bims-histon Biomed News
on Histones
Issue of 2020‒05‒31
forty-eight papers selected by
Benjamin Weekley
University of Southern California

  1. Cell Rep. 2020 May 26. pii: S2211-1247(20)30629-X. [Epub ahead of print]31(8): 107676
      The human genome encodes millions of regulatory elements, of which only a small fraction are active within a given cell type. Little is known about the global impact of chromatin remodelers on regulatory DNA landscapes and how this translates to gene expression. We use precision genome engineering to reawaken homozygously inactivated SMARCA4, a central ATPase of the human SWI/SNF chromatin remodeling complex, in lung adenocarcinoma cells. Here, we combine DNase I hypersensitivity, histone modification, and transcriptional profiling to show that SMARCA4 dramatically increases both the number and magnitude of accessible chromatin sites genome-wide, chiefly by unmasking sites of low regulatory factor occupancy. By contrast, transcriptional changes are concentrated within well-demarcated remodeling domains wherein expression of specific genes is gated by both distal element activation and promoter chromatin configuration. Our results provide a perspective on how global chromatin remodeling activity is translated to gene expression via regulatory DNA.
    Keywords:  BAF complex; SMARCA4; cancer epigenetics; chromatin accessibility; chromatin domains; chromatin remodeling; gene regulation; lung adenocarcinoma; topologically associating domains
  2. Transl Med Aging. 2020 ;4 22-31
      In multi-cellular organisms, the control of gene expression is key not only for development, but also for adult cellular homeostasis, and deregulation of gene expression correlates with aging. A key layer in the study of gene regulation mechanisms lies at the level of chromatin: cellular chromatin states (i.e. the 'epigenome') can tune transcriptional profiles, and, in line with the prevalence of transcriptional alterations with aging, accumulating evidence suggests that the chromatin landscape is altered with aging across cell types and species. However, although alterations in the chromatin make-up of cells are considered to be a hallmark of aging, little is known of the genomic loci that are specifically affected by age-related chromatin state remodeling and of their biological significance. Here, we report the analysis of genome-wide profiles of core histone H3 occupancy in aging male mouse tissues (i.e. heart, liver, cerebellum and olfactory bulb) and primary cultures of neural stem cells. We find that, although no drastic changes in H3 levels are observed, local changes in H3 occupancy occur with aging across tissues and cells with both regions of increased or decreased occupancy. These changes are compatible with a general increase in chromatin accessibility at pro-inflammatory genes and may thus mechanistically underlie known shift in gene expression programs during aging.
  3. Cell Mol Life Sci. 2020 May 23.
      Squamous cell carcinoma (SCC) is an aggressive malignancy that can originate from various organs. TP63 is a master regulator that plays an essential role in epidermal differentiation. It is also a lineage-dependent oncogene in SCC. ΔNp63α is the prominent isoform of TP63 expressed in epidermal cells and SCC, and overexpression promotes SCC development through a variety of mechanisms. Recently, ΔNp63α was highlighted to act as an epidermal-specific pioneer factor that binds closed chromatin and enhances chromatin accessibility at epidermal enhancers. ΔNp63α coordinates chromatin-remodeling enzymes to orchestrate the tissue-specific enhancer landscape and three-dimensional high-order architecture of chromatin. Moreover, ΔNp63α establishes squamous-like enhancer landscapes to drive oncogenic target expression during SCC development. Importantly, ΔNp63α acts as an upstream regulator of super enhancers to activate a number of oncogenic transcripts linked to poor prognosis in SCC. Mechanistically, ΔNp63α activates genes transcription through physically interacting with a number of epigenetic modulators to establish enhancers and enhance chromatin accessibility. In contrast, ΔNp63α also represses gene transcription via interacting with repressive epigenetic regulators. ΔNp63α expression is regulated at multiple levels, including transcriptional, post-transcriptional, and post-translational levels. In this review, we summarize recent advances of p63 in epigenomic and transcriptional control, as well as the mechanistic regulation of p63.
    Keywords:  Basal cell; Epigenetic reprogramming; Histone modification; Oncogene addiction; SWI/SNF complex; Ubiquitin–proteasome system
  4. PLoS Genet. 2020 May 28. 16(5): e1008681
      A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.
  5. Nucleic Acids Res. 2020 May 27. pii: gkaa441. [Epub ahead of print]
      Functional crosstalk between histone modifications and chromatin remodeling has emerged as a key regulatory mode of transcriptional control during cell fate decisions, but the underlying mechanisms are not fully understood. Here we discover an HRP2-DPF3a-BAF epigenetic pathway that coordinates methylated histone H3 lysine 36 (H3K36me) and ATP-dependent chromatin remodeling to regulate chromatin dynamics and gene transcription during myogenic differentiation. Using siRNA screening targeting epigenetic modifiers, we identify hepatoma-derived growth factor-related protein 2 (HRP2) as a key regulator of myogenesis. Knockout of HRP2 in mice leads to impaired muscle regeneration. Mechanistically, through its HIV integrase binding domain (IBD), HRP2 associates with the BRG1/BRM-associated factor (BAF) chromatin remodeling complex by interacting directly with the BAF45c (DPF3a) subunit. Through its Pro-Trp-Trp-Pro (PWWP) domain, HRP2 preferentially binds to H3K36me2. Consistent with the biochemical studies, ChIP-seq analyses show that HRP2 colocalizes with DPF3a across the genome and that the recruitment of HRP2/DPF3a to chromatin is dependent on H3K36me2. Integrative transcriptomic and cistromic analyses, coupled with ATAC-seq, reveal that HRP2 and DPF3a activate myogenic genes by increasing chromatin accessibility through recruitment of BRG1, the ATPase subunit of the BAF complex. Taken together, these results illuminate a key role for the HRP2-DPF3a-BAF complex in the epigenetic coordination of gene transcription during myogenic differentiation.
  6. Commun Biol. 2020 May 25. 3(1): 262
      Histones modulate gene expression by chromatin compaction, regulating numerous processes such as differentiation. However, the mechanisms underlying histone degradation remain elusive. Human embryonic stem cells (hESCs) have a unique chromatin architecture characterized by low levels of trimethylated histone H3 at lysine 9 (H3K9me3), a heterochromatin-associated modification. Here we assess the link between the intrinsic epigenetic landscape and ubiquitin-proteasome system of hESCs. We find that hESCs exhibit high expression of the ubiquitin-conjugating enzyme UBE2K. Loss of UBE2K upregulates the trimethyltransferase SETDB1, resulting in H3K9 trimethylation and repression of neurogenic genes during differentiation. Besides H3K9 trimethylation, UBE2K binds histone H3 to induce its polyubiquitination and degradation by the proteasome. Notably, ubc-20, the worm orthologue of UBE2K, also regulates histone H3 levels and H3K9 trimethylation in Caenorhabditis elegans germ cells. Thus, our results indicate that UBE2K crosses evolutionary boundaries to promote histone H3 degradation and reduce H3K9me3 repressive marks in immortal cells.
  7. PLoS One. 2020 ;15(5): e0233630
      Characterization of distinct histone methylation and acetylation binding patterns in promoters and prediction of novel regulatory regions remains an important area of genomic research, as it is hypothesized that distinct chromatin signatures may specify unique genomic functions. However, methods that have been proposed in the literature are either descriptive in nature or are fully parametric and hence more restrictive in pattern discovery. In this article, we propose a two-step non-parametric statistical inference procedure to characterize unique histone modification patterns and apply it to analyzing the binding patterns of four histone marks, H3K4me2, H3K4me3, H3K9ac, and H4K20me1, in human B-lymphoblastoid cells. In the first step, we used a functional principal component analysis method to represent the concatenated binding patterns of these four histone marks around the transcription start sites as smooth curves. In the second step, we clustered these curves to reveal several unique classes of binding patterns. These uncovered patterns were used in turn to scan the whole-genome to predict novel and alternative promoters. Our analyses show that there are three distinct promoter binding patterns of active genes. Further, 19654 regions not within known gene promoters were found to overlap with human ESTs, CpG islands, or common SNPs, indicative of their potential role in gene regulation, including being potential novel promoter regions.
  8. Molecules. 2020 May 27. pii: E2496. [Epub ahead of print]25(11):
      Epigenetic research has rapidly evolved into a dynamic field of genome biology. Chromatin regulation has been proved to be an essential aspect for all genomic processes, including DNA repair. Chromatin structure is modified by enzymes and factors that deposit, erase, and interact with epigenetic marks such as DNA and histone modifications, as well as by complexes that remodel nucleosomes. In this review we discuss recent advances on how the chromatin state is modulated during this multi-step process of damage recognition, signaling, and repair. Moreover, we examine how chromatin is regulated when different pathways of DNA repair are utilized. Furthermore, we review additional modes of regulation of DNA repair, such as through the role of global and localized chromatin states in maintaining expression of DNA repair genes, as well as through the activity of epigenetic enzymes on non-nucleosome substrates. Finally, we discuss current and future applications of the mechanistic interplays between chromatin regulation and DNA repair in the context cancer treatment.
    Keywords:  DNA damage; DNA repair; chromatin remodeling; epigenomics
  9. Chemosphere. 2020 May 19. pii: S0045-6535(20)31326-6. [Epub ahead of print]256 127133
      Atmospheric fine particulate matter exposure (PM2.5) can increase the incidence and mortality of heart disease, and raise the risk of fetal congenital heart defect, which have recently drawn much attention. In this study, C57BL/6 mice were exposed to PM2.5 (approximately equivalent to 174 μg/m3) by intratracheal instillation during the gestation. After birth, 10 weeks old offspring mice were divided into four groups: male exposed group (ME), female exposed group (FE), male control group (MC), female control group (FC). The pathological injury, pro-inflammatory cytokines, histone acetylation levels, and expressions of GATA-binding protein 4 (GATA4) and downstream genes were investigated. The results showed that exposure to PM2.5 in utero increased pathological damage and TNF-α and IL-6 levels in hearts of offspring mice, and effects in ME were more serious than FE. Notably, GATA4 protein levels in hearts in ME were significantly lower than that of MC, accompanied by down-regulation of histone acetyltransferase (HAT)-p300 and up-regulation of histone deacetylase-SIRT3. As GATA4 downstream genes, ratios of β-MHC gene expression to α-MHC significantly raised in ME relative to the MC. Results of chromatin immunoprecipitation (ChIP)-qPCR assay found that binding levels of acetylated histone 3 lysine 9 (H3K9ac) in GATA4 promoter region in the hearts of ME or FE were markedly decreased compared with their corresponding control groups. It suggested that maternal exposure to PM2.5 may cause cardiac injury in the offspring, heart damage of male mice was worse than female mice, in which process HAT-p300, H3K9ac, transcription factor GATA4 may play an important regulation role.
    Keywords:  Exposure in utero; Fine particulate matter; GATA4; Heart injury; Histone acetylation; Mice
  10. Stem Cell Res Ther. 2020 May 24. 11(1): 196
      BACKGROUND: Mesendodermal formation during early gastrulation requires the expression of lineage-specific genes, while the regulatory mechanisms during this process have not yet been fully illustrated. TATA box-binding protein (TBP) and TBP-like factors are general transcription factors responsible for the transcription initiation by recruiting the preinitiation complex to promoter regions. However, the role of TBP family members in the regulation of mesendodermal specification remains largely unknown.METHODS: We used an in vitro mesendodermal differentiation system of human embryonic stem cells (hESCs), combining with the microarray and quantitative polymerase chain reaction (qRT-PCR) analysis, loss of function and gain of function to determine the function of the TBP family member TBP-related factor 3 (TRF3) during mesendodermal differentiation of hESCs. The chromatin immunoprecipitation (ChIP) and biochemistry analysis were used to determine the binding of TRF3 to the promoter region of key mesendodermal genes.
    RESULTS: The mesendodermal differentiation of hESCs was confirmed by the microarray gene expression profile, qRT-PCR, and immunocytochemical staining. The expression of TRF3 mRNA was enhanced during mesendodermal differentiation of hESCs. The TRF3 deficiency did not affect the pluripotent marker expression, alkaline phosphatase activity, and cell cycle distribution of undifferentiated hESCs or the expression of early neuroectodermal genes during neuroectodermal differentiation. During the mesendodermal differentiation, the expression of pluripotency markers decreased in both wild-type and TRF3 knockout (TRF3-/-) cells, while the TRF3 deficiency crippled the expression of the mesendodermal markers. The reintroduction of TRF3 into the TRF3-/- hESCs rescued inhibited mesendodermal differentiation. Mechanistically, the TRF3 binding profile was significantly shifted to the mesendodermal specification during mesendodermal differentiation of hESCs based on the ChIP-seq data. Moreover, ChIP and ChIP-qPCR analysis showed that TRF3 was enriched at core promoter regions of mesendodermal developmental genes, EOMESODERMIN, BRACHYURY, mix paired-like homeobox, and GOOSECOID homeobox, during mesendodermal differentiation of hESCs.
    CONCLUSIONS: These results reveal that the TBP family member TRF3 is dispensable in the undifferentiated hESCs and the early neuroectodermal differentiation. However, it directs mesendodermal lineage commitment of hESCs via specifically promoting the transcription of key mesendodermal transcription factors. These findings provide new insights into the function and mechanisms of the TBP family member in hESC early lineage specification.
    Keywords:  BRACHYURY (T); EOMESODERMIN (EOMES); GOOSECOID homeobox (GSC); Human embryonic stem cells (hESCs); Mesendodermal differentiation; Mix paired-like homeobox (MIXL1); TATA box-binding protein-related factor 3 (TRF3)
  11. Epigenetics Chromatin. 2020 May 27. 13(1): 24
      BACKGROUND: The YEATS domain is a highly conserved protein structure that interacts with acetylated and crotonylated lysine residues in N-terminal tails of histones. The budding yeast genome encodes three YEATS domain proteins (Taf14, Yaf9, and Sas5) that are all the subunits of different complexes involved in histone acetylation, gene transcription, and chromatin remodeling. As the strains deficient in all these three genes are inviable, it has been proposed that the YEATS domain is essential in yeast. In this study we investigate in more detail the requirement of YEATS domain proteins for yeast survival and the possible roles of Taf14 YEATS domain in the regulation of gene transcription.RESULTS: We found that YEATS domains are not essential for the survival of Saccharomyces cerevisiae cells. Although the full deletion of all YEATS proteins is lethal in yeast, we show that the viability of cells can be restored by the expression of the YEATS-less version of Taf14 protein. We also explore the in vivo functions of Taf14 protein and show that the primary role of its YEATS domain is to stabilize the transcription pre-initiation complex (PIC). Our results indicate that Taf14-mediated interactions become crucial for PIC formation in rpb9Δ cells, where the recruitment of TFIIF to the PIC is hampered. Although H3 K9 residue has been identified as the interaction site of the Taf14 YEATS domain in vitro, we found that it is not the only interaction target in vivo.
    CONCLUSIONS: Lethality of YEATS-deficient cells can be rescued by the expression of truncated Taf14 protein lacking the entire YEATS domain, indicating that the YEATS domains are not required for cell survival. The YEATS domain of Taf14 participates in PIC stabilization and acetylated/crotonylated H3K9 is not the critical target of the Taf14 YEATS domain in vivo.
    Keywords:  Pre-initiation complex (PIC); RNA polymerase II; TFIID; TFIIF; Taf14; Transcription; YEATS
  12. Nat Chem. 2020 May 29.
      Elucidating the physiological binding partners of histone post-translational modifications (hPTMs) is key to understanding fundamental epigenetic regulatory pathways. Determining such interactomes will enable the study of how perturbations of these interactions affect disease. Here we use a synthetic biology approach to set a series of hPTM-controlled photo-affinity traps in native chromatin. Using quantitative proteomics, the local interactomes of these chemically customized chromatin landscapes are determined. We show that the approach captures transiently interacting factors such as methyltransferases and demethylases, as well as previously reported and novel hPTM reader proteins. We also apply this in situ proteomics approach to a recently disclosed cancer-associated histone mutation, H3K4M, revealing a number of perturbed interactions with the mutated tail. Collectively our studies demonstrate that modifying and interrogating native chromatin with chemical precision is a powerful tool for exploring epigenetic regulation and dysregulation at the molecular level.
  13. Oncogene. 2020 May 29.
      The epigenetic environment plays an important role in DNA damage recognition and repair, both at DNA double-strand breaks and at deprotected telomeres. To increase understanding on how DNA damage responses (DDR) at deprotected telomeres are regulated by modification and remodeling of telomeric chromatin we screened 38 methyltransferases for their ability to promote telomere dysfunction-induced genomic instability. As top hit we identified MMSET, a histone methyltransferase (HMT) causally linked to multiple myeloma and Wolf-Hirschhorn syndrome. We show that MMSET promotes non-homologous end-joining (NHEJ) at deprotected telomeres through Ligase4-dependent classical NHEJ, and does not contribute to Ligase3-dependent alternative NHEJ. Moreover, we show that this is dependent on the catalytic activity of MMSET, enabled by its SET-domain. Indeed, in absence of MMSET H3K36-dimethylation (H3K36me2) decreases, both globally and at subtelomeric regions. Interestingly, the level of MMSET-dependent H3K36me2 directly correlates with NHEJ-efficiency. We show that MMSET depletion does not impact on recognition of deprotected telomeres by the DDR-machinery or on subsequent recruitment of DDR-factors acting upstream or at the level of DNA repair pathway choice. Our data are most consistent with an important role for H3K36me2 in more downstream steps of the DNA repair process. Moreover, we find additional H3K36me2-specific HMTs to contribute to NHEJ at deprotected telomeres, further emphasizing the importance of H3K36me2 in DNA repair.
  14. Front Cell Dev Biol. 2020 ;8 331
      The nucleosome is the principal structural unit of chromatin. Although many studies focus on individual histone post-translational modifications (PTMs) in isolation, it is important to recognize that multiple histone PTMs can function together or cross-regulate one another within the nucleosome context. In addition, different modifications or histone-binding surfaces can synergize to stabilize the binding of nuclear factors to nucleosomes. To facilitate these types of studies, we present here a step-by-step protocol for isolating high yields of mononucleosomes for biochemical analyses. Furthermore, we discuss differences and variations of the basic protocol used in different publications and characterize the relative abundance of selected histone PTMs and chromatin-binding proteins in the different chromatin fractions obtained by this method.
    Keywords:  MNase; chromatin-binding proteins; combinatorial histone modifications; histone variant; immunoprecipitation; mononucleosome IP; nucleosome
  15. Clin Epigenetics. 2020 May 25. 12(1): 73
      Therapeutic intervention of proteins participating in chromatin-mediated signaling with small-molecules is a novel option to reprogram expression networks for restraining disease states. Protein methyltransferases form the prominent family of such proteins regulating gene expression via epigenetic mechanisms thereby representing novel targets for pharmacological intervention. Disruptor of telomeric silencing, hDot1L is the only non-SET domain containing histone methyltransferase that methylates histone H3 at lysine 79. H3K79 methylation mediated by hDot1L plays a crucial role in mixed lineage leukemia (MLL) pathosis. MLL fusion protein mediated mistargeting of DOT1L to aberrant gene locations results in ectopic H3K79 methylation culminating in aberrant expression of leukemogenic genes like HOXA9 and MEIS1. hDOT1L has thus been proposed as a potential target for therapeutic intervention in MLL. This review presents the general overview of hDOT1L and its functional role in distinct biological processes. Furthermore, we discuss various therapeutic strategies against hDOT1L as a promising drug target to vanquish therapeutically challenging MLL.
    Keywords:  Chromatin; DNA repair; Gene expression; Histone methylation; Histone methyltransferases; Mixed lineage leukemia
  16. Int J Biol Macromol. 2020 May 23. pii: S0141-8130(20)33334-1. [Epub ahead of print]
      Affinity for G-quadruplex (G4) structures may be a common feature of transcription-facilitating histone chaperons (HCs). This assumption is based on previous unmatched studies of HCs FACT, nucleolin (NCL), BRD3, and ATRX. We verified this assumption and considered its implications for the therapeutic applications of synthetic (exogenous) G4s and the biological significance of genomic G4s. First, we questioned whether exogenous G4s that recognize cell-surface NCL and could trap other HCs in the nucleus are usable as anticancer agents. We performed in vitro binding assays and selected leading multi-targeted G4s. They exhibited minor effects on cell viability. The presumed NCL-regulated intracellular transport of G4s was inefficient or insufficient for tumor-specific G4 delivery. Next, to clarify whether G4s in the human genome could recruit HCs, we compared available HC ChIP-seq data with G4-seq/G4-ChIP-seq data. Several G4s, including the well-known c-Myc quadruplex structure, were found to be colocalized with HC occupancy sites in cancer cell lines. As evidenced by our molecular modeling data, c-Myc G4 might interfere with the HC function of BRD3 but is unlikely to prevent the BRD3-driven assembly of the chromatin remodeling complex. The c-Myc case illustrates the intricate role of genomic G4s in chromatin remodeling, nucleosome remodeling, and transcription.
    Keywords:  C-Myc; G-quadruplexes; Histone chaperons
  17. Cell Mol Life Sci. 2020 May 26.
      Modification of the cancer-associated chromatin landscape in response to therapeutic DNA damage influences gene expression and contributes to cell fate. The central histone mark H2Bub1 results from addition of a single ubiquitin on lysine 120 of histone H2B and is an important regulator of gene expression. Following treatment with a platinum-based chemotherapeutic, there is a reduction in global levels of H2Bub1 accompanied by an increase in levels of the tumor suppressor p53. Although total H2Bub1 decreases following DNA damage, H2Bub1 is enriched downstream of transcription start sites of specific genes. Gene-specific H2Bub1 enrichment was observed at a defined group of genes that clustered into cancer-related pathways and correlated with increased gene expression. H2Bub1-enriched genes encompassed fifteen p53 target genes including PPM1D, BTG2, PLK2, MDM2, CDKN1A and BBC3, genes related to ERK/MAPK signalling, those participating in nucleotide excision repair including XPC, and genes involved in the immune response and platinum drug resistance including POLH. Enrichment of H2Bub1 at key cancer-related genes may function to regulate gene expression and influence the cellular response to therapeutic DNA damage.
    Keywords:  Cisplatin; DNA damage; H2Bub1; Histone modification; Monoubiquitination; p53
  18. Genes Dev. 2020 May 28.
      Chromatin modifiers play critical roles in epidermal development, but the functions of histone deacetylases in this context are poorly understood. The class I HDAC, HDAC3, is of particular interest because it plays divergent roles in different tissues by partnering with tissue-specific transcription factors. We found that HDAC3 is expressed broadly in embryonic epidermis and is required for its orderly stepwise stratification. HDAC3 protein stability in vivo relies on NCoR and SMRT, which function redundantly in epidermal development. However, point mutations in the NCoR and SMRT deacetylase-activating domains, which are required for HDAC3's enzymatic function, permit normal stratification, indicating that HDAC3's roles in this context are largely independent of its histone deacetylase activity. HDAC3-bound sites are significantly enriched for predicted binding motifs for critical epidermal transcription factors including AP1, GRHL, and KLF family members. Our results suggest that among these, HDAC3 operates in conjunction with KLF4 to repress inappropriate expression of Tgm1, Krt16, and Aqp3 In parallel, HDAC3 suppresses expression of inflammatory cytokines through a Rela-dependent mechanism. These data identify HDAC3 as a hub coordinating multiple aspects of epidermal barrier acquisition.
    Keywords:  HDAC3; KLF4; NCoR; SMRT; chromatin; epidermal barrier; epidermis; epigenetic; histone deacetylase; mouse
  19. Biochem Biophys Res Commun. 2020 May 21. pii: S0006-291X(20)30983-9. [Epub ahead of print]
      SET domain with lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), recently suggested to exert a critical role among kidney disorders, whereas its role in diabetes associated IRI co-morbidity remains complete elusive. The present study aimed to understand the role of SET7/9 and histone methylation in regulation of inflammatory signaling under IRI in diabetes mellitus and non-diabetic rats. Our results demonstrated that IRI caused renal dysfunction via increased blood urea nitrogen (BUN) levels in ND and DM rats. The NF-κB mediated inflammatory cascade like increased p-NF-κB, reduced IκBα levels followed by enhanced leukocyte infiltration as shown by increased MCP-1 expressions. IRI results in increased histone H3 methylation at lysine 4 and 36 (H3K4Me2, H3K36Me2), and decreased histone H3 methylation at lysine 9. Additionally, IRI increased the protein and mRNA expression of H3K4Me2 specific histone methyltransferase-SET7/9 in DM and ND rats. The abovementioned results remain prominent in DM rats compared to ND rats followed by IRI. Further, treatment with a novel SET7/9 inhibitor; cyproheptadine, significantly improved renal functioning via reducing the BUN levels in ND and DM rats. Hence, this study demonstrated the role of SET7/9 in mediating active transcription via H3K4Me2, ultimately regulated the NFκB-mediated inflammatory cascade. Therefore, SET7/9 can be explored as novel target for drug development against IRI under DM and ND conditions.
    Keywords:  Cyproheptadine; Diabetes; Histone methylation; Ischemia renal injury; Kidney
  20. Cell Death Differ. 2020 May 23.
      Senescence is accompanied with histones level alteration; however, the roles and the mechanisms of histone reduction in cellular senescence are largely unknown. Protein arginine methyltransferase 1 (PRMT1) is the major enzyme that generates monomethyl and asymmetrical dimethyl arginine. Here we showed that abrogation of PRMT1-mediated senescence was accompanied with decreasing histone H4 level. Consistently, under multiple classic senescence models, H4 decreasing was also been found prior to the other 3 core histones. Noticeably, asymmetric demethylation of histone H4 at arginine 3 (H4R3me2as), catalyzed by PRMT1, was decreased prior to histone H4. In addition, we showed that the PRMT1-mediated H4R3me2as maintained H4 stability. Reduction of H4R3me2as level increased the interaction between proteasome activator PA200 and histone H4, which catalyzes the poly-ubiquitin-independent degradation of H4. Moreover, H4 degradation promoted nucleosome decomposition, resulting in increased senescence-associated genes transcription. Significantly, H4 was restored by 3 well-informed anti-aging drugs (metformin, rapamycin, and resveratrol) much earlier than other senescence markers detected under H2O2-induced senescence. Thus, we uncovered a novel function of H4R3me2as in modulation of cellular senescence via regulating H4 stability. This finding also points to the value of histone H4 as a senescence indicator and a potential anti-aging drug screening marker.
  21. Proc Natl Acad Sci U S A. 2020 May 27. pii: 201913261. [Epub ahead of print]
      Despite its popularity, chromatin immunoprecipitation followed by sequencing (ChIP-seq) remains a tedious (>2 d), manually intensive, low-sensitivity and low-throughput approach. Here, we combine principles of microengineering, surface chemistry, and molecular biology to address the major limitations of standard ChIP-seq. The resulting technology, FloChIP, automates and miniaturizes ChIP in a beadless fashion while facilitating the downstream library preparation process through on-chip chromatin tagmentation. FloChIP is fast (<2 h), has a wide dynamic range (from 106 to 500 cells), is scalable and parallelized, and supports antibody- or sample-multiplexed ChIP on both histone marks and transcription factors. In addition, FloChIP's interconnected design allows for straightforward chromatin reimmunoprecipitation, which allows this technology to also act as a microfluidic sequential ChIP-seq system. Finally, we ran FloChIP for the transcription factor MEF2A in 32 distinct human lymphoblastoid cell lines, providing insights into the main factors driving collaborative DNA binding of MEF2A and into its role in B cell-specific gene regulation. Together, our results validate FloChIP as a flexible and reproducible automated solution for individual or sequential ChIP-seq.
    Keywords:  ChIP-seq; epigenetics; microfluidics; transcription factor
  22. Nat Commun. 2020 May 26. 11(1): 2639
      Homologous recombination (HR) is important for error-free DNA double strand break repair and maintenance of genomic stability. However, upregulated HR is also used by cancer cells to promote therapeutic resistance. Therefore, inducing HR deficiency (HRD) is a viable strategy to sensitize HR proficient cancers to DNA targeted therapies in order to overcome therapeutic resistance. A bromodomain containing protein, BRD9, was previously reported to regulate chromatin remodeling and transcription. Here, we discover that following DNA damage, the bromodomain of BRD9 binds acetylated K515 on RAD54 and facilitates RAD54's interaction with RAD51, which is essential for HR. BRD9 is overexpressed in ovarian cancer and depleting BRD9 sensitizes cancer cells to olaparib and cisplatin. In addition, inhibitor of BRD9, I-BRD9, acts synergistically with olaparib in HR-proficient cancer cells. Overall, our results elucidate a role for BRD9 in HR and identify BRD9 as a potential therapeutic target to promote synthetic lethality and overcome chemoresistance.
  23. Oncogene. 2020 May 26.
      Small cell lung cancer (SCLC) is a highly aggressive malignancy with poor outcomes associated with resistance to cisplatin-based chemotherapy. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2), which silences transcription through trimethylation of histone H3 lysine 27 (H3K27me3) and has emerged as an important therapeutic target with inhibitors targeting its methyltransferase activity under clinical investigation. Here, we show that EZH2 has a non-catalytic and PRC2-independent role in stabilizing DDB2 to promote nucleotide excision repair (NER) and govern cisplatin resistance in SCLC. Using a synthetic lethality screen, we identified important regulators of cisplatin resistance in SCLC cells, including EZH2. EZH2 depletion causes cellular cisplatin and UV hypersensitivity in an epistatic manner with DDB1-DDB2. EZH2 complexes with DDB1-DDB2 and promotes DDB2 stability by impairing its ubiquitination independent of methyltransferase activity or PRC2, thereby facilitating DDB2 localization to cyclobutane pyrimidine dimer crosslinks to govern their repair. Furthermore, targeting EZH2 for depletion with DZNep strongly sensitizes SCLC cells and tumors to cisplatin. Our findings reveal a non-catalytic and PRC2-independent function for EZH2 in promoting NER through DDB2 stabilization, suggesting a rationale for targeting EZH2 beyond its catalytic activity for overcoming cisplatin resistance in SCLC.
  24. Methods Mol Biol. 2020 ;2151 93-107
      DNA-binding proteins play critical roles in many major processes such as development and sexual biology of Schistosoma mansoni and are important for the pathogenesis of schistosomiasis. Chromatin immunoprecipitation (ChIP) experiments followed by sequencing (ChIP-seq) are useful to characterize the association of genomic regions with posttranslational chemical modifications of histone proteins. Challenges in the standard ChIP protocol have motivated recent enhancements in this approach, such as reducing the number of cells required and increasing the resolution. In this chapter, we describe the latest advances made by our group in the ChIP methods to improve the standard ChIP protocol to reduce the number of input cells required and to increase the resolution and robustness of ChIP in S. mansoni.
    Keywords:  ChIPmentation; Development; Histone modifications; Native chromatin immunoprecipitation; Schistosomiasis; Trematode
  25. Int J Mol Sci. 2020 May 21. pii: E3634. [Epub ahead of print]21(10):
      Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFβ1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFβ1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFβ1-induced and uninduced PLOD2 expression.
    Keywords:  KRAB; PLOD2; cancer; epigenetic editing; fibrosis; gene repression
  26. Nat Commun. 2020 May 25. 11(1): 2606
      Nucleoporin proteins (Nups) have been proposed to mediate spatial and temporal chromatin organization during gene regulation. Nevertheless, the molecular mechanisms in mammalian cells are not well understood. Here, we report that Nucleoporin 153 (NUP153) interacts with the chromatin architectural proteins, CTCF and cohesin, and mediates their binding across cis-regulatory elements and TAD boundaries in mouse embryonic stem (ES) cells. NUP153 depletion results in altered CTCF and cohesin binding and differential gene expression - specifically at the bivalent developmental genes. To investigate the molecular mechanism, we utilize epidermal growth factor (EGF)-inducible immediate early genes (IEGs). We find that NUP153 controls CTCF and cohesin binding at the cis-regulatory elements and POL II pausing during the basal state. Furthermore, efficient IEG transcription relies on NUP153. We propose that NUP153 links the nuclear pore complex (NPC) to chromatin architecture allowing genes that are poised to respond rapidly to developmental cues to be properly modulated.
  27. Genet Mol Biol. 2020 ;pii: S1415-47572020000400706. [Epub ahead of print]43(2): e20190338
      Regulation of target proteins by the ubiquitin-proteasome system (UPS) is common in a wide range of cellular events, including transcriptional regulation, cell cycle progression, differentiation, and tumorigenesis. Ubiquitin-specific protease 7 (USP7) has been implicated in tumor development and metastasis in various malignancies through the regulation of target protein stability. In this study, we found that the enhancer of zeste homolog 2 (EZH2), which catalyzes the methylation at lysine 27 of histone H3, is a target of USP7 and is stabilized by USP7-mediated deubiquitination. In prostate cancer cells, the transcriptional repression function of EZH2 was inhibited by USP7-knockdown. Furthermore, ectopic introduction of EZH2 restored the cell migration, invasion, and sphere-forming potential of prostate cancer cells, which had been decreased by USP7-knockdown. Moreover, combined treatment with the USP7-specific inhibitor P5091 and EZH2 inhibitors, such as GSK126, EPZ6438, and DZNep, induced synergistic inhibitory effects on cell migration, invasion, and sphere-forming potential in prostate cancer cells. Collectively, our findings revealed that the promotion of the malignancy-associated characteristics of prostate cancer cells by USP7 was in part due to EZH2 stabilization. Thus, we suggest that simultaneous treatment with a USP7 inhibitor and an EZH2 inhibitor could be a rational strategy for treating EZH2-dependent cancers.
  28. Nat Biotechnol. 2020 May 25.
      With the exception of lamina-associated domains, the radial organization of chromatin in mammalian cells remains largely unexplored. Here we describe genomic loci positioning by sequencing (GPSeq), a genome-wide method for inferring distances to the nuclear lamina all along the nuclear radius. GPSeq relies on gradual restriction digestion of chromatin from the nuclear lamina toward the nucleus center, followed by sequencing of the generated cut sites. Using GPSeq, we mapped the radial organization of the human genome at 100-kb resolution, which revealed radial patterns of genomic and epigenomic features and gene expression, as well as A and B subcompartments. By combining radial information with chromosome contact frequencies measured by Hi-C, we substantially improved the accuracy of whole-genome structure modeling. Finally, we charted the radial topography of DNA double-strand breaks, germline variants and cancer mutations and found that they have distinctive radial arrangements in A and B subcompartments. We conclude that GPSeq can reveal fundamental aspects of genome architecture.
  29. Oncogene. 2020 May 25.
      Chromatin remodeling factors contribute to establish aberrant gene expression programs in cancer cells and therefore represent valuable targets for therapeutic intervention. BPTF (Bromodomain PhD Transcription Factor), a core subunit of the nucleosome remodeling factor (NURF), modulates c-MYC oncogenic activity in pancreatic cancer. Here, we analyze the role of BPTF in c-MYC-driven B-cell lymphomagenesis using the Eμ-Myc transgenic mouse model of aggressive B-cell lymphoma. We find that BPTF is required for normal B-cell differentiation without evidence of haploinsufficiency. In contrast, deletion of one Bptf allele is sufficient to delay lymphomagenesis in Eμ-Myc mice. Tumors arising in a Bptf heterozygous background display decreased c-MYC levels and pathway activity, together with increased activation of the NF-κB pathway, a molecular signature characteristic of human diffuse large B-cell lymphoma (DLBCL). In human B-cell lymphoma samples, we find a strong correlation between BPTF and c-MYC mRNA and protein levels, together with an anti-correlation between BPTF and NF-κB pathway activity. Our results indicate that BPTF is a relevant therapeutic target in B-cell lymphomas and that, upon its inhibition, cells acquire distinct oncogenic dependencies.
  30. Cells. 2020 May 21. pii: E1280. [Epub ahead of print]9(5):
      Tumor necrosis factor-α (TNF-α) plays a significant role in inflammation and cancer-related apoptosis. We identified a TNF-α-mediated epigenetic mechanism of apoptotic cell death regulation in estrogen receptor-α (ERα)-positive human breast cancer cells. To assess the apoptotic effect of TNF-α, annexin V/ propidium iodide (PI) double staining, cell viability assays, and Western blotting were performed. To elucidate this mechanism, histone deacetylase (HDAC) activity assay and immunoprecipitation (IP) were conducted; the mechanism was subsequently confirmed through chromatin IP (ChIP) assays. Finally, we assessed HDAC3-ERα-mediated apoptotic cell death after TNF-α treatment in ERα-positive human breast cancer (MCF-7) cells via the transcriptional activation of p53 target genes using luciferase assay and quantitative reverse transcription PCR. The TNF-α-induced selective apoptosis in MCF-7 cells was negatively regulated by the HDAC3-ERα complex in a caspase-7-dependent manner. HDAC3 possessed a p53-binding element, thus suppressing the transcriptional activity of its target genes. In contrast, MCF-7 cell treatment with TNF-α led to dissociation of the HDAC3-ERα complex and substitution of the occupancy on the promoter by the p53-p300 complex, thus accelerating p53 target gene expression. In this process, p53 stabilization was accompanied by its acetylation. This study showed that p53-mediated apoptosis in ERα-positive human breast cancer cells was negatively regulated by HDAC3-ERα in a caspase-7-dependent manner. Therefore, these proteins have potential application in therapeutic strategies.
    Keywords:  ERα; HDAC3; TNF-α; apoptosis; p53
  31. PLoS Genet. 2020 May 26. 16(5): e1008770
      Hormone-dependent activation of enhancers includes histone hyperacetylation and mediator recruitment. Histone hyperacetylation is mostly explained by a bimodal switch model, where histone deacetylases (HDACs) disassociate from chromatin, and histone acetyl transferases (HATs) are recruited. This model builds on decades of research on steroid receptor regulation of transcription. Yet, the general concept of the bimodal switch model has not been rigorously tested genome wide. We have used a genomics approach to study enhancer hyperacetylation by the thyroid hormone receptor (TR), described to operate as a bimodal switch. H3 acetylation, HAT and HDAC ChIP-seq analyses of livers from hypo- and hyperthyroid wildtype, TR deficient and NCOR1 disrupted mice reveal three types of thyroid hormone (T3)-regulated enhancers. One subset of enhancers is bound by HDAC3-NCOR in the absence of hormone and constitutively occupy TR and HATs irrespective of T3 levels, suggesting a poised enhancer state in absence of hormone. In presence of T3, HDAC3-NCOR1 dissociates from these enhancers leading to histone hyperacetylation, suggesting a histone acetylation rheostat function of HDACs at poised enhancers. Another subset of enhancers, not occupied by HDACs, is hyperacetylated in a T3-dependent manner, where TR is recruited to chromatin together with HATs. Lastly, a subset of enhancers, is not occupied directly by TR yet require TR for histone hyperacetylation. This indirect enhancer activation involves co-association with TR bound enhancers within super-enhancers or topological associated domains. Collectively, this demonstrates various mechanisms controlling hormone-dependent transcription and adds significant details to the otherwise simple bimodal switch model.
  32. Clin Epigenetics. 2020 May 24. 12(1): 72
      Increasing evidence has suggested that epigenetic and metabolic alterations in cancer cells are highly intertwined. As the master epigenetic regulator, enhancer of zeste homolog 2 (EZH2) suppresses gene transcription mainly by catalyzing the trimethylation of histone H3 at lysine 27 (H3K27me3) and exerts highly enzymatic activity in cancer cells. Cancer cells undergo the profound metabolic reprogramming and manifest the distinct metabolic profile. The emerging studies have explored that EZH2 is involved in altering the metabolic profiles of tumor cells by multiple pathways, which cover glucose, lipid, and amino acid metabolism. Meanwhile, the stability and methyltransferase activity of EZH2 can be also affected by the metabolic activity of tumor cells through various mechanisms, including post-translational modification. In this review, we have summarized the correlation between EZH2 and cellular metabolic activity during tumor progression and drug treatment. Finally, as a promising target, we proposed a novel strategy through a combination of EZH2 inhibitors with metabolic regulators for future cancer therapy.
    Keywords:  EZH2; Histone modification; Metabolism; Tumor therapy
  33. J Neurooncol. 2020 May 29.
      PURPOSE: Malignant gliomas remain significant challenges in clinic and pose dismal prognosis on patients. In this study, we focused on growth arrest-specific 5 (GAS5), a tumor suppressive long non-coding RNA in glioma, explored its crosstalk with miR-424, and examined their biological functions in glioma.METHODS: Expressions of GAS5 and miR-424 were measured using qRT-PCR. The regulation of GAS5 on miR-424 expression was examined in GAS5-overexpressing glioma cells by combining methylation-specific PCR, western blotting, and RNA immunoprecipitation. Functional significance of GAS5 and miR-424 on in vitro cell proliferation, apoptosis, migration, invasion, and in vivo tumor growth was examined using colony formation, flow cytometry, wound healing, transwell assay, and the xenograft model, respectively. The potential targeting of AKT3 by miR-424 was investigated using luciferase reporter assay.
    RESULTS: GAS5 and miR-424 were significantly down-regulated in glioma cells. GAS5 directly interacted with enhancer of zeste homolog 2 (EZH2), stimulated the formation of polycomb repressive complex 2 (PRC2), reduced the levels of DNA methyltransferases (Dnmts), alleviated promoter methylation of miR-424, and promoted miR-424 expression. Functionally, GAS5, by up-regulating miR-424, inhibited cell proliferation, migration, and invasion, while increased apoptosis of glioma cells in vitro, and suppressed xenograft growth in vivo. miR-424 directly inhibited AKT3 and altered the expressions of AKT3 targets, cyclinD1, c-Myc, Bax, and Bcl-2, which might contribute to its tumor suppressive activities.
    CONCLUSIONS: GAS5, by inhibiting methylation and boosting expression of miR-424, inhibits AKT3 signaling and suppresses multiple malignant phenotypes. Therefore, stimulating GAS5/miR-424 signaling may benefit the treatment of glioma.
    Keywords:  Glioma; Methylation; PRC2; lncRNA-GAS5; miR-424
  34. Curr Top Dev Biol. 2020 ;pii: S0070-2153(20)30035-1. [Epub ahead of print]139 35-60
      For decades, the early development of the Xenopus embryo has been an essential model system to study the gene regulatory mechanisms that govern cellular specification. At the top of the hierarchy of gene regulatory networks, maternally deposited transcription factors initiate this process and regulate the expression of zygotic genes that give rise to three distinctive germ layer cell types (ectoderm, mesoderm, and endoderm), and subsequent generation of organ precursors. The onset of germ layer specification is also closely coupled with changes associated with chromatin modifications. This review will examine the timing of maternal transcription factors initiating the zygotic genome activation, the epigenetic landscape of embryonic chromatin, and the network structure that governs the process.
    Keywords:  Cis-regulatory modules; Enhancers; Feed forward loop; Gene regulatory network; Germ layers; Maternal transcription factors; Promoters; Xenopus; Zygotic genome activation
  35. Adv Exp Med Biol. 2020 ;1239 199-231
      Although originally characterized as a cytoplasmic protein, myosin of various classes also performs key functions in the nucleus. We review the data concerning the nuclear localization, mechanism of entry, and functional interactions of myosin I, II, V, VI, X, XVI, and XVIII. To date, the first-characterized "nuclear myosin I" (or, in the prevailing nomenclature, myosin IC isoform B) remains the best-studied nuclear myosin, although results are rapidly accumulating that illuminate the roles of other myosin classes, and an outline of a unified picture of myosin functions in the nucleus is beginning to emerge. Reflecting the state of knowledge in this field, the review concentrates on the mechanisms mediating and regulating import of myosin IC into the nucleus and its role, alongside myosin V and VI, in transcription. Myosin functions in chromatin dynamics, epigenetic mechanisms, intranuclear motility, and nuclear export of RNA and protein are also addressed. Partners and regulators of myosin, such as nuclear actin, kinases, and phosphatases are briefly covered. Problem areas are identified and testable hypotheses are offered with an aim of focusing the research efforts on overcoming the gaps on the way toward a systems-level understanding of processes involving nuclear myosins and their place in cell physiology as a whole.
    Keywords:  Chromatin; Intranuclear motility; Localization; Myosin; Myosin IC; Nuclear actin; Nuclear export; Nuclear import; Nuclear myosin; Nucleolus; Nucleus; RNA processing; Transcription
  36. Cells. 2020 May 27. pii: E1340. [Epub ahead of print]9(6):
      Nuclear lamins (NLs) are essential components of the animal cell nucleus involved in the regulation of a plethora of molecular and cellular processes. These include the nuclear envelope assembly and stability, mechanotransduction and chromatin organization, transcription, DNA replication, damage repair, and genomic integrity maintenance. Mutations in NLs can lead to the development of a wide range of distinct disease phenotypes, laminopathies, consisting of cardiac, neuromuscular, metabolic and premature aging syndromes. In addition, alterations in the expression of nuclear lamins were associated with different types of neoplastic diseases. Despite the importance and critical roles that NLs play in the diverse cellular activities, we only recently started to uncover the complexity of regulatory mechanisms governing their expression, localization and functions. This integrative review summarizes and discusses the recent findings on the emerging roles of ubiquitin and ubiquitin-like modifiers (ULMs) in the regulation of NLs, highlighting the intriguing molecular associations and cross-talks occurring between NLs and these regulatory molecules under physiological conditions and in the disease states.
    Keywords:  autophagy; laminopathies; nuclear lamins; proteasome; ubiquitin; ubiquitin-like modifiers
  37. Curr Pharm Biotechnol. 2020 May 28.
      INTRODUCTION: Resistance to corticosteroid is an essential mechanism in uncontrolled asthma as the corticosteroid is the mainstay of therapy. There are recent reports that epigenetic factors play a crucial role in the regulation of steroid action. Overexpression of P glycoprotein (P-gp) and reduce expression of Histone deacetylase 2 (HDAC2) has been linked to regulating the steroid action in other diseases like Nephrotic Syndrome (NS). However, their role in uncontrolled asthma is still not clear and warrant further investigation. We evaluated the expression and activity of P-gp and HDAC2 in patients with controlled asthma (CA) and uncontrolled asthma (UA).METHODS: A total of 60 CA (mean age 51.72±17.02 years, male=38), and 38 of UA (mean age=53.55±11.90 years, male=17) were recruited. The level of control was defined according to ( Global Initiative for Asthma) GINA 2016 criteria. The mRNA expression of HDAC2 and P-gp was studied by quantitative real-time polymerase chain reaction(PCR)the functional activity of P-gp was evaluated by commercially available kit via flow cytometry, and HDAC2 enzymatic activity was measured by commercially available kit by enzyme-linked immunosorbent assay (ELISA).
    RESULTS: P-gp expression and the functionality, was significantly higher in UA group of patients as compared to CA group of patients (p<0.005), moreover HDAC2 expression was significantly reduced in UA patients as compared to CA patients, (p<0.005). The enzymatic activity of HDAC2 was also significantly reduced in UA patients as compared to CA patients(p<0.005).
    CONCLUSION: P-gp overexpression and HDAC2 under expression play an essential role in uncontrolled asthma by impairing the response to corticosteroid.
    Keywords:  Controlled asthma; Flow Cytometry; Gene expression; HDAC2; P-glycoprotein; Steroid responsiveness; Uncontrolled Asthma
  38. Mol Brain. 2020 May 29. 13(1): 85
      Genetic and epigenetic factors contribute to the development of the spinal cord. Failure in correct exertion of the developmental programs, including neurulation, neural tube closure and neurogenesis of the diverse spinal cord neuronal subtypes results in defects of variable severity. We here report on the histone methyltransferase Disruptor of Telomeric 1 Like (DOT1L), which mediates histone H3 lysine 79 (H3K79) methylation. Conditional inactivation of DOT1L using Wnt1-cre as driver (Dot1l-cKO) showed that DOT1L expression is essential for spinal cord neurogenesis and localization of diverse neuronal subtypes, similar to its function in the development of the cerebral cortex and cerebellum. Transcriptome analysis revealed that DOT1L deficiency favored differentiation over progenitor proliferation. Dot1l-cKO mainly decreased the numbers of dI1 interneurons expressing Lhx2. In contrast, Lhx9 expressing dI1 interneurons did not change in numbers but localized differently upon Dot1l-cKO. Similarly, loss of DOT1L affected localization but not generation of dI2, dI3, dI5, V0 and V1 interneurons. The resulting derailed interneuron patterns might be responsible for increased cell death, occurrence of which was restricted to the late developmental stage E18.5. Together our data indicate that DOT1L is essential for subtype-specific neurogenesis, migration and localization of dorsal and ventral interneurons in the developing spinal cord, in part by regulating transcriptional activation of Lhx2.
    Keywords:  DOT1L; Epigenetics; Interneuron; Localization; Methyltransferase; Specification; Spinal cord
  39. Oncol Rep. 2020 May 28.
      Changes in histone H3 lysine 9 trimethylation (H3K9me3) may be related to the development of drug‑resistant acute myeloid leukaemia (AML); insights into the network of H3K9me3 may improve patient prognosis. Patient data were derived from the Gene Expression Omnibus (GEO) database and data from AML cells treated with chidamide, a novel benzamide chemical class of histone deacetylase inhibitor (HDACi), in vitro were derived from ChIP‑seq. Patients and AML cell data were analysed using GEO2R, GOseq, KOBAS, the STRING database and Cytoscape 3.5.1. We identified several genes related to the upregulation or downregulation of H3K9me3 in AML patients; some of these genes were related to apoptosis, autophagy, and the pathway of cell longevity. AML cells treated with chidamide in vitro showed the same gene changes. The protein interactions in the network did not have significantly more interactions than expected, suggesting the need for more research to identify these interactions. One compelling result from the protein interaction study was that sirtuin 1 (SIRT1) may have an indirect interaction with lysine‑specific demethylase 4A (KDM4A). These results help explain alterations of H3K9me3 in AML that may direct further studies aimed at improving patient prognosis. These results may also provide a basis for chidamide as a treatment strategy for AML patients in the future.
  40. ACS Appl Mater Interfaces. 2020 May 26.
      Bmi1 is associated with advanced prognosis of acute myeloid leukemia (AML), and polyethylenimine (PEI)-stabilized Bmi1 siRNA-entrapped human serum albumin (HSA) nanocarriers (PEI@HSANCs) were used to protect siRNA from degradation and also to control epigenetic regulation-based AML therapy. The nanoform increased the transfection efficiency of Bmi1 siRNA through caveolae-mediated endocytosis and enhanced Bax translocation into the mitochondria. It enhanced the caspase 3-mediated apoptosis through the Bax activation and Bcl-2 inhibition. The molecular analysis reveals the downregulation of polycomb proteins, Bmi1 and EzH2, along with inhibition of H3K27me3 and H2AK119ub1. The signaling cascade revealed downregulation of Bmi1 through ubiquitin-mediated degradation and is reversed by a proteasome inhibitor. Further mechanistic studies established a crucial role of transcription factor, C-Myb and Bmi1, as its direct targets for maintenance and progression of AML. Chromatin immunoprecipitation (ChIP) assay confirmed Bmi1 as a direct target of C-Myb as it binds to promoter sequence of Bmi1 between -235 to +43 and -111 to +43. The in vivo studies performed in the AML xenograft model evidence a decrease in the population of leukemic stem cells marker (CD45+) and an increase in the myeloid differentiating marker expression (CD11b+) in the bone marrow after the Bmi1 siRNA nanoconjugated therapy. Activation of apoptotic pathways and withdrawal of epigenetic repression through a ubiquitin proteasomal pathway potentiating a novel antileukemic therapy were established.
    Keywords:  Bmi1 siRNA; RNAi; acute myeloid leukemia; epigenetics; polycomb; ubiquitin ligase
  41. PLoS Genet. 2020 May 29. 16(5): e1008798
      Alterations in epigenetic silencing have been associated with ageing and tumour formation. Although substantial efforts have been made towards understanding the mechanisms of gene silencing, novel regulators in this process remain to be identified. To systematically search for components governing epigenetic silencing, we developed a genome-wide silencing screen for yeast (Saccharomyces cerevisiae) silent mating type locus HMR. Unexpectedly, the screen identified the mismatch repair (MMR) components Pms1, Mlh1, and Msh2 as being required for silencing at this locus. We further found that the identified genes were also required for proper silencing in telomeres. More intriguingly, the MMR mutants caused a redistribution of Sir2 deacetylase, from silent mating type loci and telomeres to rDNA regions. As a consequence, acetylation levels at histone positions H3K14, H3K56, and H4K16 were increased at silent mating type loci and telomeres but were decreased in rDNA regions. Moreover, knockdown of MMR components in human HEK293T cells increased subtelomeric DUX4 gene expression. Our work reveals that MMR components are required for stable inheritance of gene silencing patterns and establishes a link between the MMR machinery and the control of epigenetic silencing.
  42. Clin Epigenetics. 2020 May 29. 12(1): 74
      BACKGROUND: The histone 3 lysine 4 (H3K4) monomethylase KMT2C is mutated across several cancer types; however, the effects of mutations on epigenome organization, gene expression, and cell growth are not clear. A frequently recurring mutation in colorectal cancer (CRC) with microsatellite instability is a single nucleotide deletion within the exon 38 poly-A(9) repeat (c.8390delA) which results in frameshift preceding the functional carboxy-terminal SET domain. To study effects of KMT2C expression in CRC cells, we restored one allele to wild type KMT2C in the two CRC cell lines RKO and HCT116, which both are homozygous c.8390delA mutant.RESULTS: Gene editing resulted in increased KMT2C expression, increased H3K4me1 levels, altered gene expression profiles, and subtle negative effects on cell growth, where higher dependence and stronger effects of KMT2C expression were observed in RKO compared to HCT116 cells. Surprisingly, we found that the two RKO and HCT116 CRC cell lines have distinct baseline H3K4me1 epigenomic profiles. In RKO cells, a flatter genome-wide H3K4me1 profile was associated with more increased H3K4me1 deposition at enhancers, reduced cell growth, and more differential gene expression relative to HCT116 cells when KMT2C was restored. Profiling of H3K4me1 did not indicate a highly specific regulation of gene expression as KMT2C-induced H3K4me1 deposition was found globally and not at a specific enhancer sub-set in the engineered cells. Although we observed variation in differentially regulated gene sets between cell lines and individual clones, differentially expressed genes in both cell lines included genes linked to known cancer signaling pathways, estrogen response, hypoxia response, and aspects of immune system regulation.
    CONCLUSIONS: Here, KMT2C restoration reduced CRC cell growth and reinforced genome-wide H3K4me1 deposition at enhancers; however, the effects varied depending upon the H3K4me1 status of KMT2C deficient cells. Results indicate that KMT2C inactivation may promote colorectal cancer development through transcriptional dysregulation in several pathways with known cancer relevance.
    Keywords:  Cancer; H3K4me1; KMT2C; MLL3
  43. J Cancer Res Clin Oncol. 2020 May 28.
      PURPOSE: Breast cancer is the leading cause of cancer death in females. Histone modifications have been shown to have an influence on the gene expression. This study focusses on the histone modifications H3K9ac and H3K4me3 in breast cancer and their impact on survival METHODS: H3K4me3 and H3K9ac expression was immunohistochemically examined in 235 tissue samples.RESULTS: Positive estrogen receptor status was correlated with a higher IRS of the nuclear (p = 0.033), and of the cytoplasmic H3K4me3 staining (p = 0.009). H3K9ac intensity was associated to the Her2 status (p = 0.045) and to poor prognosis in cells with positive Ki67 status (p = 0.013). A high intensity of nuclear H3K4me3 staining was found to be correlated with a lower 10-year-survival (p = 0.026) and with lower breast cancer-specific survival (p = 0.004). High percentage score (> 190) of H3K9ac expression was correlated to worse breast cancer-specific survival (p = 0.005). Shorter progression-free survival was found in patients with nuclear (p = 0.013) and cytoplasmic H3K4me3expression (p = 0.024) and H3K9ac expression (p = 0.023).
    CONCLUSION: This analysis provides new evidence of histone modifications in breast cancer. High H3K4me3 and H3K9ac expression was correlated with survival rates. Further investigation of histone modifications in breast cancer could lead to a more profound understanding of the molecular mechanisms of cancer development and could result in new therapeutic strategies.
    Keywords:  Breast cancer; Epigenetic; Histone modification; survival
  44. BMC Res Notes. 2020 May 24. 13(1): 254
      OBJECTIVES: UHRF1 is a multi-domain protein that recognizes both histone and DNA modification marks on chromatin. UHRF1 is involved in various cellular processes that lead to tumorigenesis and thus attracted considerable attention as a potential anti-cancer drug target. The SRA domain is a unique to the UHRF family. SRA domain recognizes 5-methylcytosine in hemimethylated DNA and necessary for maintenance DNA methylation mediated by DNMT1. Small molecules capable of interacting with the SRA domain may reduce aberrant methylation levels by preventing the interaction of 5-methylcytosine with the SRA domain and thereby blocking substrate access to the catalytic center of DNMT1. The data were collected to identify and predict an initial set of small molecules that are expected to bind to the SRA domain.DATA DESCRIPTION: Nearly 2.4 million molecules from various chemical libraries were screened with the SRA domain of UHRF1 using Schrodinger's Small Molecule Drug Discovery Suite. The data is available in the form of a methodology presentation, MS Excel files listing the top hits, and Maestro pose viewer files that provide visualization of how the identified ligands interact with the SRA domain.
    Keywords:  Cancer; DNA methylation; DNMT1; SRA domain; Structure-based screening; UHRF1; UHRF1 inhibitor; Virtual screening
  45. Nitric Oxide. 2020 May 26. pii: S1089-8603(20)30038-0. [Epub ahead of print]
      Inorganic dietary nitrate plays vital roles in biological functions via the exogenous NO3-/NO2-/NO pathway under hypoxia and ischemia. We previously verified the antioxidative effects of inorganic nitrate in a mouse model of total body irradiation (TBI). Accordingly, in this study, we evaluated the effects of inorganic nitrate on prevention of TBI-induced colon injury and dysbiosis of the gut microbiome. Nitrate significantly rescued the abnormal biological indexes (body weight, white blood cell, red blood cell, platelet, hemoglobin level and intestinal canal lengths) induced by TBI. Then, we detected oxidative stress and DNA damage indexes (phospho-histone H2AX and p53 binding protein 1), which were both increased by irradiation (IR) and alleviated by nitrate. IR-induced apoptosis and senescence were ameliorated by inorganic nitrate. The distribution of the gut microbiome differed for mice with TBI and those receiving inorganic nitrate. The average abundance of Lactobacillus significantly increased, and that of Bacteroidales decreased at the genus level in the nitrate group compared with that in the IR alone group. At 30 days after TBI, the abundances of Bacteroides and Faecalibaculum decreased, whereas that of Lactobacillus increased in the IR + nitrate group compared with that in the IR alone group. Inorganic nitrate efficiently prevents TBI-induced colon epithelium injury and maintains the homeostasis of the gut microbiome. Thus, our results showed that inorganic nitrate might be a promising treatment for TBI induced colon injury.
    Keywords:  Colonic mucosa; Gut microbiome; Nitrate; Oxidative stress injury; Total body irradiation
  46. Int J Mol Sci. 2020 May 26. pii: E3762. [Epub ahead of print]21(11):
      The deubiquitination of histone H2A on lysine 119 by 2A-DUB/MYSM1, BAP1, USP16, and other enzymes is required for key cellular processes, including transcriptional activation, apoptosis, and cell cycle control, during normal hematopoiesis and tissue development, and in tumor cells. Based on our finding that MYSM1 colocalizes with γH2AX foci in human peripheral blood mononuclear cells, leukemia cells, and melanoma cells upon induction of DNA double-strand breaks with topoisomerase inhibitor etoposide, we applied a mass spectrometry-based proteomics approach to identify novel 2A-DUB/MYSM1 interaction partners in DNA-damage responses. Differential display of MYSM1 binding proteins significantly enriched after exposure of 293T cells to etoposide revealed an interacting network of proteins involved in DNA damage and replication, including factors associated with poor melanoma outcome. In the context of increased DNA-damage in a variety of cell types in Mysm1-deficient mice, in bone marrow cells upon aging and in UV-exposed Mysm1-deficient skin, our current mass spectrometry data provide additional evidence for an interaction between MYSM1 and key DNA replication and repair factors, and indicate a potential function of 2A-DUB/MYSM1 in DNA repair processes.
    Keywords:  DNA damage; HELLS; MYSM1; apoptosis; cancer; histone deubiquitinase; homologous recombination (HR); melanoma; p53; γH2AX
  47. Bone. 2020 May 20. pii: S8756-3282(20)30220-9. [Epub ahead of print]137 115440
      There is mounting evidence in the literature that mesenchymal stromal/stem cell (MSC) like populations derived from different tissues, undergo epigenetic changes during aging, leading to compromised connective tissue integrity and function. This body of work has linked the biological aging of MSC to changes in their epigenetic signatures affecting growth, lifespan, self-renewal and multi-potential, due to deregulation of processes such as cellular senescence, oxidative stress, DNA damage, telomere shortening and DNA damage. This review addresses recent findings examining DNA methylation, histone modifications and miRNA changes in aging MSC populations. Moreover, we explore how epigenetic factors alter cellular pathways and associated biological networks, contributing to the MSC aging phenotype. Finally we discuss the crucial areas requiring a greater understanding of these processes, in order to piece together a global picture of the changing epigenetic landscape in MSC during aging.
    Keywords:  Aging; DNA methylation; Epigenetics; Histome modifications; MSC; Mesenchymal Stem/Stromal Cells; Skeleton; miRNA
  48. Curr Biol. 2020 May 26. pii: S0960-9822(20)30560-1. [Epub ahead of print]
      Maternally inherited RNA and proteins control much of embryonic development. The effect of such maternal information beyond embryonic development is largely unclear. Here, we report that maternal contribution of histone H3.3 assembly complexes can prevent the expression of late-onset anatomical, physiologic, and behavioral abnormalities of C. elegans. We show that mutants lacking hira-1, an evolutionarily conserved H3.3-deposition factor, have severe pleiotropic defects that manifest predominantly at adulthood. These late-onset defects can be maternally rescued, and maternally derived HIRA-1 protein can be detected in hira-1(-/-) progeny. Mitochondrial stress likely contributes to the late-onset defects, given that hira-1 mutants display mitochondrial stress, and the induction of mitochondrial stress results in at least some of the hira-1 late-onset abnormalities. A screen for mutants that mimic the hira-1 mutant phenotype identified PQN-80-a HIRA complex component, known as UBN1 in humans-and XNP-1-a second H3.3 chaperone, known as ATRX in humans. pqn-80 and xnp-1 abnormalities are also maternally rescued. Furthermore, mutants lacking histone H3.3 have a late-onset defect similar to a defect of hira-1, pqn-80, and xnp-1 mutants. These data demonstrate that H3.3 assembly complexes provide non-DNA-based heritable information that can markedly influence adult phenotype. We speculate that similar maternal effects might explain the missing heritability of late-onset human diseases, such as Alzheimer's disease, Parkinson's disease, and type 2 diabetes.
    Keywords:  ATRX; C. elegans; H3.3; HIRA-1; late-onset; maternal effect; mitochondrial stress