bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2020‒04‒26
fifty-three papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit


  1. Nat Commun. 2020 Apr 24. 11(1): 2020
    Corona RI, Seo JH, Lin X, Hazelett DJ, Reddy J, Fonseca MAS, Abassi F, Lin YG, Mhawech-Fauceglia PY, Shah SP, Huntsman DG, Gusev A, Karlan BY, Berman BP, Freedman ML, Gayther SA, Lawrenson K.
      The functional consequences of somatic non-coding mutations in ovarian cancer (OC) are unknown. To identify regulatory elements (RE) and genes perturbed by acquired non-coding variants, here we establish epigenomic and transcriptomic landscapes of primary OCs using H3K27ac ChIP-seq and RNA-seq, and then integrate these with whole genome sequencing data from 232 OCs. We identify 25 frequently mutated regulatory elements, including an enhancer at 6p22.1 which associates with differential expression of ZSCAN16 (P = 6.6 × 10-4) and ZSCAN12 (P = 0.02). CRISPR/Cas9 knockout of this enhancer induces downregulation of both genes. Globally, there is an enrichment of single nucleotide variants in active binding sites for TEAD4 (P = 6 × 10-11) and its binding partner PAX8 (P = 2×10-10), a known lineage-specific transcription factor in OC. In addition, the collection of cis REs associated with PAX8 comprise the most frequently mutated set of enhancers in OC (P = 0.003). These data indicate that non-coding somatic mutations disrupt the PAX8 transcriptional network during OC development.
    DOI:  https://doi.org/10.1038/s41467-020-15951-0
  2. iScience. 2020 Apr 24. pii: S2589-0042(20)30218-2. [Epub ahead of print]23(4): 101034
    Luo Z, Wang X, Jiang H, Wang R, Chen J, Chen Y, Xu Q, Cao J, Gong X, Wu J, Yang Y, Li W, Han C, Cheng CY, Rosenfeld MG, Sun F, Song X.
      Three-dimensional chromatin structures undergo dynamic reorganization during mammalian spermatogenesis; however, their impacts on gene regulation remain unclear. Here, we focused on understanding the structure-function regulation of meiotic chromosomes by Hi-C and other omics techniques in mouse spermatogenesis across five stages. Beyond confirming recent reports regarding changes in compartmentalization and reorganization of topologically associating domains (TADs), we further demonstrated that chromatin loops are present prior to and after, but not at, the pachytene stage. By integrating Hi-C and RNA-seq data, we showed that the switching of A/B compartments between spermatogenic stages is tightly associated with meiosis-specific mRNAs and piRNAs expression. Moreover, our ATAC-seq data indicated that chromatin accessibility per se is not responsible for the TAD and loop diminishment at pachytene. Additionally, our ChIP-seq data demonstrated that CTCF and cohesin remain bound at TAD boundary regions throughout meiosis, suggesting that dynamic reorganization of TADs does not require CTCF and cohesin clearance.
    Keywords:  Genomics; Male Reproductive Endocrinology; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2020.101034
  3. Mol Oncol. 2020 Apr 24.
    Baumgart SJ, Nevedomskaya E, Lesche R, Newman R, Mumberg D, Haendler B.
      Prostate cancer (PCa) is one of the most frequent tumor types in the male Western population. Early- and late-stage PCa are dependent on androgen signaling, and inhibitors of the androgen receptor (AR) axis represent the standard therapy. Here, we studied in detail the global impact of darolutamide, a newly approved AR antagonist, on the transcriptome and AR-bound cistrome in two PCa cell models. Darolutamide strongly depleted the AR from gene regulatory regions and abolished AR-driven transcriptional signaling. Enhancer activation was blocked at the chromatin level as evaluated by H3K27 acetylation (H3K27ac), H3K4 monomethylation (H3K4me1), and FOXA1, MED1 and BRD4 binding. We identified genomic regions with high affinities for the AR in androgen-stimulated, but also in androgen-depleted conditions. A similar AR affinity pattern was observed in healthy and PCa tissue samples. High FOXA1, BRD4, H3K27ac and H3K4me1 levels were found to mark regions showing AR binding in the hormone-depleted setting. Conversely, low FOXA1, BRD4 and H3K27ac levels were observed at regulatory sites that responded strongly to androgen stimulation, and AR interactions at these sites was blocked by darolutamide. Beside marked loss of AR occupancy, FOXA1 recruitment to chromatin was also clearly reduced after darolutamide treatment. We furthermore identified numerous androgen-regulated super-enhancers (SEs) that were associated with hallmark androgen and cell proliferation-associated gene sets. Importantly, these SEs are also active in PCa tissues and sensitive to darolutamide treatment in our models. Our findings demonstrate that darolutamide is a potent AR antagonist blocking genome-wide AR enhancer and SE activation, and downstream transcription. We also show the existence of a dynamic AR cistrome that depends on the androgen levels and on high AR affinity regions present in PCa cell lines and also in tissue samples.
    Keywords:  FOXA1; androgen receptor; cistrome; histone acetylation; prostate cancer; super-enhancer
    DOI:  https://doi.org/10.1002/1878-0261.12693
  4. Cell Rep. 2020 Apr 21. pii: S2211-1247(20)30430-7. [Epub ahead of print]31(3): 107530
    Lau SF, Chen C, Fu WY, Qu JY, Cheung TH, Fu AKY, Ip NY.
      Impairment of microglial clearance activity contributes to beta-amyloid (Aβ) pathology in Alzheimer's disease (AD). While the transcriptome profile of microglia directs microglial functions, how the microglial transcriptome can be regulated to alleviate AD pathology is largely unknown. Here, we show that injection of interleukin (IL)-33 in an AD transgenic mouse model ameliorates Aβ pathology by reprogramming microglial epigenetic and transcriptomic profiles to induce a microglial subpopulation with enhanced phagocytic activity. These IL-33-responsive microglia (IL-33RMs) express a distinct transcriptome signature that is highlighted by increased major histocompatibility complex class II genes and restored homeostatic signature genes. IL-33-induced remodeling of chromatin accessibility and PU.1 transcription factor binding at the signature genes of IL-33RM control their transcriptome reprogramming. Specifically, disrupting PU.1-DNA interaction abolishes the microglial state transition and Aβ clearance that is induced by IL-33. Thus, we define a PU.1-dependent transcriptional pathway that drives the IL-33-induced functional state transition of microglia, resulting in enhanced Aβ clearance.
    Keywords:  MHC-II; PU.1; beta-amyloid; chemotaxis; chromatin accessibility; disease-associated microglia; epigenetics; interleukins; phagocytosis; single-cell RNA-sequencing
    DOI:  https://doi.org/10.1016/j.celrep.2020.107530
  5. Front Genet. 2020 ;11 338
    Ye B, Yang G, Li Y, Zhang C, Wang Q, Yu G.
      ZNF143, a human homolog of the transcriptional activator Staf, is a C2H2-type protein consisting of seven zinc finger domains. As a transcription factor (TF), ZNF143 is sequence specifically binding to chromatin and activates the expression of protein-coding and non-coding genes on a genome scale. Although it is ubiquitous expressed, its expression in cancer cells and tissues is usually higher than that in normal cells and tissues. Therefore, abnormal expression of ZNF143 is related to cancer cell survival, proliferation, differentiation, migration, and invasion, suggesting that new small molecules can be designed by targeting ZNF143 as it may be a good potential biomarker and therapeutic target for related cancers. However, the mechanism on how ZNF143 regulates its targeting gene remains unclear. Recently, with the development of chromatin conformation capture (3C) and its derivatives, and high-throughput sequencing technology, new findings have been obtained in the study of ZNF143. Pioneering studies have showed that ZNF143 binds directly to promoters and contributes to chromatin interactions connecting promoters to distal regulatory elements, such as enhancers. Further, it has proved that ZNF143 is involved in CCCTC-binding factor (CTCF) in establishing the conserved chromatin loops by cooperating with cohesin and other partners. These results indicate that ZNF143 is a key loop formation factor. In addition, we report ZNF143 is dynamically bound to chromatin during the cell cycle demonstrated that it is a potential mitotic bookmarking factor. It may be associated with CTCF for mitosis-to-G1 phase transition and chromatin loop re-establishment in early G1 phase. In the future, researchers could further clarify the fine mechanism of ZNF143 in mediating chromatin loops with the help of CUT&RUN (CUT&Tag) and Cut-C technology. Thus, in this review, we summarize the research progress of TF ZNF143 in detail and also predict the potential functions of ZNF143 in cell fate and identity based on our recent discoveries.
    Keywords:  ZNF143; biomarker; chromatin organization; loop; transcription factor
    DOI:  https://doi.org/10.3389/fgene.2020.00338
  6. Cancer Cell. 2020 Apr 14. pii: S1535-6108(20)30162-8. [Epub ahead of print]
    Di Genua C, Valletta S, Buono M, Stoilova B, Sweeney C, Rodriguez-Meira A, Grover A, Drissen R, Meng Y, Beveridge R, Aboukhalil Z, Karamitros D, Belderbos ME, Bystrykh L, Thongjuea S, Vyas P, Nerlov C.
      Acute erythroid leukemia (AEL) commonly involves both myeloid and erythroid lineage transformation. However, the mutations that cause AEL and the cell(s) that sustain the bilineage leukemia phenotype remain unknown. We here show that combined biallelic Cebpa and Gata2 zinc finger-1 (ZnF1) mutations cooperatively induce bilineage AEL, and that the major leukemia-initiating cell (LIC) population has a neutrophil-monocyte progenitor (NMP) phenotype. In pre-leukemic NMPs Cebpa and Gata2 mutations synergize by increasing erythroid transcription factor (TF) expression and erythroid TF chromatin access, respectively, thereby installing ectopic erythroid potential. This erythroid-permissive chromatin conformation is retained in bilineage LICs. These results demonstrate that synergistic transcriptional and epigenetic reprogramming by leukemia-initiating mutations can generate neomorphic pre-leukemic progenitors, defining the lineage identity of the resulting leukemia.
    Keywords:  CEBPA; GATA2; acute erythroid leukemia; acute myeloid leukemia; chromatin accessibility; leukemia-initiating cell; lineage priming; oncogene co-operation
    DOI:  https://doi.org/10.1016/j.ccell.2020.03.022
  7. Cancer Res. 2020 Apr 20. pii: canres.3226.2019. [Epub ahead of print]
    Zhang L, Huo Q, Ge C, Zhao F, Zhou Q, Chen X, Tian H, Chen T, Xie H, Cui Y, Yao M, Li H, Li J.
      Zinc finger protein 143 (ZNF143) belongs to the zinc finger protein family and possesses transcription factor activity by binding sequence-specific DNA. The exact biological role of ZNF143 in hepatocellular carcinoma (HCC) has not been investigated. Here we report that ZNF143 is overexpressed in HCC tissues and its overexpression correlates with poor prognosis. Gain- and loss-of-function experiments showed that ZNF143 promoted HCC cell proliferation, colony formation, and tumor growth in vitro and in vivo. ZNF143 accelerated HCC cell-cycle progression by activating cell division cycle 6 (CDC6). Mechanistically, ZNF143 promoted expression of CDC6 by directly activating transcription of histone demethylase mineral dust-induced gene (MDIG), which in turn reduced H3K9me3 enrichment in the CDC6 promoter region. Consistently, ZNF143 expression correlated significantly with MDIG and CDC6 expression in HCC. Collectively, we propose a model for a ZNF143-MDIG-CDC6 oncoprotein axis that provides novel insight into ZNF143 which may serve as a therapeutic target in HCC.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-19-3226
  8. J Comput Biol. 2020 Apr 20.
    Ye Y, Yang Z, Lei J.
       During mammalian embryo development, reprogramming of DNA methylation plays important roles in the erasure of parental epigenetic memory and the establishment of naive pluripotent cells. Multiple enzymes that regulate the processes of methylation and demethylation work together to shape the pattern of genome-scale DNA methylation and guide the process of cell differentiation. Recent availability of methylome information from single-cell whole genome bisulfite sequencing (scBS-seq) provides an opportunity to study DNA methylation dynamics in the whole genome in individual cells, which reveal the heterogeneous methylation distributions of enhancers in embryo stem cells. In this study, we developed a computational model of enhancer methylation inheritance to study the dynamics of genome-scale DNA methylation reprogramming during exit from pluripotency. The model enables us to track genome-scale DNA methylation reprogramming at single-cell level during the embryo development process and reproduce the DNA methylation heterogeneity reported by scBS-seq. Model simulations show that DNA methylation heterogeneity is an intrinsic property driven by cell division along the development process, and the collaboration between neighboring enhancers is required for heterogeneous methylation. Our study suggests that the mechanism of genome-scale oscillation might not be necessary for the DNA methylation heterogenity during exit from pluripotency.
    Keywords:  DNA methylation; embryo development; genome-scale oscillation; heterogeneity; stochastic simulation
    DOI:  https://doi.org/10.1089/cmb.2019.0413
  9. Science. 2020 Apr 23. pii: eabb0074. [Epub ahead of print]
    Michael AK, Grand RS, Isbel L, Cavadini S, Kozicka Z, Kempf G, Bunker RD, Schenk AD, Graff-Meyer A, Pathare GR, Weiss J, Matsumoto S, Burger L, Schübeler D, Thomä NH.
      Transcription factors (TFs) regulate gene expression through chromatin where nucleosomes restrict DNA access. To study how TFs bind nucleosome-occupied motifs we focused on the reprogramming factors OCT4 and SOX2. We determined TF engagement throughout a nucleosome at base-pair resolution in vitro, enabling cryo-EM structure determination at two preferred positions. Depending on motif location, OCT4-SOX2 differentially distort nucleosomal DNA. At one position, OCT4-SOX2 removes DNA from Histone H2A/Histone H3 (H2A/H3); however, at an inverted motif, the TFs only induce local DNA distortions. OCT4 uses one of its two DNA binding domains to engage DNA in both structures, reading-out a partial motif. These findings explain site specific nucleosome engagement by the pluripotency factors OCT4-SOX2 and reveal how TFs distort nucleosomes to access chromatinized motifs.
    DOI:  https://doi.org/10.1126/science.abb0074
  10. Nucleic Acids Res. 2020 Apr 20. pii: gkaa232. [Epub ahead of print]
    Wu Y, Wang X, Xu F, Zhang L, Wang T, Fu X, Jin T, Zhang W, Ye L.
      High-mobility group AT-hook 2 (HMGA2) is an architectural transcription factor that plays essential roles in embryonic development and cancer progression. However, the mechanism of HMGA2 regulation remains largely uncharacterized. Here, we demonstrate that HMGA2 can be modulated by hepatitis B X-interacting protein (HBXIP), an oncogenic transcriptional coactivator, in esophageal squamous cell carcinoma (ESCC). HMGA2 expression was positively associated with HBXIP expression in clinical ESCC tissues, and their high levels were associated with advanced tumor stage and reduced overall and disease-free survival. We found that oncogenic HBXIP could posttranslationally upregulate HMGA2 protein level in ESCC cells. HBXIP induced HMGA2 acetylation at the lysine 26 (K26), resulting in HMGA2 protein accumulation. In this process, HBXIP increased the acetyltransferase p300/CBP-associated factor (PCAF) phosphorylation and activation via the Akt pathway, then PCAF directly interacted with HMGA2, leading to HMGA2 acetylation in the cells. HMGA2 K26 acetylation enhanced its DNA binding capacity and blocked its ubiquitination and then inhibited proteasome-dependent degradation. Functionally, HBXIP-stabilized HMGA2 could promote ESCC cell growth in vitro and in vivo. Strikingly, aspirin suppressed ESCC growth by inhibiting HBXIP and HMGA2. Collectively, our findings disclose a new mechanism for the posttranslational regulation of HMGA2 mediated by HBXIP in ESCC.
    DOI:  https://doi.org/10.1093/nar/gkaa232
  11. Mol Cell. 2020 Apr 10. pii: S1097-2765(20)30199-4. [Epub ahead of print]
    Gillespie MA, Palii CG, Sanchez-Taltavull D, Shannon P, Longabaugh WJR, Downes DJ, Sivaraman K, Espinoza HM, Hughes JR, Price ND, Perkins TJ, Ranish JA, Brand M.
      Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). However, despite years of studies, our knowledge about the protein copy number of TFs in the nucleus is limited. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first gene regulatory network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs' cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that, in the nucleus, co-repressors are dramatically more abundant than co-activators at the protein level, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.
    Keywords:  absolute quantification; cell fate; erythropoiesis; gene regulatory network; hematopoiesis; protein stoichiometry; proteomics; stem cells; targeted mass spectrometry; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2020.03.031
  12. Mol Cell. 2020 Apr 03. pii: S1097-2765(20)30193-3. [Epub ahead of print]
    Guo R, Jiang C, Zhang Y, Govande A, Trudeau SJ, Chen F, Fry CJ, Puri R, Wolinsky E, Schineller M, Frost TC, Gebre M, Zhao B, Giulino-Roth L, Doench JG, Teng M, Gewurz BE.
      Epstein-Barr virus (EBV) is associated with multiple human malignancies. To evade immune detection, EBV switches between latent and lytic programs. How viral latency is maintained in tumors or in memory B cells, the reservoir for lifelong EBV infection, remains incompletely understood. To gain insights, we performed a human genome-wide CRISPR/Cas9 screen in Burkitt lymphoma B cells. Our analyses identified a network of host factors that repress lytic reactivation, centered on the transcription factor MYC, including cohesins, FACT, STAGA, and Mediator. Depletion of MYC or factors important for MYC expression reactivated the lytic cycle, including in Burkitt xenografts. MYC bound the EBV genome origin of lytic replication and suppressed its looping to the lytic cycle initiator BZLF1 promoter. Notably, MYC abundance decreases with plasma cell differentiation, a key lytic reactivation trigger. Our results suggest that EBV senses MYC abundance as a readout of B cell state and highlights Burkitt latency reversal therapeutic targets.
    Keywords:  DNA looping; MYC; herpesvirus; latency reversal; lytic reactivation; tumor virus
    DOI:  https://doi.org/10.1016/j.molcel.2020.03.025
  13. Epigenetics Chromatin. 2020 Apr 22. 13(1): 22
    Reske JJ, Wilson MR, Chandler RL.
      BACKGROUND: Chromatin dysregulation is associated with developmental disorders and cancer. Numerous methods for measuring genome-wide chromatin accessibility have been developed in the genomic era to interrogate the function of chromatin regulators. A recent technique which has gained widespread use due to speed and low input requirements with native chromatin is the Assay for Transposase-Accessible Chromatin, or ATAC-seq. Biologists have since used this method to compare chromatin accessibility between two cellular conditions. However, approaches for calculating differential accessibility can yield conflicting results, and little emphasis is placed on choice of normalization method during differential ATAC-seq analysis, especially when global chromatin alterations might be expected.RESULTS: Using an in vivo ATAC-seq data set generated in our recent report, we observed differences in chromatin accessibility patterns depending on the data normalization method used to calculate differential accessibility. This observation was further verified on published ATAC-seq data from yeast. We propose a generalized workflow for differential accessibility analysis using ATAC-seq data. We further show this workflow identifies sites of differential chromatin accessibility that correlate with gene expression and is sensitive to differential analysis using negative controls.
    CONCLUSIONS: We argue that researchers should systematically compare multiple normalization methods before continuing with differential accessibility analysis. ATAC-seq users should be aware of the interpretations of potential bias within experimental data and the assumptions of the normalization method implemented.
    Keywords:  ATAC-seq; Bioinformatics; Chromatin accessibility; Differential accessibility; Genomics; Normalization
    DOI:  https://doi.org/10.1186/s13072-020-00342-y
  14. Genome Biol. 2020 Apr 20. 21(1): 94
    Yu W, Uzun Y, Zhu Q, Chen C, Tan K.
      Single-cell chromatin accessibility sequencing has become a powerful technology for understanding epigenetic heterogeneity of complex tissues. However, there is a lack of open-source software for comprehensive processing, analysis, and visualization of such data generated using all existing experimental protocols. Here, we present scATAC-pro for quality assessment, analysis, and visualization of single-cell chromatin accessibility sequencing data. scATAC-pro computes a range of quality control metrics for several key steps of experimental protocols, with a flexible choice of methods. It generates summary reports for both quality assessment and downstream analysis. scATAC-pro is available at https://github.com/tanlabcode/scATAC-pro.
    Keywords:  Bioinformatics; Chromatin accessibility; Genomics; Single cell
    DOI:  https://doi.org/10.1186/s13059-020-02008-0
  15. Mol Cell Biol. 2020 Apr 20. pii: MCB.00460-19. [Epub ahead of print]
    Feng W, Chen S, Wang J, Wang X, Chen H, Ning W, Zhang Y.
      RNA helicase DHX33 was found to regulate the transcription of multiple genes involved in cancer development. But the underlying molecular mechanism remains unclear. Here in this study, we found DHX33 associated extensively with gene promoters at CG rich region. Its deficiency reduced the loading of active RNA polymerase II at gene promoters. Furthermore, we observed a functional interaction between DHX33, AP-2β and DNA demethylation protein Gadd45a (growth arrest and DNA damage inductile protein 45a) at specific gene promoters. DHX33 is required to recruit GADD45a, thereby causes local DNA demethylation through further recruiting ten-eleven-translocation (Tet methylcytosine dioxygenase) enzyme, as manifested by reduced 5-hydroxymethyl cytosine levels for a subset of genes after DHX33 deficiency. This process might involve R-loop formation in GC skew as a guidance signal at promoter sites. Our study for the first time provides the original evidence for DHX33 to alter epigenetic marks and regulate specific gene transcription through interaction with Gadd45a.
    DOI:  https://doi.org/10.1128/MCB.00460-19
  16. Stem Cell Reports. 2020 Apr 15. pii: S2213-6711(20)30116-8. [Epub ahead of print]
    Kwon JB, Vankara A, Ettyreddy AR, Bohning JD, Gersbach CA.
      Engineered CRISPR/Cas9-based transcriptional activators can potently and specifically activate endogenous fate-determining genes to direct differentiation of pluripotent stem cells. Here, we demonstrate that endogenous activation of the PAX7 transcription factor results in stable epigenetic remodeling and differentiates human pluripotent stem cells into skeletal myogenic progenitor cells. Compared with exogenous overexpression of PAX7 cDNA, we find that endogenous activation results in the generation of more proliferative myogenic progenitors that can maintain PAX7 expression over multiple passages in serum-free conditions while preserving the capacity for terminal myogenic differentiation. Transplantation of human myogenic progenitors derived from endogenous activation of PAX7 into immunodeficient mice resulted in a greater number of human dystrophin+ myofibers compared with exogenous PAX7 overexpression. RNA-sequencing analysis also revealed transcriptome-wide differences between myogenic progenitors generated via CRISPR-based endogenous activation of PAX7 and exogenous PAX7 cDNA overexpression. These studies demonstrate the utility of CRISPR/Cas9-based transcriptional activators for controlling cell-fate decisions.
    Keywords:  CRISPR; differentiation; epigenome editing; iPSCs; myoblasts; myogenesis; pluripotent; satellite cells
    DOI:  https://doi.org/10.1016/j.stemcr.2020.03.026
  17. Methods Mol Biol. 2020 ;2154 197-215
    Bar C, Valdes VJ, Ezhkova E.
      Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a method designed to detect interactions between chromatin and the proteins bound to it. This method has been widely used for characterizing epigenetic landscapes in many cell types; however, a limiting factor has been the requirement of a high number of cells. Here, we describe a protocol for ChIP in epidermal cells from a newborn mouse, purified by fluorescence-activated cell sorting (FACS). This protocol has been optimized specifically for prefixed, low cell numbers, resulting in enough immunoprecipitated DNA suitable for genome-wide analysis.
    Keywords:  ChIP-seq; Epidermal cells; FACS purified cells; Low cell number ChIP
    DOI:  https://doi.org/10.1007/978-1-0716-0648-3_17
  18. Cell. 2020 Apr 13. pii: S0092-8674(20)30348-2. [Epub ahead of print]
    Sladitschek HL, Fiuza UM, Pavlinic D, Benes V, Hufnagel L, Neveu PA.
      Single-cell RNA sequencing (scRNA-seq) provides a leap forward in resolving cellular diversity and developmental trajectories but fails to comprehensively delineate the spatial organization and precise cellular makeup of individual embryos. Here, we reconstruct from scRNA-seq and light sheet imaging data a canonical digital embryo that captures the genome-wide gene expression trajectory of every single cell at every cell division in the 18 lineages up to gastrulation in the ascidian Phallusia mammillata. By using high-coverage scRNA-seq, we devise a computational framework that stratifies single cells of individual embryos into cell types without prior knowledge. Unbiased transcriptome data analysis mapped each cell's physical position and lineage history, yielding the complete history of gene expression at the genome-wide level for every single cell in a developing embryo. A comparison of individual embryos reveals both extensive reproducibility between symmetric embryo sides and a large inter-embryonic variability due to small differences in embryogenesis timing.
    Keywords:  ascidian; cell fate specification; cell type classification; embryogenesis; gene expression noise; light sheet imaging; lineage reconstruction; single-cell RNA sequencing; spatial reconstruction
    DOI:  https://doi.org/10.1016/j.cell.2020.03.055
  19. Plant J. 2020 Apr 23.
    An Z, Yin L, Liu Y, Peng M, Shen WH, Dong A.
      Histones are highly basic proteins involved in packaging DNA into chromatin, and histone modifications are fundamental in epigenetic regulation in eukaryotes. Among the numerous chromatin modifiers identified in Arabidopsis (Arabidopsis thaliana), MORF RELATED GENE 1 (MRG1) and MRG2 have redundant functions in reading histone H3 lysine 36 trimethylation (H3K36me3). Here, we show that MRG2 binds histone chaperones belonging to the NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) family, including NAP1-RELATED PROTEIN 1 (NRP1) and NRP2. Characterization of the loss-of-function mutants mrg1 mrg2, nrp1 nrp2 and mrg1 mrg2 nrp1 nrp2 revealed that MRG1/MRG2 and NRP1/NRP2 regulate flowering time through fine-tuning transcription of floral genes by distinct molecular mechanisms. In particular, the physical interaction between NRP1/NRP2 and MRG1/MRG2 inhibited the binding of MRG1/MRG2 to the transcription factor CONSTANS (CO), leading to a transcriptional repression of FLOWERING LOCUS T (FT) through impeded H4K5 acetylation (H4K5ac) within the FT chromatin. In contrast, NRP1/2 and MRG1/MRG2 act together, likely in a multiprotein complex manner, in promoting transcription of FLOWERING LOCUS C (FLC) via increase of both H4K5ac and H3K9ac in the FLC chromatin. Because the expression pattern of FLC represents the major category of differentially expressed genes identified by genome-wide RNA-seq analysis in the mrg1 mrg2, nrp1 nrp2 and mrg1 mrg2 nrp1 nrp2 mutants, it is reasonable to speculate that the NRP1/2-MRG1/MRG2 complex may be involved in transcriptional activation of genes beyond FLC and flowering time control.
    Keywords:  Arabidopsis thaliana; flowering; histone chaperone; histone methylation reader; transcription
    DOI:  https://doi.org/10.1111/tpj.14780
  20. Epigenetics Chromatin. 2020 Apr 22. 13(1): 21
    Pongor LS, Gross JM, Vera Alvarez R, Murai J, Jang SM, Zhang H, Redon C, Fu H, Huang SY, Thakur B, Baris A, Marino-Ramirez L, Landsman D, Aladjem MI, Pommier Y.
      BACKGROUND: Next-generation sequencing allows genome-wide analysis of changes in chromatin states and gene expression. Data analysis of these increasingly used methods either requires multiple analysis steps, or extensive computational time. We sought to develop a tool for rapid quantification of sequencing peaks from diverse experimental sources and an efficient method to produce coverage tracks for accurate visualization that can be intuitively displayed and interpreted by experimentalists with minimal bioinformatics background. We demonstrate its strength and usability by integrating data from several types of sequencing approaches.RESULTS: We have developed BAMscale, a one-step tool that processes a wide set of sequencing datasets. To demonstrate the usefulness of BAMscale, we analyzed multiple sequencing datasets from chromatin immunoprecipitation sequencing data (ChIP-seq), chromatin state change data (assay for transposase-accessible chromatin using sequencing: ATAC-seq, DNA double-strand break mapping sequencing: END-seq), DNA replication data (Okazaki fragments sequencing: OK-seq, nascent-strand sequencing: NS-seq, single-cell replication timing sequencing: scRepli-seq) and RNA-seq data. The outputs consist of raw and normalized peak scores (multiple normalizations) in text format and scaled bigWig coverage tracks that are directly accessible to data visualization programs. BAMScale also includes a visualization module facilitating direct, on-demand quantitative peak comparisons that can be used by experimentalists. Our tool can effectively analyze large sequencing datasets (~ 100 Gb size) in minutes, outperforming currently available tools.
    CONCLUSIONS: BAMscale accurately quantifies and normalizes identified peaks directly from BAM files, and creates coverage tracks for visualization in genome browsers. BAMScale can be implemented for a wide set of methods for calculating coverage tracks, including ChIP-seq and ATAC-seq, as well as methods that currently require specialized, separate tools for analyses, such as splice-aware RNA-seq, END-seq and OK-seq for which no dedicated software is available. BAMscale is freely available on github (https://github.com/ncbi/BAMscale).
    Keywords:  ATAC-seq; ChIP-seq; Expression; Histone modifications; NS-seq; RNA-seq; Replication origins; Replication timing; SLFN11
    DOI:  https://doi.org/10.1186/s13072-020-00343-x
  21. Nucleic Acids Res. 2020 Apr 24. pii: gkaa264. [Epub ahead of print]
    Song Q, Lee J, Akter S, Rogers M, Grene R, Li S.
      Recent advances in genomic technologies have generated data on large-scale protein-DNA interactions and open chromatin regions for many eukaryotic species. How to identify condition-specific functions of transcription factors using these data has become a major challenge in genomic research. To solve this problem, we have developed a method called ConSReg, which provides a novel approach to integrate regulatory genomic data into predictive machine learning models of key regulatory genes. Using Arabidopsis as a model system, we tested our approach to identify regulatory genes in data sets from single cell gene expression and from abiotic stress treatments. Our results showed that ConSReg accurately predicted transcription factors that regulate differentially expressed genes with an average auROC of 0.84, which is 23.5-25% better than enrichment-based approaches. To further validate the performance of ConSReg, we analyzed an independent data set related to plant nitrogen responses. ConSReg provided better rankings of the correct transcription factors in 61.7% of cases, which is three times better than other plant tools. We applied ConSReg to Arabidopsis single cell RNA-seq data, successfully identifying candidate regulatory genes that control cell wall formation. Our methods provide a new approach to define candidate regulatory genes using integrated genomic data in plants.
    DOI:  https://doi.org/10.1093/nar/gkaa264
  22. Nat Commun. 2020 Apr 24. 11(1): 1971
    Jew B, Alvarez M, Rahmani E, Miao Z, Ko A, Garske KM, Sul JH, Pietiläinen KH, Pajukanta P, Halperin E.
      We present Bisque, a tool for estimating cell type proportions in bulk expression. Bisque implements a regression-based approach that utilizes single-cell RNA-seq (scRNA-seq) or single-nucleus RNA-seq (snRNA-seq) data to generate a reference expression profile and learn gene-specific bulk expression transformations to robustly decompose RNA-seq data. These transformations significantly improve decomposition performance compared to existing methods when there is significant technical variation in the generation of the reference profile and observed bulk expression. Importantly, compared to existing methods, our approach is extremely efficient, making it suitable for the analysis of large genomic datasets that are becoming ubiquitous. When applied to subcutaneous adipose and dorsolateral prefrontal cortex expression datasets with both bulk RNA-seq and snRNA-seq data, Bisque replicates previously reported associations between cell type proportions and measured phenotypes across abundant and rare cell types. We further propose an additional mode of operation that merely requires a set of known marker genes.
    DOI:  https://doi.org/10.1038/s41467-020-15816-6
  23. Nat Commun. 2020 Apr 20. 11(1): 1886
    Sun L, Jing Y, Liu X, Li Q, Xue Z, Cheng Z, Wang D, He H, Qian W.
      In higher eukaryotes, heterochromatin is mainly composed of transposable elements (TEs) silenced by epigenetic mechanisms. But, the silencing of certain heterochromatin-associated TEs is disrupted by heat stress. By comparing genome-wide high-resolution chromatin packing patterns under normal or heat conditions obtained through Hi-C analysis, we show here that heat stress causes global rearrangement of the 3D genome in Arabidopsis thaliana. Contacts between pericentromeric regions and distal chromosome arms, as well as proximal intra-chromosomal interactions along the chromosomes, are enhanced. However, interactions within pericentromeres and those between distal intra-chromosomal regions are decreased. Many inter-chromosomal interactions, including those within the KNOT, are also reduced. Furthermore, heat activation of TEs exhibits a high correlation with the reduction of chromosomal interactions involving pericentromeres, the KNOT, the knob, and the upstream and downstream flanking regions of the activated TEs. Together, our results provide insights into the relationship between TE activation and 3D genome reorganization.
    DOI:  https://doi.org/10.1038/s41467-020-15809-5
  24. Sci Rep. 2020 Apr 21. 10(1): 6751
    McAninch D, Mäkelä JA, La HM, Hughes JN, Lovell-Badge R, Hobbs RM, Thomas PQ.
      SOX3 is a transcription factor expressed within the developing and adult nervous system where it mostly functions to help maintain neural precursors. Sox3 is also expressed in other locations, notably within the spermatogonial stem/progenitor cell population in postnatal testis. Independent studies have shown that Sox3 null mice exhibit a spermatogenic block as young adults, the mechanism of which remains poorly understood. Using a panel of spermatogonial cell marker genes, we demonstrate that Sox3 is expressed within the committed progenitor fraction of the undifferentiated spermatogonial pool. Additionally, we use a Sox3 null mouse model to define a potential role for this factor in progenitor cell function. We demonstrate that Sox3 expression is required for transition of undifferentiated cells from a GFRα1+ self-renewing state to the NGN3 + transit-amplifying compartment. Critically, using chromatin immunoprecipitation, we demonstrate that SOX3 binds to a highly conserved region in the Ngn3 promoter region in vivo, indicating that Ngn3 is a direct target of SOX3. Together these studies indicate that SOX3 functions as a pro-commitment factor in spermatogonial stem/progenitor cells.
    DOI:  https://doi.org/10.1038/s41598-020-63290-3
  25. Circ Res. 2020 Apr 21.
    Beisaw A, Kuenne C, Günther S, Dallmann J, Wu CC, Bentsen M, Looso M, Stainier D.
      Rationale: The adult human heart is an organ with low regenerative potential. Heart failure following acute myocardial infarction is a leading cause of death due to the inability of cardiomyocytes to proliferate and replenish lost cardiac muscle. While the zebrafish has emerged as a powerful model to study endogenous cardiac regeneration, the molecular mechanisms by which cardiomyocytes respond to damage by disassembling sarcomeres, proliferating, and repopulating the injured area remain unclear. Furthermore, we are far from understanding the regulation of the chromatin landscape and epigenetic barriers that must be overcome for cardiac regeneration to occur. Objective: To identify transcription factor regulators of the chromatin landscape which promote cardiomyocyte regeneration in zebrafish, and investigate their function.Methods and Results: Using the Assay for Transposase-Accessible Chromatin coupled to high-throughput sequencing (ATAC-Seq), we first find that the regenerating cardiomyocyte chromatin accessibility landscape undergoes extensive changes following cryoinjury, and that AP-1 binding sites are the most highly enriched motifs in regions that gain accessibility during cardiac regeneration. Furthermore, using bioinformatic and gene expression analyses, we find that the AP-1 response in regenerating adult zebrafish cardiomyocytes is largely different from the response in adult mammalian cardiomyocytes. Using a cardiomyocyte-specific dominant negative approach, we show that blocking AP-1 function leads to defects in cardiomyocyte proliferation as well as decreased chromatin accessibility at the fbxl22 and ilk loci, which regulate sarcomere disassembly and cardiomyocyte protrusion into the injured area, respectively. We further show that overexpression of the AP-1 family members Junb and Fosl1 can promote changes in mammalian cardiomyocyte behavior in vitro. Conclusions: AP-1 transcription factors play an essential role in the cardiomyocyte response to injury by regulating chromatin accessibility changes, thereby allowing the activation of gene expression programs that promote cardiomyocyte dedifferentiation, proliferation, and protrusion into the injured area.
    Keywords:  ATAC-Seq; cardiomyocyte protrusion; chromatin dynamics; sarcomere disassembly; zebrafish
    DOI:  https://doi.org/10.1161/CIRCRESAHA.119.316167
  26. Bioinformatics. 2020 Apr 23. pii: btaa269. [Epub ahead of print]
    Franzén O, Björkegren JLM.
      SUMMARY: Single cell RNA sequencing is a technology to measure gene expression in single cells. It has enabled discovery of new cell types and established cell type atlases of tissues and organs. The widespread adoption of single cell RNA-seq has created a need for user-friendly software for data analysis. We have developed a web server, alona, that incorporates several of the most popular single cell analysis algorithms into a flexible pipeline. alona can perform quality filtering, normalization, batch correction, clustering, cell type annotation, and differential gene expression analysis. Data are visualized in the web browser using an interface based on JavaScript, allowing the user to query genes of interest and visualize the cluster structure. alona accepts a compressed gene expression matrix and identifies cell clusters with a graph-based clustering strategy. Cell types are identified from a comprehensive collection of marker genes or by specifying a custom set of marker genes.AVAILABILITY AND IMPLEMENTATION: The service runs at https://alona.panglaodb.se and the Python package can be downloaded from https://oscar-franzen.github.io/adobo/.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btaa269
  27. Oncogene. 2020 Apr 20.
    Cassandri M, Butera A, Amelio I, Lena AM, Montanaro M, Mauriello A, Anemona L, Candi E, Knight RA, Agostini M, Melino G.
      Breast cancer is the second leading cause of cancer-related deaths among women, largely due to the progression of a significant fraction of primary tumours to the metastatic stage. Here, we show that zinc-finger protein 750 (ZNF750) opposes the migration and invasion of breast cancer cells by repressing a prometastatic transcriptional programme, which includes genes involved in focal adhesion and extracellular matrix interactions, such as LAMB3 and CTNNAL1. Mechanistically, ZNF750 recruits the epigenetic modifiers KDM1A and HDAC1 to the promoter regions of LAMB3 and CTNNAL1, influencing histone marks and transactivating these genomic sites. Gene expression analysis in cancer patient datasets indicated that ZNF750 and its targets were negative prognostic factors in breast cancer. Together, our findings shed light on the molecular mechanism by which ZNF750 regulates cell migration and invasion, suggesting a role in breast cancer metastasis.
    DOI:  https://doi.org/10.1038/s41388-020-1277-5
  28. Nat Commun. 2020 Apr 24. 11(1): 1987
    Liu D, Shoag JE, Poliak D, Goueli RS, Ravikumar V, Redmond D, Vosoughi A, Fontugne J, Pan H, Lee D, Thomas D, Salari K, Wang Z, Romanel A, Te A, Lee R, Chughtai B, Olumi AF, Mosquera JM, Demichelis F, Elemento O, Rubin MA, Sboner A, Barbieri CE.
      Benign prostatic hyperplasia (BPH), a nonmalignant enlargement of the prostate, is among the most common diseases affecting aging men, but the underlying molecular features remain poorly understood, and therapeutic options are limited. Here we employ a comprehensive molecular investigation of BPH, including genomic, transcriptomic and epigenetic profiling. We find no evidence of neoplastic features in BPH: no evidence of driver genomic alterations, including low coding mutation rates, mutational signatures consistent with aging tissues, minimal copy number alterations, and no genomic rearrangements. At the epigenetic level, global hypermethylation is the dominant process. Integrating transcriptional and methylation signatures identifies two BPH subgroups with distinct clinical features and signaling pathways, validated in two independent cohorts. Finally, mTOR inhibitors emerge as a potential subtype-specific therapeutic option, and men exposed to mTOR inhibitors show a significant decrease in prostate size. We conclude that BPH consists of distinct molecular subgroups, with potential for subtype-specific precision therapy.
    DOI:  https://doi.org/10.1038/s41467-020-15913-6
  29. Sci Rep. 2020 Apr 20. 10(1): 6590
    McIntyre ABR, Gokhale NS, Cerchietti L, Jaffrey SR, Horner SM, Mason CE.
      Many cellular mRNAs contain the modified base m6A, and recent studies have suggested that various stimuli can lead to changes in m6A. The most common method to map m6A and to predict changes in m6A between conditions is methylated RNA immunoprecipitation sequencing (MeRIP-seq), through which methylated regions are detected as peaks in transcript coverage from immunoprecipitated RNA relative to input RNA. Here, we generated replicate controls and reanalyzed published MeRIP-seq data to estimate reproducibility across experiments. We found that m6A peak overlap in mRNAs varies from ~30 to 60% between studies, even in the same cell type. We then assessed statistical methods to detect changes in m6A peaks as distinct from changes in gene expression. However, from these published data sets, we detected few changes under most conditions and were unable to detect consistent changes across studies of similar stimuli. Overall, our work identifies limits to MeRIP-seq reproducibility in the detection both of peaks and of peak changes and proposes improved approaches for analysis of peak changes.
    DOI:  https://doi.org/10.1038/s41598-020-63355-3
  30. J Biol Chem. 2020 Apr 24. pii: jbc.RA119.011685. [Epub ahead of print]
    Dudakovic A, Samsonraj RM, Paradise CR, Galeano-Garces C, Mol MO, Galeano-Garces D, Zan P, Galvan ML, Hevesi M, Pichurin O, Thaler R, Begun DL, Kloen P, Karperien M, Larson AN, Westendorf JJ, Cool SM, van Wijnen AJ.
      Bone-stimulatory therapeutics include bone morphogenetic proteins (e.g. BMP2), parathyroid hormone, and antibody-based suppression of WNT antagonists. Inhibition of the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2) is both bone-anabolic and osteo-protective. EZH2 inhibition stimulates key components of bone-stimulatory signaling pathways, including the BMP2 signaling cascade. Because of high costs and adverse effects associated with BMP2 use, here we investigated whether BMP2 dosing can be reduced by co-treatment with EZH2 inhibitors. Co-administration of BMP2 with the EZH2 inhibitor GSK126 enhanced differentiation of murine (MC3T3) osteoblasts, reflected by increased alkaline phosphatase activity, alizarin red staining, and expression of bone-related marker genes (e.g. Bglap and Phospho1). Strikingly, co-treatment with BMP2 (10 ng/ml) and GSK126 (5 μM) was synergistic and was as effective as 50 ng/ml BMP2 at inducing MC3T3 osteoblastogenesis. Similarly, the BMP2-GSK126 co-treatment stimulated osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells, reflected by induction of key osteogenic markers (e.g. Osterix/SP7 and IBSP). A combination of BMP2 (300 ng local) and GSK126 (5 μg local and 5 days of 50 mg/kg systemic) yielded more consistent bone healing than single treatments with either compound in a mouse calvarial critical-sized defect model according to results from μCT, histomorphometry, and surgical grading of qualitative X-rays. We conclude that EZH2 inhibition facilitates BMP2-mediated induction of osteogenic differentiation of progenitor cells and maturation of committed osteoblasts. We propose that epigenetic priming, coupled with bone anabolic agents, enhances osteogenesis and could be leveraged in therapeutic strategies to improve bone mass.
    Keywords:  BMP2; Ezh2; bone morphogenetic protein (BMP); chromatin regulation; enhancer of zeste homolog; epigenetics; histone methylation; mesenchymal stem cells (MSCs); methyltransferase; osteoblast; osteogenesis
    DOI:  https://doi.org/10.1074/jbc.RA119.011685
  31. Nucleic Acids Res. 2020 Apr 20. pii: gkaa261. [Epub ahead of print]
    Kantidze OL, Razin SV.
      The detailed principles of the hierarchical folding of eukaryotic chromosomes have been revealed during the last two decades. Along with structures composing three-dimensional (3D) genome organization (chromatin compartments, topologically associating domains, chromatin loops, etc.), the molecular mechanisms that are involved in their establishment and maintenance have been characterized. Generally, protein-protein and protein-DNA interactions underlie the spatial genome organization in eukaryotes. However, it is becoming increasingly evident that weak interactions, which exist in biological systems, also contribute to the 3D genome. Here, we provide a snapshot of our current understanding of the role of the weak interactions in the establishment and maintenance of the 3D genome organization. We discuss how weak biological forces, such as entropic forces operating in crowded solutions, electrostatic interactions of the biomolecules, liquid-liquid phase separation, DNA supercoiling, and RNA environment participate in chromosome segregation into structural and functional units and drive intranuclear functional compartmentalization.
    DOI:  https://doi.org/10.1093/nar/gkaa261
  32. Neuron. 2020 Apr 10. pii: S0896-6273(20)30232-4. [Epub ahead of print]
    Huang AY, Woo J, Sardar D, Lozzi B, Bosquez Huerta NA, Lin CJ, Felice D, Jain A, Paulucci-Holthauzen A, Deneen B.
      Astrocytes play essential roles in brain function by supporting synaptic connectivity and associated circuits. How these roles are regulated by transcription factors is unknown. Moreover, there is emerging evidence that astrocytes exhibit regional heterogeneity, and the mechanisms controlling this diversity remain nascent. Here, we show that conditional deletion of the transcription factor nuclear factor I-A (NFIA) in astrocytes in the adult brain results in region-specific alterations in morphology and physiology that are mediated by selective DNA binding. Disruptions in astrocyte function following loss of NFIA are most pronounced in the hippocampus, manifested by impaired interactions with neurons, coupled with diminution of learning and memory behaviors. These changes in hippocampal astrocytes did not affect basal neuronal properties but specifically inhibited synaptic plasticity, which is regulated by NFIA in astrocytes through calcium-dependent mechanisms. Together, our studies reveal region-specific transcriptional dependencies for astrocytes and identify astrocytic NFIA as a key transcriptional regulator of hippocampal circuits.
    Keywords:  Astrocyte; Hippocampal Circuits; NFIA; Regional Diversity; Transcription Factor
    DOI:  https://doi.org/10.1016/j.neuron.2020.03.025
  33. Cell Rep. 2020 Apr 21. pii: S2211-1247(20)30432-0. [Epub ahead of print]31(3): 107532
    Ma Q, Yang F, Mackintosh C, Jayani RS, Oh S, Jin C, Nair SJ, Merkurjev D, Ma W, Allen S, Wang D, Almenar-Queralt A, Garcia-Bassets I.
      Cisplatin is an antineoplastic drug administered at suboptimal and intermittent doses to avoid life-threatening effects. Although this regimen shortly improves symptoms in the short term, it also leads to more malignant disease in the long term. We describe a multilayered analysis ranging from chromatin to translation-integrating chromatin immunoprecipitation sequencing (ChIP-seq), global run-on sequencing (GRO-seq), RNA sequencing (RNA-seq), and ribosome profiling-to understand how cisplatin confers (pre)malignant features by using a well-established ovarian cancer model of cisplatin exposure. This approach allows us to segregate the human transcriptome into gene modules representing distinct regulatory principles and to characterize that the most cisplatin-disrupted modules are associated with underlying events of super-enhancer plasticity. These events arise when cancer cells initiate without ultimately ending the program of drug-stimulated death. Using a PageRank-based algorithm, we predict super-enhancer regulator ISL1 as a driver of this plasticity and validate this prediction by using CRISPR/dCas9-KRAB inhibition (CRISPRi) and CRISPR/dCas9-VP64 activation (CRISPRa) tools. Together, we propose that cisplatin reprograms cancer cells when inducing them to undergo near-to-death experiences.
    Keywords:  GRO-seq; PageRank; Seurat; cisplatin; drug resistance; gene dysregulation; multi-omic approach; ovarian cancer; ribosome profiling; super-enhancer
    DOI:  https://doi.org/10.1016/j.celrep.2020.107532
  34. J Oncol. 2020 ;2020 8978930
    Cao X, Liu L, Cao X, Cui Y, Zou C, Chen A, Qiu Y, Quan M, Ren K, Chen X, Cao J.
      Background: Whether DNA methyltransferase 1 (DNMT1)/miR-34a/FoxM1 signaling promotes the stemness of liver cancer stem cells (LCSCs) remains unclear. This study aimed to assess whether methylation-based silencing of miR-34a by DNMT1 contributes to stemness features via FoxM1 upregulation in LCSCs.Methods: The CD133+ subgroup of MHCC97H cells sorted by MACS was used as LCSCs. DNMT1, BMI1, SOX2, and OCT4 mRNA levels, and miR-34a amounts were determined by qRT-PCR. DNMT1, CD44, and FoxM1 proteins were analyzed by immunoblot. Sphere and colony formation abilities were detected by respective assays. CD133+ cell percentages were assessed by flow cytometry. In vivo oncogenicity was evaluated using a tumor xenograft model in mice. The effects of DNMT1/miR-34a signaling on the stemness of LCSCs were examined by knockdown or overexpression of DNMT1 and/or transfection of miR-34a mimic or inhibitor using lentivirus-delivery systems. FoxM1 association with miR-34a was detected by a reporter assay.
    Results: We here showed that LCSCs exhibited elevated DNMT1 activity and expression, lower miR-34a expression with higher promoter methylation, and stronger stemness, compared with the parental liver cancer cells. DNMT1 knockdown repressed DNMT1, increased miR-34a amounts by promoter demethylation, and reduced stemness in LCSCs, whereas DNMT1 overexpression had the opposite effects in liver cancer cells. Transfection with miR-34a mimic repressed the stemness of LCSCs, while miR-34a inhibitor significantly downregulated miR-34a and enhanced stemness, without affecting DNMT1 in liver cancer cells. MiR-34a mimic rescued the effects of DNMT1 overexpression on the stemness of LCSCs, without affecting DNMT1 expression. Finally, FOXM1 was identified as a direct target by miR-34a in LCSCs.
    Conclusions: We revealed that aberrant activation of DNMT1 causes miR-34a promoter methylation and suppression, leading to FoxM1 upregulation by disinhibition and promotion of LCSC stemness. These findings suggest that blockage of DNMT1/miR-34a-mediated FOXM1 upregulation might suppress liver cancer by targeting LCSCs.
    DOI:  https://doi.org/10.1155/2020/8978930
  35. Nat Protoc. 2020 Apr 20.
    Wienert B, Wyman SK, Yeh CD, Conklin BR, Corn JE.
      DISCOVER-seq (discovery of in situ Cas off-targets and verification by sequencing) is a broadly applicable approach for unbiased CRISPR-Cas off-target identification in cells and tissues. It leverages the recruitment of DNA repair factors to double-strand breaks (DSBs) after genome editing with CRISPR nucleases. Here, we describe a detailed experimental protocol and analysis pipeline with which to perform DISCOVER-seq. The principle of this method is to track the precise recruitment of MRE11 to DSBs by chromatin immunoprecipitation followed by next-generation sequencing. A customized open-source bioinformatics pipeline, BLENDER (blunt end finder), then identifies off-target sequences genome wide. DISCOVER-seq is capable of finding and measuring off-targets in primary cells and in situ. The two main advantages of DISCOVER-seq are (i) low false-positive rates because DNA repair enzyme binding is required for genome edits to occur and (ii) its applicability to a wide variety of systems, including patient-derived cells and animal models. The whole protocol, including the analysis, can be completed within 2 weeks.
    DOI:  https://doi.org/10.1038/s41596-020-0309-5
  36. Nat Commun. 2020 Apr 24. 11(1): 2027
    Zhang S, Kim B, Zhu X, Gui X, Wang Y, Lan Z, Prabhu P, Fond K, Wang A, Guo F.
      The mechanisms by which oligodendroglia modulate CNS angiogenesis remain elusive. Previous in vitro data suggest that oligodendroglia regulate CNS endothelial cell proliferation and blood vessel formation through hypoxia inducible factor alpha (HIFα)-activated Wnt (but not VEGF) signaling. Using in vivo genetic models, we show that HIFα in oligodendroglia is necessary and sufficient for angiogenesis independent of CNS regions. At the molecular level, HIFα stabilization in oligodendroglia does not perturb Wnt signaling but rather activates VEGF. At the functional level, genetically blocking oligodendroglia-derived VEGF but not Wnt significantly decreases oligodendroglial HIFα-regulated CNS angiogenesis. Blocking astroglia-derived Wnt signaling reduces astroglial HIFα-regulated CNS angiogenesis. Together, our in vivo data demonstrate that oligodendroglial HIFα regulates CNS angiogenesis through Wnt-independent and VEGF-dependent signaling. These findings suggest an alternative mechanistic understanding of CNS angiogenesis by postnatal glial cells and unveil a glial cell type-dependent HIFα-Wnt axis in regulating CNS vessel formation.
    DOI:  https://doi.org/10.1038/s41467-020-15656-4
  37. Gut. 2020 Apr 23. pii: gutjnl-2019-320205. [Epub ahead of print]
    Barcena-Varela M, Paish H, Alvarez L, Uriarte I, Latasa MU, Santamaria E, Recalde M, Garate M, Claveria A, Colyn L, Arechederra M, Iraburu MJ, Milkiewicz M, Milkiewicz P, Sangro B, Robinson SM, French J, Pardo-Saganta A, Oyarzabal J, Prosper F, Rombouts K, Oakley F, Mann J, Berasain C, Avila MA, G Fernandez-Barrena M.
      OBJECTIVE: Hepatic stellate cells (HSC) transdifferentiation into myofibroblasts is central to fibrogenesis. Epigenetic mechanisms, including histone and DNA methylation, play a key role in this process. Concerted action between histone and DNA-mehyltransferases like G9a and DNMT1 is a common theme in gene expression regulation. We aimed to study the efficacy of CM272, a first-in-class dual and reversible G9a/DNMT1 inhibitor, in halting fibrogenesis.DESIGN: G9a and DNMT1 were analysed in cirrhotic human livers, mouse models of liver fibrosis and cultured mouse HSC. G9a and DNMT1 expression was knocked down or inhibited with CM272 in human HSC (hHSC), and transcriptomic responses to transforming growth factor-β1 (TGFβ1) were examined. Glycolytic metabolism and mitochondrial function were analysed with Seahorse-XF technology. Gene expression regulation was analysed by chromatin immunoprecipitation and methylation-specific PCR. Antifibrogenic activity and safety of CM272 were studied in mouse chronic CCl4 administration and bile duct ligation (BDL), and in human precision-cut liver slices (PCLSs) in a new bioreactor technology.
    RESULTS: G9a and DNMT1 were detected in stromal cells in areas of active fibrosis in human and mouse livers. G9a and DNMT1 expression was induced during mouse HSC activation, and TGFβ1 triggered their chromatin recruitment in hHSC. G9a/DNMT1 knockdown and CM272 inhibited TGFβ1 fibrogenic responses in hHSC. TGFβ1-mediated profibrogenic metabolic reprogramming was abrogated by CM272, which restored gluconeogenic gene expression and mitochondrial function through on-target epigenetic effects. CM272 inhibited fibrogenesis in mice and PCLSs without toxicity.
    CONCLUSIONS: Dual G9a/DNMT1 inhibition by compounds like CM272 may be a novel therapeutic strategy for treating liver fibrosis.
    Keywords:  fibrogenesis; gene regulation; glucose metabolism
    DOI:  https://doi.org/10.1136/gutjnl-2019-320205
  38. Nat Commun. 2020 Apr 24. 11(1): 2025
    Mattesen TB, Rasmussen MH, Sandoval J, Ongen H, Árnadóttir SS, Gladov J, Martinez-Cardus A, Castro de Moura M, Madsen AH, Laurberg S, Dermitzakis ET, Esteller M, Andersen CL, Bramsen JB.
      Transcriptional characterization and classification has potential to resolve the inter-tumor heterogeneity of colorectal cancer and improve patient management. Yet, robust transcriptional profiling is difficult using formalin-fixed, paraffin-embedded (FFPE) samples, which complicates testing in clinical and archival material. We present MethCORR, an approach that allows uniform molecular characterization and classification of fresh-frozen and FFPE samples. MethCORR identifies genome-wide correlations between RNA expression and DNA methylation in fresh-frozen samples. This information is used to infer gene expression information in FFPE samples from their methylation profiles. MethCORR is here applied to methylation profiles from 877 fresh-frozen/FFPE samples and comparative analysis identifies the same two subtypes in four independent cohorts. Furthermore, subtype-specific prognostic biomarkers that better predicts relapse-free survival (HR = 2.66, 95%CI [1.67-4.22], P value < 0.001 (log-rank test)) than UICC tumor, node, metastasis (TNM) staging and microsatellite instability status are identified and validated using DNA methylation-specific PCR. The MethCORR approach is general, and may be similarly successful for other cancer types.
    DOI:  https://doi.org/10.1038/s41467-020-16000-6
  39. Genome Res. 2020 Apr 20.
    Hsiao CJ, Tung P, Blischak JD, Burnett JE, Barr KA, Dey KK, Stephens M, Gilad Y.
      Cellular heterogeneity in gene expression is driven by cellular processes, such as cell cycle and cell-type identity, and cellular environment such as spatial location. The cell cycle, in particular, is thought to be a key driver of cell-to-cell heterogeneity in gene expression, even in otherwise homogeneous cell populations. Recent advances in single-cell RNA-sequencing (scRNA-seq) facilitate detailed characterization of gene expression heterogeneity and can thus shed new light on the processes driving heterogeneity. Here, we combined fluorescence imaging with scRNA-seq to measure cell cycle phase and gene expression levels in human induced pluripotent stem cells (iPSCs). By using these data, we developed a novel approach to characterize cell cycle progression. Although standard methods assign cells to discrete cell cycle stages, our method goes beyond this and quantifies cell cycle progression on a continuum. We found that, on average, scRNA-seq data from only five genes predicted a cell's position on the cell cycle continuum to within 14% of the entire cycle and that using more genes did not improve this accuracy. Our data and predictor of cell cycle phase can directly help future studies to account for cell cycle-related heterogeneity in iPSCs. Our results and methods also provide a foundation for future work to characterize the effects of the cell cycle on expression heterogeneity in other cell types.
    DOI:  https://doi.org/10.1101/gr.247759.118
  40. Cancer Immunol Res. 2020 Apr 22. pii: canimm.0866.2019. [Epub ahead of print]
    Abou Alaiwi S, Nassar AH, Xie W, Bakouny Z, Berchuck JE, Braun DA, Baca SC, Nuzzo PV, Flippot R, Mouhieddine TH, Spurr LF, Li YY, Li T, Flaifel A, Steinharter JA, Margolis CA, Vokes NI, Du H, Shukla SA, Cherniack AD, Sonpavde G, Haddad RI, Awad MM, Giannakis M, Hodi FS, Liu XS, Signoretti S, Kadoch C, Freedman ML, Kwiatkowski DJ, Van Allen EM, Choueiri TK.
      Prior data have variably implicated the inactivation of the mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) complex with increased tumor sensitivity to immune checkpoint inhibitors (ICIs). Herein, we examined the association between mSWI/SNF variants and clinical outcomes to ICIs. We correlated somatic loss-of-function (LOF) variants in a pre-defined set of mSWI/SNF genes (ARID1A, ARID1B, SMARCA4, SMARCB1, PBRM1, and ARID2) with clinical outcomes in cancer patients treated with systemic ICIs. We identified 676 patients from Dana Farber Cancer Institute (DFCI) and 848 patients from a publicly available database from Memorial Sloan Kettering Cancer Center (MSKCC) who met the inclusion criteria. Multivariable analyses were conducted and adjusted for available baseline factors and tumor mutational burden (TMB). Median follow-up was 19.6 (17.6-22.0) months and 28.0 (25.0-29.0) months for the DFCI and MSKCC cohorts, respectively. Seven solid tumor subtypes were examined. In the DFCI cohort, LOF variants of mSWI/SNF did not predict improved overall survival (OS), time to treatment failure (TTF), or disease control rate (DCR). Only renal cell carcinoma patients with mSWI/SNF LOF showed significantly improved OS and TTF with adjusted hazard ratios (95% CI) of 0.33 (0.16-0.7) and 0.49 (0.27-0.88), respectively, and this was mostly driven by PRBM1. In the MSKCC cohort, where only OS was captured, LOF mSWI/SNF did not correlate with improved outcomes across any tumor subtype. We did not find a consistent association between mSWI/SNF LOF variants and improved clinical outcomes to ICIs, suggesting that mSWI/SNF variants should not be considered as biomarkers of response to ICIs.
    DOI:  https://doi.org/10.1158/2326-6066.CIR-19-0866
  41. Oncol Rep. 2020 Apr 06.
    Joo JS, Cho SY, Rou WS, Kim JS, Kang SH, Lee ES, Moon HS, Kim SH, Sung JK, Kwon IS, Eun HS, Lee BS.
      TEA Domain Transcription Factors (TEADs) are important in development and serve essential roles in tumorigenesis; however, the role of TEAD2 expression in hepatocellular carcinoma (HCC) has not been widely examined. The present study was conducted to investigate the expression status of TEAD2 in HCC and to evaluate whether the expression of TEAD2 is associated with the prognosis of patients with HCC. mRNA expression data was retrieved for Hippo pathway genes of 50 normal control and 377 HCC samples from The Cancer Genome Atlas data portal. Gene set enrichment, GeneNeighbors, ClassNeighbors and survival analyses were then performed based on the gene expression levels. The mRNA expression of TEAD2 and VGLL4 was significantly higher in HCC compared with the normal control samples, and the mRNA expression of TEAD2 was higher in advanced stages than in early stages. Specifically, survival analysis revealed that higher mRNA expression of TEAD2 was significantly associated with a less favorable overall survival rate (P=0.0067) and there was a trend towards significance between higher mRNA expression of VGLL4 and poor overall survival rate (P=0.051). According to the gene set enrichment analysis, patients with higher mRNA expression of TEAD2 and VGLL4 had strongly enhanced epithelial‑mesenchymal transition and angiogenesis, which are associated with tumor progression. In conclusion, increased mRNA expression of TEAD2 is associated with a poor prognosis in patients with HCC. TEAD2 may serve as a prognostic factor for HCC and a novel therapeutic target.
    DOI:  https://doi.org/10.3892/or.2020.7578
  42. Oncogene. 2020 Apr 20.
    Zhou Y, Zhang J, Li H, Huang T, Wong CC, Wu F, Wu M, Weng N, Liu L, Cheng ASL, Yu J, Wong N, Lo KW, Tang PMK, Kang W, To KF.
      Hippo signaling functions to limit cellular growth, but the aberrant nuclear accumulation of its downstream YAP1 leads to carcinogenesis. YAP1/TEAD complex activates the oncogenic downstream transcription, such as CTGF and c-Myc. How YAP1 is protected in the cytoplasm from ubiquitin-mediated degradation remains elusive. In this study, a member of Angiomotin (Motin) family, AMOTL1 (Angiomotin Like 1), was screened out as the only one to promote YAP1 nuclear accumulation by several clinical cohorts, which was further confirmed by the cellular functional assays. The interaction between YAP1 and AMOTL1 was suggested by co-immunoprecipitation and immunofluorescent staining. The clinical significance of the AMOTL1-YAP1-CTGF axis in gastric cancer (GC) was analyzed by multiple clinical cohorts. Moreover, the therapeutic effect of targeting the oncogenic axis was appraised by drug-sensitivity tests and xenograft-formation assays. The upregulation of AMOTL1 is associated with unfavorable clinical outcomes of GC, and knocking down AMOTL1 impairs its oncogenic properties. The cytoplasmic interaction between AMOTL1 and YAP1 protects each other from ubiquitin-mediated degradation. AMOTL1 promotes YAP1 translocation into the nuclei to activate the downstream expression, such as CTGF. Knocking down AMOTL1, YAP1, and CTGF enhances the therapeutic efficacies of the first-line anticancer drugs. Taken together, AMOTL1 plays an oncogenic role in gastric carcinogenesis through interacting with YAP1 and promoting its nuclear accumulation. A combination of AMOTL1, YAP1, and CTGF expression might serve as a surrogate of Hippo activation status. The co-activation of the AMOTL1/YAP1-CTGF axis is associated with poor clinical outcomes of GC patients, and targeting this oncogenic axis may enhance the chemotherapeutic effects.
    DOI:  https://doi.org/10.1038/s41388-020-1293-5
  43. Cell Syst. 2020 Apr 22. pii: S2405-4712(20)30110-1. [Epub ahead of print]10(4): 363-378.e12
    Schuh L, Saint-Antoine M, Sanford EM, Emert BL, Singh A, Marr C, Raj A, Goyal Y.
      Non-genetic transcriptional variability is a potential mechanism for therapy resistance in melanoma. Specifically, rare subpopulations of cells occupy a transient pre-resistant state characterized by coordinated high expression of several genes and survive therapy. How might these rare states arise and disappear within the population? It is unclear whether the canonical models of probabilistic transcriptional pulsing can explain this behavior, or if it requires special, hitherto unidentified mechanisms. We show that a minimal model of transcriptional bursting and gene interactions can give rise to rare coordinated high expression states. These states occur more frequently in networks with low connectivity and depend on three parameters. While entry into these states is initiated by a long transcriptional burst that also triggers entry of other genes, the exit occurs through independent inactivation of individual genes. Together, we demonstrate that established principles of gene regulation are sufficient to describe this behavior and argue for its more general existence. A record of this paper's transparent peer review process is included in the Supplemental Information.
    Keywords:  drug resistance; gene expression; melanoma; network; non-genetic; stochasticity
    DOI:  https://doi.org/10.1016/j.cels.2020.03.004
  44. BMC Bioinformatics. 2020 Apr 19. 21(1): 149
    Freedman AH, Gaspar JM, Sackton TB.
      BACKGROUND: Typical experimental design advice for expression analyses using RNA-seq generally assumes that single-end reads provide robust gene-level expression estimates in a cost-effective manner, and that the additional benefits obtained from paired-end sequencing are not worth the additional cost. However, in many cases (e.g., with Illumina NextSeq and NovaSeq instruments), shorter paired-end reads and longer single-end reads can be generated for the same cost, and it is not obvious which strategy should be preferred. Using publicly available data, we test whether short-paired end reads can achieve more robust expression estimates and differential expression results than single-end reads of approximately the same total number of sequenced bases.RESULTS: At both the transcript and gene levels, 2 × 40 paired-end reads unequivocally provide expression estimates that are more highly correlated with 2 × 125 than 1 × 75 reads; in nearly all cases, those correlations are also greater than for 1 × 125, despite the greater total number of sequenced bases for the latter. Across an array of metrics, differential expression tests based upon 2 × 40 consistently outperform those using 1 × 75.
    CONCLUSION: Researchers seeking a cost-effective approach for gene-level expression analysis should prefer short paired-end reads over a longer single-end strategy. Short paired-end reads will also give reasonably robust expression estimates and differential expression results at the isoform level.
    Keywords:  Differential expression; RNA-seq; Short read sequencing
    DOI:  https://doi.org/10.1186/s12859-020-3484-z
  45. Nucleic Acids Res. 2020 Apr 20. pii: gkaa236. [Epub ahead of print]
    Rydenfelt M, Klinger B, Klünemann M, Blüthgen N.
      Extracting signalling pathway activities from transcriptome data is important to infer mechanistic origins of transcriptomic dysregulation, for example in disease. A popular method to do so is by enrichment analysis of signature genes in e.g. differentially regulated genes. Previously, we derived signatures for signalling pathways by integrating public perturbation transcriptome data and generated a signature database called SPEED (Signalling Pathway Enrichment using Experimental Datasets), for which we here present a substantial upgrade as SPEED2. This web server hosts consensus signatures for 16 signalling pathways that are derived from a large number of transcriptomic signalling perturbation experiments. When providing a gene list of e.g. differentially expressed genes, the web server allows to infer signalling pathways that likely caused these genes to be deregulated. In addition to signature lists, we derive 'continuous' gene signatures, in a transparent and automated fashion without any fine-tuning, and describe a new algorithm to score these signatures.
    DOI:  https://doi.org/10.1093/nar/gkaa236
  46. Nucleic Acids Res. 2020 Apr 24. pii: gkaa263. [Epub ahead of print]
    Tan ZW, Fei G, Paulo JA, Bellaousov S, Martin SES, Duveau DY, Thomas CJ, Gygi SP, Boutz PL, Walker S.
      Intron detention in precursor RNAs serves to regulate expression of a substantial fraction of genes in eukaryotic genomes. How detained intron (DI) splicing is controlled is poorly understood. Here, we show that a ubiquitous post-translational modification called O-GlcNAc, which is thought to integrate signaling pathways as nutrient conditions fluctuate, controls detained intron splicing. Using specific inhibitors of the enzyme that installs O-GlcNAc (O-GlcNAc transferase, or OGT) and the enzyme that removes O-GlcNAc (O-GlcNAcase, or OGA), we first show that O-GlcNAc regulates splicing of the highly conserved detained introns in OGT and OGA to control mRNA abundance in order to buffer O-GlcNAc changes. We show that OGT and OGA represent two distinct paradigms for how DI splicing can control gene expression. We also show that when DI splicing of the O-GlcNAc-cycling genes fails to restore O-GlcNAc homeostasis, there is a global change in detained intron levels. Strikingly, almost all detained introns are spliced more efficiently when O-GlcNAc levels are low, yet other alternative splicing pathways change minimally. Our results demonstrate that O-GlcNAc controls detained intron splicing to tune system-wide gene expression, providing a means to couple nutrient conditions to the cell's transcriptional regime.
    DOI:  https://doi.org/10.1093/nar/gkaa263
  47. Nucleic Acids Res. 2020 Apr 23. pii: gkaa252. [Epub ahead of print]
    Leporcq C, Spill Y, Balaramane D, Toussaint C, Weber M, Bardet AF.
      Transcription factors (TFs) regulate the expression of gene expression. The binding specificities of many TFs have been deciphered and summarized as position-weight matrices, also called TF motifs. Despite the availability of hundreds of known TF motifs in databases, it remains non-trivial to quickly query and visualize the enrichment of known TF motifs in genomic regions of interest. Towards this goal, we developed TFmotifView, a web server that allows to study the distribution of known TF motifs in genomic regions. Based on input genomic regions and selected TF motifs, TFmotifView performs an overlap of the genomic regions with TF motif occurrences identified using a dynamic P-value threshold. TFmotifView generates three different outputs: (i) an enrichment table and scatterplot calculating the significance of TF motif occurrences in genomic regions compared to control regions, (ii) a genomic view of the organisation of TF motifs in each genomic region and (iii) a metaplot summarizing the position of TF motifs relative to the center of the regions. TFmotifView will contribute to the integration of TF motif information with a wide range of genomic datasets towards the goal to better understand the regulation of gene expression by transcription factors. TFmotifView is freely available at http://bardet.u-strasbg.fr/tfmotifview/.
    DOI:  https://doi.org/10.1093/nar/gkaa252
  48. Biochemistry. 2020 Apr 24.
    Bokhovchuk F, Mesrouze Y, Meyerhofer M, Zimmermann C, Fontana P, Erdmann D, Jemth P, Chene P.
      The Hippo pathway is an evolutionarily conserved signalling pathway that is involved in the control of organ size and development. The TEAD transcription factors are the most downstream elements of the Hippo pathway, and their transcriptional activity is regulated via the interaction with different co-regulators such as YAP. The structure of the YAP:TEAD complex shows that YAP binds to TEAD via two distinct secondary structure elements, an α-helix and an Ω-loop, and site-directed mutagenesis experiments revealed that the Ω-loop is the "hot spot" of this interaction. While substantial knowledge has been gained on how YAP and TEAD interact with each other, little is known about the mechanism leading to the formation of a complex between these two proteins. Here we combine site-directed mutagenesis with pre-steady-state kinetic measurements to show that the association between these proteins follows an apparent one-step binding mechanism. Furthermore, linear free energy relationships and a Φ analysis suggest that binding-induced folding of the YAP α-helix to TEAD occurs independently of and before formation of the Ω-loop interface. Thus, the binding-induced folding of YAP appears not to conform to the concomitant formation of tertiary structure (nucleation-condensation) usually observed for coupled binding and folding reactions. Our findings demonstrate how a mechanism reminiscent of the classical framework (diffusion-collision) mechanism of protein folding may operate in disorder-to-order transitions involving intrinsically disordered proteins.
    DOI:  https://doi.org/10.1021/acs.biochem.0c00217
  49. Methods Mol Biol. 2020 ;2154 217-230
    Mardaryev AN, Fessing MY.
      Spatial genome organization in the cell nucleus plays a crucial role in the control of genome functions. Our knowledge about spatial genome organization is relying on the advances in gene imaging technologies and the biochemical approaches based on the spatial dependent ligation of the genomic regions. Fluorescent in situ hybridization using specific fluorescent DNA and RNA probes in cells and tissues with the spatially preserved nuclear and genome architecture (3D-FISH) provides a powerful tool for the further advancement of our knowledge about genome structure and functions. Here we describe the 3D-FISH protocols allowing for such an analysis in mammalian tissue in situ including in the skin. These protocols include DNA probe amplification and labeling; tissue fixation; preservation and preparation for hybridization; hybridization of the DNA probes with genomic DNA in the tissue; and post-hybridization tissue sample processing.
    Keywords:  3-D FISH analysis; Epigenetics; Fluorescent in situ hybridization; Spatial genome organization
    DOI:  https://doi.org/10.1007/978-1-0716-0648-3_18
  50. J Cell Physiol. 2020 Apr 23.
    Zhang Q, Zuo H, Yu S, Lin Y, Chen S, Liu H, Chen Z.
      Runt-related transcription factor 2 (Runx2) has been shown to regulate osteoblast differentiation by directly or indirectly regulating numerous osteoblast-related genes. However, our understanding of the transcriptional mechanisms of RUNX2 is mainly restricted to its transactivation, while the mechanism underlying its inhibitory effect during osteoblast differentiation remains largely unknown. Here, we incorporated the anti-RUNX2 chromatin immunoprecipitation (ChIP) sequencing in MC3T3-E1 cells and RNA-sequencing of parietal bone from Runx2 heterozygous mutant mice, to identify the putative genes negatively regulated by RUNX2. We identified HtrA serine peptidase 1 (Htra1) as a target gene and found ten candidate Htra1 enhancers potentially regulated by RUNX2, among which seven were verified by dual-luciferase assays. Furthermore, we investigated the motifs in the vicinity of RUNX2-binding sites and identified early growth response 1 (EGR1) as a potential partner transcription factor (TF) potentially regulating Htra1 expression, which was subsequently confirmed by Re-ChIP assays. RUNX2 and EGR1 co-repressed Htra1 and increased the expression levels of other osteoblast marker genes, such as osterix, osteocalcin, and osteoprotegerin at the messenger RNA and protein level. Moreover, Alizarin red staining combined with alkaline phosphatase (ALP) staining showed decreased calcified nodules and ALP activity in the siRUNX2+siEGR1 group compared with siRUNX2 group. Our findings revealed the detailed mechanism of the inhibitory function of RUNX2 towards its downstream genes, along with its partner TFs, to promote osteoblast differentiation.
    Keywords:  Egr1; Htra1; Runx2; enhancer; osteogenic differentiation
    DOI:  https://doi.org/10.1002/jcp.29704
  51. Biophys J. 2020 Apr 04. pii: S0006-3495(20)30264-2. [Epub ahead of print]
    Moller J, de Pablo JJ.
      Chromatin can be viewed as a hierarchically structured fiber that regulates gene expression. It consists of a complex network of DNA and proteins whose characteristic dynamical modes facilitate compaction and rearrangement in the cell nucleus. These modes stem from chromatin's fundamental unit, the nucleosome, and their effects are propagated across length scales. Understanding the effects of nucleosome dynamics on the chromatin fiber, primarily through post-translational modifications that occur on the histones, is of central importance to epigenetics. Within the last decade, imaging and chromosome conformation capture techniques have revealed a number of structural and statistical features of the packaged chromatin fiber at a hitherto unavailable level of resolution. Such experiments have led to increased efforts to develop polymer models that aim to reproduce, explain, and predict the contact probability scaling and density heterogeneity. At nanometer scales, available models have focused on the role of the nucleosome and epigenetic marks on local chromatin structure. At micrometer scales, existing models have sought to explain scaling laws and density heterogeneity. Less work, however, has been done to reconcile these two approaches: bottom-up and top-down models of chromatin. In this perspective, we highlight the multiscale simulation models that are driving toward an understanding of chromatin structure and function, from the nanometer to the micron scale, and we highlight areas of opportunity and some of the prospects for new frameworks that bridge these two scales. Taken together, experimental and modeling advances over the last few years have established a robust platform for the study of chromatin fiber structure and dynamics, which will be of considerable use to the chromatin community in developing an understanding of the interplay between epigenomic regulation and molecular structure.
    DOI:  https://doi.org/10.1016/j.bpj.2020.03.014
  52. Proc Natl Acad Sci U S A. 2020 Apr 20. pii: 201922422. [Epub ahead of print]
    Paolino A, Fenlon LR, Kozulin P, Haines E, Lim JWC, Richards LJ, Suárez R.
      A unique combination of transcription factor expression and projection neuron identity demarcates each layer of the cerebral cortex. During mouse and human cortical development, the transcription factor CTIP2 specifies neurons that project subcerebrally, while SATB2 specifies neuronal projections via the corpus callosum, a large axon tract connecting the two neocortical hemispheres that emerged exclusively in eutherian mammals. Marsupials comprise the sister taxon of eutherians but do not have a corpus callosum; their intercortical commissural neurons instead project via the anterior commissure, similar to egg-laying monotreme mammals. It remains unknown whether divergent transcriptional networks underlie these cortical wiring differences. Here, we combine birth-dating analysis, retrograde tracing, gene overexpression and knockdown, and axonal quantification to compare the functions of CTIP2 and SATB2 in neocortical development, between the eutherian mouse and the marsupial fat-tailed dunnart. We demonstrate a striking degree of structural and functional homology, whereby CTIP2 or SATB2 of either species is sufficient to promote a subcerebral or commissural fate, respectively. Remarkably, we reveal a substantial delay in the onset of developmental SATB2 expression in mice as compared to the equivalent stage in dunnarts, with premature SATB2 overexpression in mice to match that of dunnarts resulting in a marsupial-like projection fate via the anterior commissure. Our results suggest that small alterations in the timing of regulatory gene expression may underlie interspecies differences in neuronal projection fate specification.
    Keywords:  Bcl11b/Ctip2; corpus callosum; cortical evolution; evolutionary innovations; heterochrony
    DOI:  https://doi.org/10.1073/pnas.1922422117
  53. Cells. 2020 Apr 21. pii: E1024. [Epub ahead of print]9(4):
    Chen J, Yang Y, Li S, Yang Y, Dai Z, Wang F, Wu Z, Tso P, Wu G.
      Wnt/β-catenin is a crucial repressor of adipogenesis. We have shown that E2 promoter binding factor 1 (E2F1) suppresses Wnt/β-catenin activity through transactivation of β-catenin interacting protein 1 (CTNNBIP1), also known as inhibitor of β-catenin and TCF4 (ICAT) in human colorectal cancers. However, it remains unknown whether ICAT is required for E2F1 to promote differentiation by inhibiting β-catenin activity in pre-adipocytes. In the present study, we found that 1-methyl-3-isobutylxanthine, dexamethasone, and insulin (MDI)-induced differentiation and lipid accumulation in 3T3-L1 pre-adipocytes was reversed by activation of β-catenin triggered by CHIR99021, a GSK3β inhibitor. Intriguingly, we observed a reduced protein level of E2F1 and ICAT at a later stage of pre-adipocytes differentiation. Importantly, overexpression of ICAT in 3T3-L1 pre-adipocytes markedly promote the adipogenesis and partially reversed the inhibitory effect of CHIR99021 on MDI-induced adipogenesis and lipid accumulation by regulating adipogenic regulators and Wnt/β-catenin targets. Moreover, pre-adipocytes differentiation induced by MDI were markedly inhibited in siE2F1 or siICAT transfected 3T3-L1 cells. Gene silencing of ICAT in the E2F1 overexpressed adipocytes also inhibited the adipogenesis. These data indicated that E2F1 is a metabolic regulator with an ability to promote pre-adipocyte differentiation by activating ICAT, therefore represses Wnt/β-catenin activity in 3T3-L1 cells. We also demonstrated that ICAT overexpression did not affect oleic acid-induced lipid accumulation at the surface of Hela and HepG2 cells. In conclusion, we show that E2F1 is a critical regulator with an ability to promote differentiation and adipogenesis by activating ICAT in pre-adipocytes.
    Keywords:  3T3-L1; E2F1; ICAT; Wnt/β-catenin; adipogenesis; differentiation
    DOI:  https://doi.org/10.3390/cells9041024