the structural and dynamic basis for this differential DNA-binding specificity in the human zinc finger TF Kaiso, which exhibits high affinity for two consecutive mCpG sites in variable contexts and also for a longer, sequence-specific Kaiso binding site (KBS). By integrating structural analysis and DNA binding studies with targeted protein mutagenesis and nucleotide substitutions, we identify distinct mechanisms for readout of methylated and KBS motifs by Kaiso. We show that a key glutamate residue (E535), critical for mCpG site recognition, adopts different conformations in complexes with specific and methylated DNA. These conformational differences, together with intrinsic variations in DNA flexibility and/or solvation at TpG versus mCpG sites, contribute to different DNA affinity and sequence specificity. With methylated DNA, multiple direct contacts between E535 and the 5' mCpG site dominate the binding affinity, allowing for tolerance of different flanking DNA sequences. With KBS, Kaiso employs E535 as part of an indirect screen of the 5' flanking sequence, relying on key tyrosine-DNA interactions to stabilize an optimal DNA conformation and select against non-cognate sites. These findings demonstrate how TFs use conformational adaptation and exploit variations in DNA flexibility to achieve distinct DNA readout outcomes and target a greater variety of regulatory and epigenetic sites than previously appreciated.
    DOI:  https://doi.org/10.1021/acs.biochem.0c00253
  15. Elife. 2020 Apr 27. pii: e52648. [Epub ahead of print]9
    Kakebeen AD, Chitsazan AD, Williams MC, Saunders LM, Wills AE.
      Vertebrate appendage regeneration requires precisely coordinated remodeling of the transcriptional landscape to enable the growth and differentiation of new tissue, a process executed over multiple days and across dozens of cell types. The heterogeneity of tissues and temporally-sensitive fate decisions involved has made it difficult to articulate the gene regulatory programs enabling regeneration of individual cell types. To better understand how a regenerative program is fulfilled by neural progenitor cells (NPCs) of the spinal cord, we analyzed pax6-expressing NPCs isolated from regenerating Xenopus tropicalis tails. By intersecting chromatin accessibility data with single-cell transcriptomics, we find that NPCs place an early priority on neuronal differentiation. Late in regeneration, the priority returns to proliferation. Our analyses identify Pbx3 and Meis1 as critical regulators of tail regeneration and axon organization. Overall, we use transcriptional regulatory dynamics to present a new model for cell fate decisions and their regulators in NPCs during regeneration.
    Keywords:  developmental biology; regenerative medicine; stem cells; xenopus
    DOI:  https://doi.org/10.7554/eLife.52648
  16. Nucleic Acids Res. 2020 May 01. pii: gkaa285. [Epub ahead of print]
    Sridhar A, Orozco M, Collepardo-Guevara R.
      Intrinsically disordered proteins are crucial elements of chromatin heterogenous organization. While disorder in the histone tails enables a large variation of inter-nucleosome arrangements, disorder within the chromatin-binding proteins facilitates promiscuous binding to a wide range of different molecular targets, consistent with structural heterogeneity. Among the partially disordered chromatin-binding proteins, the H1 linker histone influences a myriad of chromatin characteristics including compaction, nucleosome spacing, transcription regulation, and the recruitment of other chromatin regulating proteins. Although it is now established that the long C-terminal domain (CTD) of H1 remains disordered upon nucleosome binding and that such disorder favours chromatin fluidity, the structural behaviour and thereby the role/function of the N-terminal domain (NTD) within chromatin is yet unresolved. On the basis of microsecond-long parallel-tempering metadynamics and temperature-replica exchange atomistic molecular dynamics simulations of different H1 NTD subtypes, we demonstrate that the NTD is completely unstructured in solution but undergoes an important disorder-to-order transition upon nucleosome binding: it forms a helix that enhances its DNA binding ability. Further, we show that the helical propensity of the H1 NTD is subtype-dependent and correlates with the experimentally observed binding affinity of H1 subtypes, suggesting an important functional implication of this disorder-to-order transition.
    DOI:  https://doi.org/10.1093/nar/gkaa285
  17. PeerJ. 2020 ;8 e8952
    Peñalosa-Ruiz G, Mulder KW, Veenstra GJC.
      Reprogramming somatic cells to induced pluripotent stem cells (iPSC) succeeds only in a small fraction of cells within the population. Reprogramming occurs in distinctive stages, each facing its own bottlenecks. It initiates with overexpression of transcription factors OCT4, SOX2, KLF4 and c-MYC (OSKM) in somatic cells such as mouse embryonic fibroblasts (MEFs). OSKM bind chromatin, silencing the somatic identity and starting the stepwise reactivation of the pluripotency programme. However, inefficient suppression of the somatic lineage leads to unwanted epigenetic memory from the tissue of origin, even in successfully generated iPSCs. Thus, it is essential to shed more light on chromatin regulators and processes involved in dissolving the somatic identity. Recent work characterised the role of transcriptional corepressors NCOR1 and NCOR2 (also known as NCoR and SMRT), showing that they cooperate with c-MYC to silence pluripotency genes during late reprogramming stages. NCOR1/NCOR2 were also proposed to be involved in silencing fibroblast identity, however it is unclear how this happens. Here, we shed light on the role of NCOR1 in early reprogramming. We show that siRNA-mediated ablation of NCOR1 and OCT4 results in very similar phenotypes, including transcriptomic changes and highly correlated high-content colony phenotypes. Both NCOR1 and OCT4 bind to promoters co-occupied by c-MYC in MEFs. During early reprogramming, downregulation of one group of somatic MEF-expressed genes requires both NCOR1 and OCT4, whereas another group of MEF-expressed genes is downregulated by NCOR1 but not OCT4. Our data suggest that NCOR1, assisted by OCT4 and c-MYC, facilitates transcriptional repression of genes with high expression in MEFs, which is necessary to bypass an early reprogramming block; this way, NCOR1 facilitates early reprogramming progression.
    Keywords:  Cell identity; Chromatin; Corepressor; Functional genomics; NCoR or NCOR1; OCT4; Pluripotency; Reprogramming; Transcriptional repression; iPS
    DOI:  https://doi.org/10.7717/peerj.8952
  18. Mol Cell. 2020 Apr 28. pii: S1097-2765(20)30230-6. [Epub ahead of print]
    Kurihara M, Kato K, Sanbo C, Shigenobu S, Ohkawa Y, Fuchigami T, Miyanari Y.
      The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with ∼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.
    Keywords:  APEX; DNA methylation; PML; Y chromosome; chromatin; phase separation
    DOI:  https://doi.org/10.1016/j.molcel.2020.04.004
  19. Cell Death Differ. 2020 Apr 28.
    Liu Z, Zhang H, Hu Y, Liu D, Li L, Li C, Wang Q, Huo J, Liu H, Xie N, Huang X, Liu Y, Chen CD, Shi Y, Zhang X.
      Medullary thymic epithelial cells (mTECs) play a central role in the establishment of T cell central immunological tolerance by promiscuously expressing tissue-restricted antigens (TRAs) and presenting them to developing T cells, leading to deletion of T cells responding to self-antigens. However, molecular mechanisms especially epigenetic regulation of mTEC homeostasis and TRA expression remain elusive. Here we show that the H3K27 demethylase Kdm6b is essential to maintain the postnatal thymic medulla by promoting mTEC survival and regulating the expression of TRA genes. Moreover, mice lacking Kdm6b developed pathological autoimmune disorders. Mechanically, Kdm6b exerted its function by reducing repressive H3K27 trimethylation (H3K27me3) at the promoters of anti-apoptotic gene Bcl2 and a set of Aire-dependent TRA genes. Thus, our findings reveal a dual role of Kdm6b in the regulation of mTEC-mediated T cell central tolerance.
    DOI:  https://doi.org/10.1038/s41418-020-0546-8
  20. BMC Bioinformatics. 2020 May 01. 21(1): 171
    Ren X, Kuan PF.
      BACKGROUND: High-throughput sequencing experiments followed by differential expression analysis is a widely used approach for detecting genomic biomarkers. A fundamental step in differential expression analysis is to model the association between gene counts and covariates of interest. Existing models assume linear effect of covariates, which is restrictive and may not be sufficient for certain phenotypes.RESULTS: We introduce NBAMSeq, a flexible statistical model based on the generalized additive model and allows for information sharing across genes in variance estimation. Specifically, we model the logarithm of mean gene counts as sums of smooth functions with the smoothing parameters and coefficients estimated simultaneously within a nested iterative method. The variance is estimated by the Bayesian shrinkage approach to fully exploit the information across all genes.
    CONCLUSIONS: Based on extensive simulations and case studies of RNA-Seq data, we show that NBAMSeq offers improved performance in detecting nonlinear effect and maintains equivalent performance in detecting linear effect compared to existing methods. The vignette and source code of NBAMSeq are available at http://bioconductor.org/packages/release/bioc/html/NBAMSeq.html.
    Keywords:  Bayesian shrinkage; Differential expression analysis; Generalized additive model; RNA-Seq; Spline model
    DOI:  https://doi.org/10.1186/s12859-020-3506-x
  21. Nucleic Acids Res. 2020 Apr 27. pii: gkaa223. [Epub ahead of print]
    Steinhaus R, Gonzalez T, Seelow D, Robinson PN.
      The temporal and spatial expression of genes is controlled by promoters and enhancers. Findings obtained over the last decade that not only promoters but also enhancers are characterized by bidirectional, divergent transcription have challenged the traditional notion that promoters and enhancers represent distinct classes of regulatory elements. Over half of human promoters are associated with CpG islands (CGIs), relatively CpG-rich stretches of generally several hundred nucleotides that are often associated with housekeeping genes. Only about 6% of transcribed enhancers defined by CAGE-tag analysis are associated with CGIs. Here, we present an analysis of enhancer and promoter characteristics and relate them to the presence or absence of CGIs. We show that transcribed enhancers share a number of CGI-dependent characteristics with promoters, including statistically significant local overrepresentation of core promoter elements. CGI-associated enhancers are longer, display higher directionality of transcription, greater expression, a lesser degree of tissue specificity, and a higher frequency of transcription-factor binding events than non-CGI-associated enhancers. Genes putatively regulated by CGI-associated enhancers are enriched for transcription regulator activity. Our findings show that CGI-associated transcribed enhancers display a series of characteristics related to sequence, expression and function that distinguish them from enhancers not associated with CGIs.
    DOI:  https://doi.org/10.1093/nar/gkaa223
  22. Nucleic Acids Res. 2020 May 01. pii: gkaa292. [Epub ahead of print]
    Kaczmarek Michaels K, Mohd Mostafa S, Ruiz Capella J, Moore CL.
      Adjusting DNA structure via epigenetic modifications, and altering polyadenylation (pA) sites at which precursor mRNA is cleaved and polyadenylated, allows cells to quickly respond to environmental stress. Since polyadenylation occurs co-transcriptionally, and specific patterns of nucleosome positioning and chromatin modifications correlate with pA site usage, epigenetic factors potentially affect alternative polyadenylation (APA). We report that the histone H3K4 methyltransferase Set1, and the histone H3K36 methyltransferase Set2, control choice of pA site in Saccharomyces cerevisiae, a powerful model for studying evolutionarily conserved eukaryotic processes. Deletion of SET1 or SET2 causes an increase in serine-2 phosphorylation within the C-terminal domain of RNA polymerase II (RNAP II) and in the recruitment of the cleavage/polyadenylation complex, both of which could cause the observed switch in pA site usage. Chemical inhibition of TOR signaling, which causes nutritional stress, results in Set1- and Set2-dependent APA. In addition, Set1 and Set2 decrease efficiency of using single pA sites, and control nucleosome occupancy around pA sites. Overall, our study suggests that the methyltransferases Set1 and Set2 regulate APA induced by nutritional stress, affect the RNAP II C-terminal domain phosphorylation at Ser2, and control recruitment of the 3' end processing machinery to the vicinity of pA sites.
    DOI:  https://doi.org/10.1093/nar/gkaa292
  23. PLoS One. 2020 ;15(4): e0232332
    Tripodi IJ, Chowdhury M, Gruca M, Dowell RD.
      The assay for transposase-accessible chromatin followed by sequencing (ATAC-seq) is an inexpensive protocol for measuring open chromatin regions. ATAC-seq is also relatively simple and requires fewer cells than many other high-throughput sequencing protocols. Therefore, it is tractable in numerous settings where other high throughput assays are challenging to impossible. Hence it is important to understand the limits of what can be inferred from ATAC-seq data. In this work, we leverage ATAC-seq to predict the presence of nascent transcription. Nascent transcription assays are the current gold standard for identifying regions of active transcription, including markers for functional transcription factor (TF) binding. We combine mapped short reads from ATAC-seq with the underlying peak sequence, to determine regions of active transcription genome-wide. We show that a hybrid signal/sequence representation classified using recurrent neural networks (RNNs) can identify these regions across different cell types.
    DOI:  https://doi.org/10.1371/journal.pone.0232332
  24. BMC Bioinformatics. 2020 May 01. 21(1): 169
    Wöste M, Leitão E, Laurentino S, Horsthemke B, Rahmann S, Schröder C.
      BACKGROUND: Analysing whole genome bisulfite sequencing datasets is a data-intensive task that requires comprehensive and reproducible workflows to generate valid results. While many algorithms have been developed for tasks such as alignment, comprehensive end-to-end pipelines are still sparse. Furthermore, previous pipelines lack features or show technical deficiencies, thus impeding analyses.RESULTS: We developed wg-blimp (whole genome bisulfite sequencing methylation analysis pipeline) as an end-to-end pipeline to ease whole genome bisulfite sequencing data analysis. It integrates established algorithms for alignment, quality control, methylation calling, detection of differentially methylated regions, and methylome segmentation, requiring only a reference genome and raw sequencing data as input. Comparing wg-blimp to previous end-to-end pipelines reveals similar setups for common sequence processing tasks, but shows differences for post-alignment analyses. We improve on previous pipelines by providing a more comprehensive analysis workflow as well as an interactive user interface. To demonstrate wg-blimp's ability to produce correct results we used it to call differentially methylated regions for two publicly available datasets. We were able to replicate 112 of 114 previously published regions, and found results to be consistent with previous findings. We further applied wg-blimp to a publicly available sample of embryonic stem cells to showcase methylome segmentation. As expected, unmethylated regions were in close proximity of transcription start sites. Segmentation results were consistent with previous analyses, despite different reference genomes and sequencing techniques.
    CONCLUSIONS: wg-blimp provides a comprehensive analysis pipeline for whole genome bisulfite sequencing data as well as a user interface for simplified result inspection. We demonstrated its applicability by analysing multiple publicly available datasets. Thus, wg-blimp is a relevant alternative to previous analysis pipelines and may facilitate future epigenetic research.
    Keywords:  Analysis pipeline; Analysis workflow; Epigenetics; Methylation; Whole-genome bisulfite sequencing
    DOI:  https://doi.org/10.1186/s12859-020-3470-5
  25. Sci Rep. 2020 Apr 27. 10(1): 7083
    Yamazaki M, Hosokawa M, Arikawa K, Takahashi K, Sakanashi C, Yoda T, Matsunaga H, Takeyama H.
      Spatial transcriptomics is useful for understanding the molecular organization of a tissue and providing insights into cellular function in a morphological context. In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability during the image acquisition and biomolecule collection processes. Here, we developed a tissue processing method for robust and reproducible RNA-seq from tissue microdissection samples. In this method, we suppressed RNA degradation in fresh-frozen tissue specimens by dehydration fixation and effectively collected a small amount of RNA molecules from microdissection samples by magnetic beads. We demonstrated the spatial transcriptome analysis of the mouse liver and brain in serial microdissection samples (100 μm in a diameter and 10 μm in thickness) produced by a microdissection punching system. Using our method, we could prevent RNA degradation at room temperature and effectively produce a sequencing library with Smart-seq2. This resulted in reproducible sequence read mapping in exon regions and the detection of more than 2000 genes compared to non-fixed samples in the RNA-seq analysis. Our method would be applied to various transcriptome analyses, providing the information for region specific gene expression in tissue specimens.
    DOI:  https://doi.org/10.1038/s41598-020-63495-6
  26. Mol Biol Cell. 2020 Apr 29. mbcE19070413
    Black M, Arumugam P, Shukla S, Pradhan A, Ustiyan V, Milewski D, Kalinichenko VV, Kalin TV.
      Forkhead box M1 (FOXM1), a nuclear transcription factor which activates cell cycle regulatory genes, is highly expressed in a majority of human cancers. The function of FOXM1 independent of nuclear transcription is unknown. In the present study, we found the FOXM1 protein inside of the mitochondria. Using site-directed mutagenesis, we generated FOXM1 mutant proteins that localized to distinct cellular compartments, uncoupling the nuclear and mitochondrial functions of FOXM1. Directing FOXM1 into the mitochondria decreased mitochondrial mass, membrane potential, respiration and electron transport chain (ETC) activity. In mitochondria, the FOXM1 directly bound to and increased the pentatricopeptide repeat domain 1 (PTCD1) protein, a mitochondrial leucine-specific tRNA binding protein that inhibits leucine-rich ETC complexes. Mitochondrial FOXM1 did not change cellular proliferation. Thus, FOXM1 translocates into mitochondria and inhibits mitochondrial respiration by increasing PTCD1. We identify a new paradigm that FOXM1 regulates mitochondrial homeostasis in a process independent of nuclear transcription.
    DOI:  https://doi.org/10.1091/mbc.E19-07-0413
  27. Cell Rep. 2020 Apr 28. pii: S2211-1247(20)30519-2. [Epub ahead of print]31(4): 107570
    Sinha S, Biswas M, Chatterjee SS, Kumar S, Sengupta A.
      Bone morphogenic protein (BMP)/transforming growth factor β (TGF-β) signaling determines mesenchymal-stromal-cell (MSC) osteolineage commitment and tissue identity. However, molecular integration of developmental signaling with MSC-intrinsic chromatin regulation remains incompletely understood. SWI/SNF-(BAF) is an ATP-dependent chromatin remodeler implicated in multi-cellular development. We show that BMPs and long-term osteogenic signals in MSCs selectively induce expression of polybromo BAF (PBAF) components Pbrm1, Arid2, and Brd7. Loss of Pbrm1/Arid2/Brd7 profoundly impairs osteolineage gene expression and osteogenesis without compromising adipogenesis. Pbrm1 loss attenuates MSC in vivo ossification. Mechanistically, Pbrm1/PBAF deficiency impairs Smad1/5/8 activation through locus-specific epi-genomic remodeling, involving Pbrm1 bromodomains, along with transcriptional downregulation of Bmpr/TgfβrII affecting BMP-early-responsive gene expression. Gain of function of BmprIβ, TgfβrII in PBAF-deficient MSCs partly restores Smad1/5/8 activation and osteogenesis. Pbrm1 loss further affects hematopoietic stem and progenitor activity through non-cell-autonomous regulation of microenvironment and niche-factor expression. Together, these findings reveal a link illustrating epi-genomic feedforward control of BMP/TGF-β signaling to transcriptional and cellular plasticity in the mesenchymal microenvironment and account for stromal-SWI/SNF in hematopoiesis.
    Keywords:  BMP/TGF-β signaling; SWI/SNF; bromodomain; chromatin remodeling; feedforward control; hematopoiesis; hematopoietic microenvironment; mesenchymal stromal cell; osteolineage differentiation; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.celrep.2020.107570
  28. Stem Cells. 2020 Apr 28.
    Jiang Q, Huang X, Hu X, Shan Z, Wu Y, Wu G, Lei L.
      Aberrant epigenetic reprogramming is one of the major barriers for somatic cell reprogramming. Although our previous study has indicated that H3K27me3 demethylase KDM6A can improve the nuclear reprogramming efficiency, the mechanism remains unclear. In this study, we demonstrate that the overexpression of Kdm6a may improve iPSC reprogramming efficiency in a demethylase enzymatic activity-dependent manner. KDM6A erased H3K27me3 on pluripotency- and metabolism-related genes, and consequently facilitated changing the gene expression profile and metabolic pattern to an intermediate state. Further, KDM6A may promote IL-6 expression, and the secreted IL-6 may further improve iPSC reprogramming efficiency. In addition, KDM6A may promote PTEN expression to decrease p-AKT and p-mTOR levels, which in turn facilitates reprogramming. Overall, our results reveal that KDM6A may promote iPSC reprogramming efficiency by accelerating changes in the gene expression profile and metabolic pattern in a demethylation-activity-dependent manner. These results may provide an insight into the relationship between epigenomics, transcriptomics, metabolomics and reprogramming. © AlphaMed Press 2020 SIGNIFICANCE STATEMENT: iPSC-based treatment allows potential therapy for multiple diseases; however, its low efficiency limits its further application. In the present study, we demonstrated that KDM6A may improve iPSC reprogramming efficiency in a demethylase enzymatic activity-dependent manner. Further, KDM6A-induced H3K27me3 distribution may alter the gene expression profile and metabolic pattern of MEFs. We ultimately found that the PTEN and IL-6 pathways contributed to improving reprogramming.
    DOI:  https://doi.org/10.1002/stem.3188
  29. Sci Rep. 2020 Apr 28. 10(1): 7157
    Tjaden B, Baum K, Marquardt V, Simon M, Trajkovic-Arsic M, Kouril T, Siebers B, Lisec J, Siveke JT, Schulte JH, Benary U, Remke M, Wolf J, Schramm A.
      N-Myc is a transcription factor that is aberrantly expressed in many tumor types and is often correlated with poor patient prognosis. Recently, several lines of evidence pointed to the fact that oncogenic activation of Myc family proteins is concomitant with reprogramming of tumor cells to cope with an enhanced need for metabolites during cell growth. These adaptions are driven by the ability of Myc proteins to act as transcriptional amplifiers in a tissue-of-origin specific manner. Here, we describe the effects of N-Myc overexpression on metabolic reprogramming in neuroblastoma cells. Ectopic expression of N-Myc induced a glycolytic switch that was concomitant with enhanced sensitivity towards 2-deoxyglucose, an inhibitor of glycolysis. Moreover, global metabolic profiling revealed extensive alterations in the cellular metabolome resulting from overexpression of N-Myc. Limited supply with either of the two main carbon sources, glucose or glutamine, resulted in distinct shifts in steady-state metabolite levels and significant changes in glutathione metabolism. Interestingly, interference with glutamine-glutamate conversion preferentially blocked proliferation of N-Myc overexpressing cells, when glutamine levels were reduced. Thus, our study uncovered N-Myc induction and nutrient levels as important metabolic master switches in neuroblastoma cells and identified critical nodes that restrict tumor cell proliferation.
    DOI:  https://doi.org/10.1038/s41598-020-64040-1
  30. Cancer Res. 2020 Apr 27. pii: canres.0435.2020. [Epub ahead of print]
    Zhou H, Blevins MA, Hsu JY, Kong D, Galbraith MD, Goodspeed A, Culp-Hill R, Oliphant MU, Ramirez D, Zhang L, Pineiro JT, Mathews Griner L, King R, Barnaeva E, Hu X, Southall NT, Ferrer M, Gustafson DL, Regan DP, D'Alessandro A, Costello JC, Patnaik S, Marugan J, Zhao R, Ford HL.
      Metastasis is the major cause of mortality for cancer patients, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The SIX1/EYA transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGF-β signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGF-C-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGF-β signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis in vivo without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacological inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-0435
  31. Nat Commun. 2020 Apr 30. 11(1): 2113
    Kotopka BJ, Smolke CD.
      Promoters play a central role in controlling gene regulation; however, a small set of promoters is used for most genetic construct design in the yeast Saccharomyces cerevisiae. Generating and utilizing models that accurately predict protein expression from promoter sequences would enable rapid generation of useful promoters and facilitate synthetic biology efforts in this model organism. We measure the gene expression activity of over 675,000 sequences in a constitutive promoter library and over 327,000 sequences in an inducible promoter library. Training an ensemble of convolutional neural networks jointly on the two data sets enables very high (R2 > 0.79) predictive accuracies on multiple sequence-activity prediction tasks. We describe model-guided design strategies that yield large, sequence-diverse sets of promoters exhibiting activities higher than those represented in training data and similar to current best-in-class sequences. Our results show the value of model-guided design as an approach for generating useful DNA parts.
    DOI:  https://doi.org/10.1038/s41467-020-15977-4
  32. Nat Commun. 2020 Apr 28. 11(1): 2061
    Wu Y, Qi T, Wang H, Zhang F, Zheng Z, Phillips-Cremins JE, Deary IJ, McRae AF, Wray NR, Zeng J, Yang J.
      Promoter-anchored chromatin interactions (PAIs) play a pivotal role in transcriptional regulation. Current high-throughput technologies for detecting PAIs, such as promoter capture Hi-C, are not scalable to large cohorts. Here, we present an analytical approach that uses summary-level data from cohort-based DNA methylation (DNAm) quantitative trait locus (mQTL) studies to predict PAIs. Using mQTL data from human peripheral blood ([Formula: see text]), we predict 34,797 PAIs which show strong overlap with the chromatin contacts identified by previous experimental assays. The promoter-interacting DNAm sites are enriched in enhancers or near expression QTLs. Genes whose promoters are involved in PAIs are more actively expressed, and gene pairs with promoter-promoter interactions are enriched for co-expression. Integration of the predicted PAIs with GWAS data highlight interactions among 601 DNAm sites associated with 15 complex traits. This study demonstrates the use of mQTL data to predict PAIs and provides insights into the role of PAIs in complex trait variation.
    DOI:  https://doi.org/10.1038/s41467-020-15587-0
  33. iScience. 2020 Apr 10. pii: S2589-0042(20)30231-5. [Epub ahead of print]23(5): 101046
    Pérez-Rico YA, Barillot E, Shkumatava A.
      CCCTC-binding factor (CTCF) is a conserved architectural protein that plays crucial roles in gene regulation and three-dimensional (3D) chromatin organization. To better understand mechanisms and evolution of vertebrate genome organization, we analyzed genome occupancy of CTCF in zebrafish utilizing an endogenously epitope-tagged CTCF knock-in allele. Zebrafish CTCF shares similar facets with its mammalian counterparts, including binding to enhancers, active promoters and repeat elements, and bipartite sequence motifs of its binding sites. However, we found that in vivo CTCF binding is not enriched at boundaries of topologically associating domains (TADs) in developing zebrafish, whereas TAD demarcation by chromatin marks did not differ from mammals. Our data suggest that general mechanisms underlying 3D chromatin organization, and in particular the involvement of CTCF in this process, differ between distant vertebrate species.
    Keywords:  Biological Sciences; Chromosome Organization; Molecular Biology
    DOI:  https://doi.org/10.1016/j.isci.2020.101046
  34. Nat Chem Biol. 2020 Apr 27.
    Shu X, Cao J, Cheng M, Xiang S, Gao M, Li T, Ying X, Wang F, Yue Y, Lu Z, Dai Q, Cui X, Ma L, Wang Y, He C, Feng X, Liu J.
      Transcriptome-wide mapping of N6-methyladenosine (m6A) at base resolution remains an issue, impeding our understanding of m6A roles at the nucleotide level. Here, we report a metabolic labeling method to detect mRNA m6A transcriptome-wide at base resolution, called 'm6A-label-seq'. Human and mouse cells could be fed with a methionine analog, Se-allyl-L-selenohomocysteine, which substitutes the methyl group on the enzyme cofactor SAM with the allyl. Cellular RNAs could therefore be metabolically modified with N6-allyladenosine (a6A) at supposed m6A-generating adenosine sites. We pinpointed the mRNA a6A locations based on iodination-induced misincorporation at the opposite site in complementary DNA during reverse transcription. We identified a few thousand mRNA m6A sites in human HeLa, HEK293T and mouse H2.35 cells, carried out a parallel comparison of m6A-label-seq with available m6A sequencing methods, and validated selected sites by an orthogonal method. This method offers advantages in detecting clustered m6A sites and holds promise to locate nuclear nascent RNA m6A modifications.
    DOI:  https://doi.org/10.1038/s41589-020-0526-9
  35. Oncogene. 2020 Apr 29.
    Sundqvist A, Vasilaki E, Voytyuk O, Bai Y, Morikawa M, Moustakas A, Miyazono K, Heldin CH, Ten Dijke P, van Dam H.
      Activator protein (AP)-1 transcription factors are essential elements of the pro-oncogenic functions of transforming growth factor-β (TGFβ)-SMAD signaling. Here we show that in multiple HER2+ and/or EGFR+ breast cancer cell lines these AP-1-dependent tumorigenic properties of TGFβ critically rely on epidermal growth factor receptor (EGFR) activation and expression of the ΔN isoform of transcriptional regulator p63. EGFR and ΔNp63 enabled and/or potentiated the activation of a subset of TGFβ-inducible invasion/migration-associated genes, e.g., ITGA2, LAMB3, and WNT7A/B, and enhanced the recruitment of SMAD2/3 to these genes. The TGFβ- and EGF-induced binding of SMAD2/3 and JUNB to these gene loci was accompanied by p63-SMAD2/3 and p63-JUNB complex formation. p63 and EGFR were also found to strongly potentiate TGFβ induction of AP-1 proteins and, in particular, FOS family members. Ectopic overexpression of FOS could counteract the decrease in TGFβ-induced gene activation after p63 depletion. p63 is also involved in the transcriptional regulation of heparin binding (HB)-EGF and EGFR genes, thereby establishing a self-amplification loop that facilitates and empowers the pro-invasive functions of TGFβ. These cooperative pro-oncogenic functions of EGFR, AP-1, p63, and TGFβ were efficiently inhibited by clinically relevant chemical inhibitors. Our findings may, therefore, be of importance for therapy of patients with breast cancers with an activated EGFR-RAS-RAF pathway.
    DOI:  https://doi.org/10.1038/s41388-020-1299-z
  36. Immunity. 2020 Apr 22. pii: S1074-7613(20)30133-3. [Epub ahead of print]
    Johnson JL, Rosenthal RL, Knox JJ, Myles A, Naradikian MS, Madej J, Kostiv M, Rosenfeld AM, Meng W, Christensen SR, Hensley SE, Yewdell J, Canaday DH, Zhu J, McDermott AB, Dori Y, Itkin M, Wherry EJ, Pardi N, Weissman D, Naji A, Prak ETL, Betts MR, Cancro MP.
      B cell subsets expressing the transcription factor T-bet are associated with humoral immune responses and autoimmunity. Here, we examined the anatomic distribution, clonal relationships, and functional properties of T-bet+ and T-bet- memory B cells (MBCs) in the context of the influenza-specific immune response. In mice, both T-bet- and T-bet+ hemagglutinin (HA)-specific B cells arose in germinal centers, acquired memory B cell markers, and persisted indefinitely. Lineage tracing and IgH repertoire analyses revealed minimal interconversion between T-bet- and T-bet+ MBCs, and parabionts showed differential tissue residency and recirculation properties. T-bet+ MBCs could be subdivided into recirculating T-betlo MBCs and spleen-resident T-bethi MBCs. Human MBCs displayed similar features. Conditional gene deletion studies revealed that T-bet expression in B cells was required for nearly all HA stalk-specific IgG2c antibodies and for durable neutralizing titers to influenza. Thus, T-bet expression distinguishes MBC subsets that have profoundly different homing, residency, and functional properties, and mediate distinct aspects of humoral immune memory.
    Keywords:  Age-associated B cells; B cell memory; BCR sequencing; Humoral immunity; T-bet(+) B cells; antibody; hemagglutinin stalk; immune repertoire profiling; influenza; tissue-resident
    DOI:  https://doi.org/10.1016/j.immuni.2020.03.020
  37. PLoS Comput Biol. 2020 Apr 27. 16(4): e1007794
    Zhang Y, Kim MS, Reichenberger ER, Stear B, Taylor DM.
      In single-cell RNA-seq (scRNA-seq) experiments, the number of individual cells has increased exponentially, and the sequencing depth of each cell has decreased significantly. As a result, analyzing scRNA-seq data requires extensive considerations of program efficiency and method selection. In order to reduce the complexity of scRNA-seq data analysis, we present scedar, a scalable Python package for scRNA-seq exploratory data analysis. The package provides a convenient and reliable interface for performing visualization, imputation of gene dropouts, detection of rare transcriptomic profiles, and clustering on large-scale scRNA-seq datasets. The analytical methods are efficient, and they also do not assume that the data follow certain statistical distributions. The package is extensible and modular, which would facilitate the further development of functionalities for future requirements with the open-source development community. The scedar package is distributed under the terms of the MIT license at https://pypi.org/project/scedar.
    DOI:  https://doi.org/10.1371/journal.pcbi.1007794
  38. Int J Mol Sci. 2020 Apr 24. pii: E3000. [Epub ahead of print]21(8):
    D'Ignazio L, Shakir D, Batie M, Muller HA, Rocha S.
      NF-κB signalling is crucial for cellular responses to inflammation but is also associated with the hypoxia response. NF-κB and hypoxia inducible factor (HIF) transcription factors possess an intense molecular crosstalk. Although it is known that HIF-1α modulates NF-κB transcriptional response, very little is understood regarding how HIF-1β contributes to NF-κB signalling. Here, we demonstrate that HIF-1β is required for full NF-κB activation in cells following canonical and non-canonical stimuli. We found that HIF-1β specifically controls TRAF6 expression in human cells but also in Drosophila melanogaster. HIF-1β binds to the TRAF6 gene and controls its expression independently of HIF-1α. Furthermore, exogenous TRAF6 expression is able to rescue all of the cellular phenotypes observed in the absence of HIF-1β. These results indicate that HIF-1β is an important regulator of NF-κB with consequences for homeostasis and human disease.
    Keywords:  ARNT; Drosophila; HIF; NF-κB TRAF6; TNF
    DOI:  https://doi.org/10.3390/ijms21083000
  39. Blood. 2020 Apr 29. pii: blood.2019003062. [Epub ahead of print]
    Fagnan A, Bagger FO, Piqué-Borràs MR, Ignacimouttou C, Caulier A, Lopez C, Robert E, Uzan B, Gelsi-Boyer V, Aid Z, Thirant C, Moll U, Tauchmann S, Kurtovic-Kozaric A, Maciejewski JP, Dierks C, Spinelli O, Salmoiraghi S, Pabst T, Shimoda K, Deleuze V, Lapillonne H, Sweeney C, Mansat-De Mas V, Leite B, Kadri Z, Malinge S, de Botton S, Micol JB, Kile BT, Carmichael CL, Iacobucci I, Mullighan CG, Carroll MP, Valent P, Bernard OA, Delabesse E, Vyas P, Birnbaum D, Anguita E, Garcon L, Soler E, Schwaller J, Mercher T.
      Acute erythroleukemia (AML-M6 or AEL) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing three genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (e.g. DNMT3A, TET2 or IDH2), and undefined cases with low mutational burden. We established an erythroid vs. myeloid transcriptomics-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, more than 25% of AEL patients, including in the genetically-undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1 binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicates that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.
    DOI:  https://doi.org/10.1182/blood.2019003062
  40. PLoS One. 2020 ;15(4): e0232271
    Baik B, Yoon S, Nam D.
      Benchmarking RNA-seq differential expression analysis methods using spike-in and simulated RNA-seq data has often yielded inconsistent results. The spike-in data, which were generated from the same bulk RNA sample, only represent technical variability, making the test results less reliable. We compared the performance of 12 differential expression analysis methods for RNA-seq data, including recent variants in widely used software packages, using both RNA spike-in and simulation data for negative binomial (NB) model. Performance of edgeR, DESeq2, and ROTS was particularly different between the two benchmark tests. Then, each method was tested under most extensive simulation conditions especially demonstrating the large impacts of proportion, dispersion, and balance of differentially expressed (DE) genes. DESeq2, a robust version of edgeR (edgeR.rb), voom with TMM normalization (voom.tmm) and sample weights (voom.sw) showed an overall good performance regardless of presence of outliers and proportion of DE genes. The performance of RNA-seq DE gene analysis methods substantially depended on the benchmark used. Based on the simulation results, suitable methods were suggested under various test conditions.
    DOI:  https://doi.org/10.1371/journal.pone.0232271
  41. Gut. 2020 Apr 27. pii: gutjnl-2019-319748. [Epub ahead of print]
    Ajani JA, Xu Y, Huo L, Wang R, Li Y, Wang Y, Pizzi MP, Scott A, Harada K, Ma L, Yao X, Jin J, Zhao W, Dong X, Badgwell BD, Shanbhag N, Tatlonghari G, Estrella JS, Roy-Chowdhuri S, Kobayashi M, Vykoukal JV, Hanash SM, Calin GA, Peng G, Lee JS, Johnson RL, Wang Z, Wang L, Song S.
      OBJECTIVE: Peritoneal carcinomatosis (PC; malignant ascites or implants) occurs in approximately 45% of advanced gastric adenocarcinoma (GAC) patients and associated with a poor survival. The molecular events leading to PC are unknown. The yes-associated protein 1 (YAP1) oncogene has emerged in many tumour types, but its clinical significance in PC is unclear. Here, we investigated the role of YAP1 in PC and its potential as a therapeutic target.METHODS: Patient-derived PC cells, patient-derived xenograft (PDX) and patient-derived orthotopic (PDO) models were used to study the function of YAP1 in vitro and in vivo. Immunofluorescence and immunohistochemical staining, RNA sequencing (RNA-Seq) and single-cell RNA-Seq (sc-RNA-Seq) were used to elucidate the expression of YAP1 and PC cell heterogeneity. LentiCRISPR/Cas9 knockout of YAP1 and a YAP1 inhibitor were used to dissect its role in PC metastases.
    RESULTS: YAP1 was highly upregulated in PC tumour cells, conferred cancer stem cell (CSC) properties and appeared to be a metastatic driver. Dual staining of YAP1/EpCAM and sc-RNA-Seq revealed that PC tumour cells were highly heterogeneous, YAP1high PC cells had CSC-like properties and easily formed PDX/PDO tumours but also formed PC in mice, while genetic knockout YAP1 significantly slowed tumour growth and eliminated PC in PDO model. Additionally, pharmacologic inhibition of YAP1 specifically reduced CSC-like properties and suppressed tumour growth in YAP1high PC cells especially in combination with cytotoxics in vivo PDX model.
    CONCLUSIONS: YAP1 is essential for PC that is attenuated by YAP1 inhibition. Our data provide a strong rationale to target YAP1 in clinic for GAC patients with PC.
    Keywords:  gastric adenocarcinoma; gene regulation; molecular oncology
    DOI:  https://doi.org/10.1136/gutjnl-2019-319748
  42. Cancer Discov. 2020 Apr 29. pii: CD-19-0789. [Epub ahead of print]
    Leon TE, Rapoz-D'Silva T, Bertoli C, Rahman S, Magnussen M, Philip B, Farah N, Richardson SE, Ahrabi S, Afonso Guerra-Assuncao J, Gupta R, Nacheva EP, Henderson S, Herrero J, Linch DC, de Bruin RAM, Mansour MR.
      Loss-of-function mutations of EZH2, the enzymatic component of PRC2, have been associated with poor outcome and chemotherapy resistance in T-cell acute lymphoblastic leukemia (T-ALL). Using isogenic T-ALL cells, with and without CRISPR/Cas9-induced EZH2-inactivating mutations, we performed a cell-based synthetic lethal drug screen. EZH2 deficient cells exhibited increased sensitivity to structurally diverse inhibitors of CHK1, an interaction that could be validated genetically. Furthermore, small molecule inhibition of CHK1 had efficacy in delaying tumor progression in isogenic EZH2 deficient, but not EZH2 wild-type T-ALL cells in vivo, as well as in a primary cell model of PRC2 mutant ALL. Mechanistically, EZH2 deficiency resulted in a gene expression signature of immature T-ALL cells, marked transcriptional upregulation of MYCN, increased replication stress, and enhanced dependency on CHK1 for cell survival. Lastly, we demonstrate this phenotype is mediated through de-repression of a distal PRC2-regulated MYCN enhancer. In conclusion, we highlight a novel and clinically exploitable pathway in high-risk EZH2 mutated T-ALL.
    DOI:  https://doi.org/10.1158/2159-8290.CD-19-0789