bims-histon Biomed News
on Histones
Issue of 2020‒06‒21
twenty-two papers selected by
Benjamin Weekley
University of Southern California


  1. Nat Commun. 2020 Jun 19. 11(1): 3140
    Lee W, Kim J, Yun JM, Ohn T, Gong Q.
      MeCP2 plays a multifaceted role in gene expression regulation and chromatin organization. Interaction between MeCP2 and methylated DNA in the regulation of gene expression is well established. However, the widespread distribution of MeCP2 suggests it has additional interactions with chromatin. Here we demonstrate, by both biochemical and genomic analyses, that MeCP2 directly interacts with nucleosomes and its genomic distribution correlates with that of H3K27me3. In particular, the methyl-CpG-binding domain of MeCP2 shows preferential interactions with H3K27me3. We further observe that the impact of MeCP2 on transcriptional changes correlates with histone post-translational modification patterns. Our findings indicate that MeCP2 interacts with genomic loci via binding to DNA as well as histones, and that interaction between MeCP2 and histone proteins plays a key role in gene expression regulation.
    DOI:  https://doi.org/10.1038/s41467-020-16907-0
  2. J Mol Biol. 2020 Jun 10. pii: S0022-2836(20)30401-0. [Epub ahead of print]
    Fourier N, Zolty M, Azriel A, Tedesco D, Levi BZ.
      The regulation of gene expression is a result of a complex interplay between chromatin remodeling, transcription factors (TFs) and signaling molecules. Cell differentiation is accompanied by chromatin remodeling of specific loci to permanently silence genes that are not essential for the differentiated cell activity. The molecular cues that recruit the chromatin remodeling machinery are not well characterized. IRF8 is an immune-cell specific TF and its expression is augmented by Interferon-γ (IFNγ). Therefore, it serves as a model gene to elucidate the molecular mechanisms governing its silencing in non-immune cells. A high-throughput shRNA library screen in IRF8 expression-restrictive cells enabled the identification of MafK as modulator of IRF8 silencing, affecting chromatin architecture. ChIP-Seq analysis revealed three MafK binding-regions (-25kb, -20kb and IRF8 6th intron) within the IRF8 locus. These MafK binding-sites are sufficient to repress a reporter-gene when cloned in genome-integrated lentiviral reporter constructs in only expression-restrictive cells. Conversely, plasmid-based constructs do not demonstrate such repressive effect. These results highlight the role of these MafK binding-sites in mediating repressed chromatin assembly. Finally, a more thorough genomic analysis was performed, using CRISPR-Cas9 to delete MafK-int6 binding-region in IRF8 expression-restrictive cells. Deleted clones exhibited an accessible chromatin conformation within the IRF8 locus that was accompanied by a significant increase in basal expression of IRF8 that was further induced by IFNγ. Taken together, we identified and characterized several MafK binding elements within the IRF8 locus that mediate repressive chromatin conformation resulting in the silencing of IRF8 expression in a cell-type specific manner.
    Keywords:  Cell-type specific expression; Chromatin structure; Gene regulation; Interferon Regulatory Factor-8, MafK
    DOI:  https://doi.org/10.1016/j.jmb.2020.06.005
  3. Parasit Vectors. 2020 Jun 17. 13(1): 314
    Liu H, Cui XY, Xu DD, Wang F, Meng LW, Zhao YM, Liu M, Shen SJ, He XH, Fang Q, Tao ZY, Jiang CZ, Zhang QF, Gu L, Xia H.
      BACKGROUND: Malaria caused by Plasmodium spp. is still a major threat to public health globally. The various approaches to developing new antimalarial agents rely on the understanding of the complex regulatory mechanisms of dynamic gene expression in the life-cycle of these malaria parasites. The nuclear members of the evolutionarily conserved actin-related protein nuclear (ARP) superfamily are the major components of nucleosome remodelling complexes. In the human malaria parasite Plasmodium falciparum, bioinformatics analysis has predicted three ARP orthologues: PfArp1, PfArp4 and PfArp6. However, little is known about the biological functions of putative PfArp4. In this study, we aimed to investigate the function and the underlying mechanisms of PfArp4 gene regulation.METHODS: A conditional gene knockdown approach was adopted by incorporating the glucosamine-inducible glmS ribozyme sequence into the 3' UTR of the PfArp4 and PfArp6 genes. The transgenic parasites PfArp4-Ty1-Ribo, PfArp6-Ty1-Ribo and pL6-PfArp4-Ty1::PfArp6-HA were generated by the CRISPR-Cas9 technique. The knockdown effect in the transgenic parasite was measured by growth curve assay and western blot (WB) analysis. The direct interaction between PfArp4 and PfArp6 was validated by co-IFA and co-IP assays. The euchromatic gene expression mediated through H2A.Z (histone H2A variant) deposition and H3K9ac modification at promoters and regulated by PfArp4, was determined by RNA-seq and ChIP-seq.
    RESULTS: The inducible knockdown of PfArp4 inhibited blood-stage development of P. falciparum. PfArp4 and PfArp6 were colocalized in the nucleus of P. falciparum parasites. PfArp4 gene knockdown altered the global transcriptome. PfArp4 protein colocalized with the histone variant H2A.Z and euchromatic marker H3K9ac in intergenic regions. The inducible downregulation of PfArp4 resulted in the depletion of H2A.Z and lower H3K9ac levels at the upstream regions of eukaryotic genes, thereby repressing the transcriptional abundance of H2A.Z-dependent genes.
    CONCLUSIONS: Our findings suggest that PfArp4 regulates the cell cycle by controlling H2A.Z deposition and affecting centromere function, contributing to the understanding the complex epigenetic regulation of gene expression and the development of P. falciparum.
    Keywords:  Arp4; Chromatin structure; Gene regulation; Malaria; Plasmodium falciparum
    DOI:  https://doi.org/10.1186/s13071-020-04139-6
  4. J Biol Chem. 2020 Jun 18. pii: jbc.RA120.013348. [Epub ahead of print]
    Liu H, Hilliard S, Kelly E, Chen CH, Saifudeen Z, El-Dahr SS.
      SIX homeobox 2 (SIX2)-positive nephron progenitor cells (NPCs) give rise to all epithelial cell types of the nephron, the filtering unit of the kidney.  NPCs have a limited lifespan and are depleted near the time of birth.   Epigenetic factors are implicated in maintenance of organ-restricted progenitors such as NPCs, but the chromatin-based mechanisms are incompletely understood. Here, using a combination of gene targeting, chromatin profiling, and single-cell RNA analysis, we examined the role of the murine histone 3 Lys-27 (H3K27) methyltransferases enhancer of zeste 1 (EZH1) and EZH2 in NPC maintenance.  We found that EZH2 expression correlates with NPC growth potential, and that EZH2 is the dominant H3K27 methyltransferase in NPCs and epithelial descendants. Surprisingly, NPCs lacking H3K27 trimethylation maintained their progenitor state but cycled slowly, leading to a smaller NPC pool and formation of fewer nephrons.  Unlike Ezh2 loss-of-function, dual inactivation of Ezh1 and Ezh2 triggered overexpression of the transcriptional repressor Hes-related family BHLH transcription factor with YRPW motif 1 (Hey1), down-regulation of Six2, and unscheduled activation of Wnt4-driven differentiation, resulting in early termination of nephrogenesis and severe renal dysgenesis.  Double-mutant NPCs also overexpressed the SIX family member, Six1. However, in this context, SIX1 failed to maintain NPC stemness.  At the chromatin level, EZH1 and EZH2 restricted accessibility to AP-1-binding motifs, and their absence promoted a regulatory landscape akin to differentiated and non-lineage cells.  We conclude that EZH2 is required for NPC renewal potential, and that tempering of the differentiation program requires cooperation of both EZH1 and EZH2.
    Keywords:  Enhancer of Zeste; Histone methylation; development; epigenetics; kidney; nephrology; nephron progenitors; polycomb
    DOI:  https://doi.org/10.1074/jbc.RA120.013348
  5. Stem Cell Reports. 2020 Jun 09. pii: S2213-6711(20)30186-7. [Epub ahead of print]
    Lezmi E, Weissbein U, Golan-Lev T, Nissim-Rafinia M, Meshorer E, Benvenisty N.
      Chromatin regulators play fundamental roles in controlling pluripotency and differentiation. We examined the effect of mutations in 703 genes from nearly 70 chromatin-modifying complexes on human embryonic stem cell (ESC) growth. While the vast majority of chromatin-associated complexes are essential for ESC growth, the only complexes that conferred growth advantage upon mutation of their members, were the repressive complexes LSD-CoREST and BHC. Both complexes include the most potent growth-restricting chromatin-related protein, ZMYM2. Interestingly, while ZMYM2 expression is rather low in human blastocysts, its expression peaks in primed ESCs and is again downregulated upon differentiation. ZMYM2-null ESCs overexpress pluripotency genes and show genome-wide promotor-localized histone H3 hyper-acetylation. These mutant cells were also refractory to differentiate in vitro and failed to produce teratomas upon injection into immunodeficient mice. Our results suggest a central role for ZMYM2 in the transcriptional regulation of the undifferentiated state and in the exit-from-pluripotency of human ESCs.
    Keywords:  chromatin; differentiation; human pluripotent stem cells; naive cells; primed cells; teratoma
    DOI:  https://doi.org/10.1016/j.stemcr.2020.05.014
  6. Curr Genet. 2020 Jun 14.
    Iyer VR.
      The incorporation of histone variants into nucleosomes has important functional consequences in all aspects of eukaryotic chromatin biology. H2A.Z is a conserved histone variant found in all eukaryotes from yeast to mammals. Recent studies in yeast have shed light on the questions of where and how nucleosomes containing this variant are situated at promoters and in relation to genes, and what its specificity implies with regard to transcription. In yeast, H2A.Z appears to be primarily incorporated into the first nucleosome in the direction of transcription initiation, either of an mRNA transcript or a divergently transcribed upstream antisense non-coding RNA. This specificity of H2A.Z is due in part to the localization at promoters of SWR1, the ATP-dependent chromatin remodeler that incorporates H2A.Z into nucleosomes. Replacement of H2A.Z with canonical H2A is dependent on the function of the transcription pre-initiation complex. The recent studies summarized in this review reveal that the directionality of H2A.Z occupancy in relation to transcription thus reflects a balance of incorporation and eviction activities, which likely have varying contributions at distinct sets of genes across the genome.
    Keywords:  H2A.Z; Histone variant; INO80; Nucleosome; SWR1
    DOI:  https://doi.org/10.1007/s00294-020-01087-7
  7. Int J Mol Sci. 2020 Jun 11. pii: E4172. [Epub ahead of print]21(11):
    Reina C, Cavalieri V.
      G-quadruplexes are four-stranded helical nucleic acid structures formed by guanine-rich sequences. A considerable number of studies have revealed that these noncanonical structural motifs are widespread throughout the genome and transcriptome of numerous organisms, including humans. In particular, G-quadruplexes occupy strategic locations in genomic DNA and both coding and noncoding RNA molecules, being involved in many essential cellular and organismal functions. In this review, we first outline the fundamental structural features of G-quadruplexes and then focus on the concept that these DNA and RNA structures convey a distinctive layer of epigenetic information that is critical for the complex regulation, either positive or negative, of biological activities in different contexts. In this framework, we summarize and discuss the proposed mechanisms underlying the functions of G-quadruplexes and their interacting factors. Furthermore, we give special emphasis to the interplay between G-quadruplex formation/disruption and other epigenetic marks, including biochemical modifications of DNA bases and histones, nucleosome positioning, and three-dimensional organization of chromatin. Finally, epigenetic roles of RNA G-quadruplexes in post-transcriptional regulation of gene expression are also discussed. Undoubtedly, the issues addressed in this review take on particular importance in the field of comparative epigenetics, as well as in translational research.
    Keywords:  DNA bases modifications; G-quadruplex; G-quartet; chromatin architecture; epigenetics; histone post-translational modifications; histone-modifying activities; noncoding RNA; nucleosome remodeling; post-transcriptional regulation
    DOI:  https://doi.org/10.3390/ijms21114172
  8. Sci Adv. 2020 Jun;6(23): eaaz7249
    Zhang Y, Chan HL, Garcia-Martinez L, Karl DL, Weich N, Slingerland JM, Verdun RE, Morey L.
      RING1B, a core Polycomb repressive complex 1 subunit, is a histone H2A ubiquitin ligase essential for development. RING1B is overexpressed in patients with luminal breast cancer (BC) and recruited to actively transcribed genes and enhancers co-occupied by the estrogen receptor α (ERα). Whether ERα-induced transcriptional programs are mediated by RING1B is not understood. We show that prolonged estrogen administration induces transcriptional output and chromatin landscape fluctuations. RING1B loss impairs full estrogen-mediated gene expression and chromatin accessibility for key BC transcription factors. These effects were mediated, in part, by RING1B enzymatic activity and nucleosome binding functions. RING1B is recruited in a cyclic manner to ERα, FOXA1, and GRHL2 cobound sites and regulates estrogen-induced enhancers and ERα recruitment. Last, ChIP exo revealed multiple binding events of these factors at single-nucleotide resolution, including RING1B occupancy approximately 10 base pairs around ERα bound sites. We propose RING1B as a key regulator of the dynamic, liganded-ERα transcriptional regulatory circuit in luminal BC.
    DOI:  https://doi.org/10.1126/sciadv.aaz7249
  9. Curr Genet. 2020 Jun 17.
    Kumar K, Moirangthem R, Kaur R.
      Histone proteins regulate cellular factors' accessibility to DNA, and histone dosage has previously been linked with DNA damage susceptibility and efficiency of DNA repair pathways. Surplus histones are known to impede the DNA repair process by interfering with the homologous recombination-mediated DNA repair in Saccharomyces cerevisiae. Here, we discuss the recent finding of association of methyl methanesulfonate (MMS) resistance with the reduced histone H4 gene dosage in the pathogenic yeast Candida glabrata. We have earlier shown that while the low histone H3 gene dosage led to MMS susceptibility, the lack of two H4-encoding ORFs, CgHHF1 and CgHHF2, led to resistance to MMS-induced DNA damage. This resistance was linked with a higher rate of homologous recombination (HR). Taking these findings further, we review the interactome analysis of histones H3 and H4 in C. glabrata. We also report that the arginine residue present at the 95th position in the C-terminal tail of histone H4 protein is required for complementation of the MMS resistance in the Cghhf1Δhhf2Δ mutant, thereby pointing out a probable role of this residue in association with HR factors. Additionally, we present evidence that reduction in H4 protein levels may constitute an important part of varied stress responses in C. glabrata. Altogether, we present an overview of histone H4 dosage, HR-mediated repair of damaged DNA and stress resistance in this opportunistic human fungal pathogen.
    Keywords:  Chromatin; Genome integrity; Histones; Homologous recombination; Human fungal pathogens; Methyl methanesulfonate (MMS); Stress resistance
    DOI:  https://doi.org/10.1007/s00294-020-01088-6
  10. Cancer Genet. 2020 May 18. pii: S2210-7762(20)30235-0. [Epub ahead of print]245 17-26
    Qian Y, Li Y, Zheng C, Lu T, Sun R, Mao Y, Yu S, Fan H, Zhang Z.
      Although it is becoming increasingly apparent that histone methyltransferases and histone demethylases play crucial roles in the cellular response to hypoxia, the impact of hypoxic environments on global patterns of histone methylation is not well demonstrated. In this study, we try to detect the global levels of histone lysine methylation in HCC cases and analyze the correlation between these modifications and the activation of hypoxia-inducible factor 1α (HIF-1α). Immunohistochemistry was used to detect the global levels of histone H3 lysine 9 dimethylation (H3K9me2), histone H3 lysine 9 trimethylation (H3K9me3), histone H3 lysine 27 trimethylation (H3K27me3) and the nuclear expression of HIF-1α in tissue arrays from 111 paraffin-embedded HCC samples. Our analyses revealed that the global levels of H3K9me2, H3K9me3 and the nuclear expression of HIF-1α were distinctly higher in HCC tissues than in peritumoral tissues. Both H3K9me2 and H3K9me3 were positively correlated with the degree of tumor differentiation and the patients' prognosis. Analysis based on the Pearson's correlation coefficient indicated a positive correlation between H3K9me2 and the nuclear expression of HIF-1α, and meanwhile, a significant correlation between the expression of H3K9me2 and H3K9me3 was also found. In addition, the combination of H3K9me2, H3K9me3 and HIF-1α, rather than one single histone modification or molecular maker, is a better prognostic maker for HCC patients. These findings provide new insights on the complex networks underlying cellular and genomic regulation in response to hypoxia and may provide novel targets for future therapies.
    Keywords:  H3K9me2; H3K9me3; HCC; HIF-1α; Prognosis
    DOI:  https://doi.org/10.1016/j.cancergen.2020.04.077
  11. Sci Rep. 2020 Jun 18. 10(1): 9903
    Spakman D, King GA, Peterman EJG, Wuite GJL.
      As the basic building blocks of chromatin, nucleosomes play a key role in dictating the accessibility of the eukaryotic genome. Consequently, nucleosomes are involved in essential genomic transactions such as DNA transcription, replication and repair. In order to unravel the mechanisms by which nucleosomes can influence, or be altered by, DNA-binding proteins, single-molecule techniques are increasingly employed. To this end, DNA molecules containing a defined series of nucleosome positioning sequences are often used to reconstitute arrays of nucleosomes in vitro. Here, we describe a novel method to prepare DNA molecules containing defined arrays of the '601' nucleosome positioning sequence by exploiting Gibson Assembly cloning. The approaches presented here provide a more accessible and efficient means to generate arrays of nucleosome positioning motifs, and facilitate a high degree of control over the linker sequences between these motifs. Nucleosomes reconstituted on such arrays are ideal for interrogation with single-molecule techniques. To demonstrate this, we use dual-trap optical tweezers, in combination with fluorescence microscopy, to monitor nucleosome unwrapping and histone localisation as a function of tension. We reveal that, although nucleosomes unwrap at ~20 pN, histones (at least histone H3) remain bound to the DNA, even at tensions beyond 60 pN.
    DOI:  https://doi.org/10.1038/s41598-020-66259-4
  12. Genome Biol. 2020 Jun 16. 21(1): 144
    Kralemann LEM, Liu S, Trejo-Arellano MS, Muñoz-Viana R, Köhler C, Hennig L.
      BACKGROUND: Stable gene repression is essential for normal growth and development. Polycomb repressive complexes 1 and 2 (PRC1&2) are involved in this process by establishing monoubiquitination of histone 2A (H2Aub1) and subsequent trimethylation of lysine 27 of histone 3 (H3K27me3). Previous work proposed that H2Aub1 removal by the ubiquitin-specific proteases 12 and 13 (UBP12 and UBP13) is part of the repressive PRC1&2 system, but its functional role remains elusive.RESULTS: We show that UBP12 and UBP13 work together with PRC1, PRC2, and EMF1 to repress genes involved in stimulus response. We find that PRC1-mediated H2Aub1 is associated with gene responsiveness, and its repressive function requires PRC2 recruitment. We further show that the requirement of PRC1 for PRC2 recruitment depends on the initial expression status of genes. Lastly, we demonstrate that removal of H2Aub1 by UBP12/13 prevents loss of H3K27me3, consistent with our finding that the H3K27me3 demethylase REF6 is positively associated with H2Aub1.
    CONCLUSIONS: Our data allow us to propose a model in which deposition of H2Aub1 permits genes to switch between repression and activation by H3K27me3 deposition and removal. Removal of H2Aub1 by UBP12/13 is required to achieve stable PRC2-mediated repression.
    Keywords:  DUB; EMF1; H2AK121ub; H2Aub; H3K27me3; LHP1; PRC1; PRC2; REF6; Responsiveness
    DOI:  https://doi.org/10.1186/s13059-020-02062-8
  13. J Mol Biol. 2020 Jun 15. pii: S0022-2836(20)30407-1. [Epub ahead of print]
    Aoki D, Awazu A, Fujii M, Uewaki JI, Hashimoto M, Tochio N, Umehara T, Tate SI.
      Facilitates chromatin transcription (FACT) is a histone chaperone that functions as a nucleosome remodeler and a chaperone. The two subunits of FACT, Spt16 and SSRP1, mediate multiple interactions between the subunits and components of the nucleosome. Among the interactions, the role of the DNA-binding domain in SSRP1 has not been characterized. We reported previously that the DNA-binding domain in Drosophila SSRP1 (dSSRP1) has multiple casein kinase II phosphorylation sites, and the DNA binding affinity of the domain changes sigmoidally in response to the degree of phosphorylation ('ultrasensitive response'). In this report, we explored the molecular mechanisms for the ultrasensitive response of the DNA-binding domain in dSSRP1 using the shortest fragment (AB-HMG, residues 434-624) responsible for nucleosome binding. AB-HMG contains two intrinsically disordered (ID) regions: the N-terminal part rich in acidic residues (AID) and the C-terminal part rich in basic residues (BID) followed by the HMG box. NMR and coarse-grained molecular dynamics simulations revealed a phosphorylation-dependent change in intramolecular contacts between the AID and BID-HMG, which is mediated by a hinge bending motion of AB-HMG to enable the ultrasensitive response. Ultrasensitivity generates two distinct forms of dSSRP1, which are high- and low-affinity nucleosome-binding forms. Drosophila FACT (dFACT) switches function according to the degree of phosphorylation of the AID in dSSRP1. We propose that dFACT in various phosphorylation states functions cooperatively to facilitate gene regulation in the context of the chromatin.
    Keywords:  NMR; coarse-grained molecular dynamics simulations; intrinsically disordered proteins; multiple phosphorylations; nucleosome chaperone
    DOI:  https://doi.org/10.1016/j.jmb.2020.06.011
  14. Cells. 2020 Jun 12. pii: E1460. [Epub ahead of print]9(6):
    Sales-Gil R, Vagnarelli P.
      Heterochromatin Protein 1 (HP1) is a highly conserved protein that has been used as a classic marker for heterochromatin. HP1 binds to di- and tri-methylated histone H3K9 and regulates heterochromatin formation, functions and structure. Besides the well-established phosphorylation of histone H3 Ser10 that has been shown to modulate HP1 binding to chromatin, several studies have recently highlighted the importance of HP1 post-translational modifications and additional epigenetic features for the modulation of HP1-chromatin binding ability and heterochromatin formation. In this review, we summarize the recent literature of HP1 post-translational modifications that have contributed to understand how heterochromatin is formed, regulated and maintained.
    Keywords:  HP1; centromeres; heterochromatin; post-translational modifications
    DOI:  https://doi.org/10.3390/cells9061460
  15. J Clin Invest. 2020 Jun 16. pii: 136155. [Epub ahead of print]
    Liu L, Ding C, Fu T, Feng Z, Lee JE, Xiao L, Xu Z, Yin Y, Guo Q, Sun Z, Sun W, Mao Y, Yang L, Zhou Z, Zhou D, Xu L, Zhu Z, Qiu Y, Ge K, Gan Z.
      Skeletal muscle depends on the precise orchestration of contractile and metabolic gene expression programs to direct fiber type specification and to ensure muscle performance. Exactly how such fiber type-specific patterns of gene expression are established and maintained remains unclear, however. Here, we demonstrate that histone mono-methyltransferase MLL4 (KMT2D), an enhancer regulator enriched in slow myofibers, plays a critical role in controlling muscle fiber identity as well as muscle performance. Skeletal muscle-specific ablation of MLL4 in mice resulted in downregulation of the slow-oxidative myofiber gene program, decreased number of type I myofibers, and diminished mitochondrial respiration, which caused reductions in muscle fat utilization and endurance capacity during exercise. Genome-wide ChIP-seq and mRNA-seq analyses revealed that MLL4 directly binds to enhancers and functions as a coactivator of the myocyte enhancer factor 2 (MEF2) to activate transcription of slow-oxidative myofiber genes. Importantly, we also found that the MLL4 regulatory circuit is associated with muscle fiber type remodeling in humans. Thus, our results uncover a pivotal role for MLL4 in specifying structural and metabolic identities of myofibers that govern muscle performance. These findings provide new therapeutic opportunities for enhancing muscle fitness to combat a variety of metabolic and muscular diseases.
    Keywords:  Epigenetics; Metabolism; Muscle Biology; Skeletal muscle; Transcription
    DOI:  https://doi.org/10.1172/JCI136155
  16. Elife. 2020 Jun 16. pii: e56178. [Epub ahead of print]9
    Farnung L, Ochmann M, Cramer P.
      Chromatin remodelling plays important roles in gene regulation during development, differentiation and in disease. The chromatin remodelling enzyme CHD4 is a component of the NuRD and ChAHP complexes that are involved in gene repression. Here we report the cryo-electron microscopy (cryo-EM) structure of Homo sapiens CHD4 engaged with a nucleosome core particle in the presence of the non-hydrolysable ATP analogue AMP-PNP at an overall resolution of 3.1 Å. The ATPase motor of CHD4 binds and distorts nucleosomal DNA at superhelical location (SHL) +2, supporting the 'twist defect' model of chromatin remodelling. CHD4 does not induce unwrapping of terminal DNA, in contrast to its homologue Chd1, which functions in gene activation. Our structure also maps CHD4 mutations that are associated with human cancer or the intellectual disability disorder Sifrim-Hitz-Weiss syndrome.
    Keywords:  biochemistry; chemical biology; human; molecular biophysics; structural biology
    DOI:  https://doi.org/10.7554/eLife.56178
  17. Elife. 2020 Jun 15. pii: e55828. [Epub ahead of print]9
    Meng F, Qian M, Peng B, Peng L, Wang X, Zheng K, Liu Z, Tang X, Zhang S, Sun S, Cao X, Pang Q, Zhao B, Ma W, Songyang Z, Xu B, Zhu WG, Xu X, Liu B.
      The DNA damage response (DDR) is a highly orchestrated process but how double-strand DNA breaks (DSBs) are initially recognized is unclear. Here, we show that polymerized SIRT6 deacetylase recognizes DSBs and potentiates the DDR in human and mouse cells. First, SIRT1 deacetylates SIRT6 at residue K33, which is important for SIRT6 polymerization and mobilization toward DSBs. Then, K33-deacetylated SIRT6 anchors to γH2AX, allowing its retention on and subsequent remodeling of local chromatin. We show that a K33R mutation that mimics hypoacetylated SIRT6 can rescue defective DNA repair as a result of SIRT1 deficiency in cultured cells. These data highlight the synergistic action between SIRTs in the spatiotemporal regulation of the DDR and DNA repair in humans and mice.
    Keywords:  biochemistry; chemical biology; human; mouse
    DOI:  https://doi.org/10.7554/eLife.55828
  18. JCI Insight. 2020 Jun 16. pii: 137127. [Epub ahead of print]
    Sanders YY, Lyu X, Zhou QJ, Xiang Z, Stanford D, Bodduluri S, Rowe SM, Thannickal VJ.
      Tissue regeneration capacity declines with aging in association with heightened oxidative stress. Expression of the oxidant-generating enzyme, NADPH oxidase 4 (Nox4) is elevated in aged mice with diminished capacity for fibrosis resolution. Bromodomain-containing protein 4 (Brd4) is a member of the bromodomain and extraterminal (BET) family of proteins that function as epigenetic "readers" of acetylated lysine groups on histones. In this study, we explored the role of Brd4 and its interaction with the p300 acetyltransferase in the regulation of Nox4, and the in-vivo efficacy of a BET inhibitor to reverse established age-associated lung fibrosis. BET inhibition interferes with the association of Brd4, p300, and acetylated histone H4K16 with the Nox4 promoter in lung fibroblasts stimulated with the pro-fibrotic cytokine, transforming growth factor-β1 (TGF-β1). This Brd4-Nox4 epigenetic axis is constitutively upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. A number of BET inhibitors, including I-BET-762, JQ1, and OTX015, downregulate Nox4 gene expression and activity. Aged mice with established and persistent lung fibrosis recovered capacity for fibrosis resolution with OTX015 treatment. This study implicates epigenetic regulation of Nox4 by Brd4 and p300, and supports BET/Brd4 inhibition as an effective strategy for the treatment of age-related fibrotic lung disease.
    Keywords:  Aging; Cell Biology; Epigenetics; Fibrosis
    DOI:  https://doi.org/10.1172/jci.insight.137127
  19. Acta Biochim Biophys Sin (Shanghai). 2020 Jun 15. pii: gmaa064. [Epub ahead of print]
    Xiang Y, Guo J, Li F, Xiong J.
      The lysine histone demethylase KDM4B is overexpressed in several types of cancers and plays dual roles in genome stability maintenance. Although KDM4B is able to recognize several histone methylations, the underlying molecular mechanism is still unknown. In this study, we purified the KDM4B chromatin-associated hybrid tudor domains (HTDs) and plant home domains (PHDs) and performed the pull-down assay to screen the tri-methyl modified histone peptides that could be efficiently recognized by KDM4B. Our results showed that both HTD alone and the combination of HTD and PHD were able to specifically bind to H3K4me3 and H4K20me3. Because H4K20me3 is essential for KDM4B's rapid recruitment to DNA damage site, we further aligned the multiple tudor peptide sequence and identified two conserved residues Y993 and W987 that are critical for KDM4B-H4K20me3 interaction. The surface plasmon resonance analysis revealed that HTD displayed a rapid H4K20me3 bind-dissociate pattern. These findings therefore provided mechanistic insights into the binding of tudor domain of KDM4B protein with H4K20me3.
    Keywords:  H4K20me3; KDM4B; tudor domain
    DOI:  https://doi.org/10.1093/abbs/gmaa064
  20. Gene X. 2020 Dec;5 100027
    Galea GL, Paradise CR, Meakin LB, Camilleri ET, Taipaleenmaki H, Stein GS, Lanyon LE, Price JS, van Wijnen AJ, Dudakovic A.
      Mechanical loading-related strains trigger bone formation by osteoblasts while suppressing resorption by osteoclasts, uncoupling the processes of formation and resorption. Osteocytes may orchestrate this process in part by secreting sclerostin (SOST), which inhibits osteoblasts, and expressing receptor activator of nuclear factor-κB ligand (RANKL/TNFSF11) which recruits osteoclasts. Both SOST and RANKL are targets of the master osteoblastic transcription factor RUNX2. Subjecting human osteoblastic Saos-2 cells to strain by four point bending down-regulates their expression of SOST and RANKL without altering RUNX2 expression. RUNX2 knockdown increases basal SOST expression, but does not alter SOST down-regulation following strain. Conversely, RUNX2 knockdown does not alter basal RANKL expression, but prevents its down-regulation by strain. Chromatin immunoprecipitation revealed RUNX2 occupies a region of the RANKL promoter containing a consensus RUNX2 binding site and its occupancy of this site decreases following strain. The expression of epigenetic acetyl and methyl writers and readers was quantified by RT-qPCR to investigate potential epigenetic bases for this change. Strain and RUNX2 knockdown both down-regulate expression of the bromodomain acetyl reader BRD2. BRD2 and RUNX2 co-immunoprecipitate, suggesting interaction within regulatory complexes, and BRD2 was confirmed to interact with the RUNX2 promoter. BRD2 also occupies the RANKL promoter and its occupancy was reduced following exposure to strain. Thus, RUNX2 may contribute to bone remodeling by suppressing basal SOST expression, while facilitating the acute strain-induced down-regulation of RANKL through a mechanosensitive epigenetic loop involving BRD2.
    Keywords:  ALP, Alkaline phosphatase; ActD, Actinomycin D; AzadC, 5-Aza-2′-deoxycytidine; BRD2; BRD2, Bromodomain-containing protein 2; CO2, Carbon Dioxide; ChIP, Chromatin immunoprecipitation; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's Modified Eagle Medium; DNA, Deoxyribonucleic Acid; Epigenetics; FACS, Fluorescence-activated cell sorting; FCS, Fetal calf serum; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; HDAC, Histone deacetylase; HPRT, Hypoxanthine Phosphoribosyltransferase 1; IU, International unit; IgG, Immunoglobulin G; Ki-67, Antigen KI-67; Mechanical strain; OPG, Osteoprotegerin/tumour necrosis factor receptor superfamily member 11B; PBS, Phosphate-Buffered Saline; PCR, polymerase chain reaction; PGE2, Prostaglandin E2; RANKL/TNFSF11, receptor activator of nuclear factor-κB ligand; RNA, Ribonucleic Acid; RT-qPCR, Quantitative reverse transcription polymerase chain reaction; RUNX2; RUNX2, Runt-related transcription factor 2; Receptor activator of nuclear factor-κB ligand; SOST, Sclerostin; Sclerostin; eGFP, enhanced green fluorescent protein; sh, Short hairpin; β2MG, Beta-2-Microglobulin
    DOI:  https://doi.org/10.1016/j.gene.2020.100027
  21. Stem Cell Res Ther. 2020 Jun 17. 11(1): 238
    Florkowska A, Meszka I, Zawada M, Legutko D, Proszynski TJ, Janczyk-Ilach K, Streminska W, Ciemerych MA, Grabowska I.
      BACKGROUND: Pluripotent stem cells present the ability to self-renew and undergo differentiation into any cell type building an organism. Importantly, a lot of evidence on embryonic stem cell (ESC) differentiation comes from in vitro studies. However, ESCs cultured in vitro do not necessarily behave as cells differentiating in vivo. For this reason, we used teratomas to study early and advanced stages of in vivo ESC myogenic differentiation and the role of Pax7 in this process. Pax7 transcription factor plays a crucial role in the formation and differentiation of skeletal muscle precursor cells during embryonic development. It controls the expression of other myogenic regulators and also acts as an anti-apoptotic factor. It is also involved in the formation and maintenance of satellite cell population.METHODS: In vivo approach we used involved generation and analysis of pluripotent stem cell-derived teratomas. Such model allows to analyze early and also terminal stages of tissue differentiation, for example, terminal stages of myogenesis, including the formation of innervated and vascularized mature myofibers.
    RESULTS: We determined how the lack of Pax7 function affects the generation of different myofiber types. In Pax7-/- teratomas, the skeletal muscle tissue occupied significantly smaller area, as compared to Pax7+/+ ones. The proportion of myofibers expressing Myh3 and Myh2b did not differ between Pax7+/+ and Pax7-/- teratomas. However, the area of Myh7 and Myh2a myofibers was significantly lower in Pax7-/- ones. Molecular characteristic of skeletal muscles revealed that the levels of mRNAs coding Myh isoforms were significantly lower in Pax7-/- teratomas. The level of mRNAs encoding Pax3 was significantly higher, while the expression of Nfix, Eno3, Mck, Mef2a, and Itga7 was significantly lower in Pax7-/- teratomas, as compared to Pax7+/+ ones. We proved that the number of satellite cells in Pax7-/- teratomas was significantly reduced. Finally, analysis of neuromuscular junction localization in samples prepared with the iDISCO method confirmed that the organization of neuromuscular junctions in Pax7-/- teratomas was impaired.
    CONCLUSIONS: Pax7-/- ESCs differentiate in vivo to embryonic myoblasts more readily than Pax7+/+ cells. In the absence of functional Pax7, initiation of myogenic differentiation is facilitated, and as a result, the expression of mesoderm embryonic myoblast markers is upregulated. However, in the absence of functional Pax7 neuromuscular junctions, formation is abnormal, what results in lower differentiation potential of Pax7-/- ESCs during advanced stages of myogenesis.
    Keywords:  Differentiation; Mouse, embryonic stem cell; Myogenesis; Neuromuscular junction; Pax7; Teratoma
    DOI:  https://doi.org/10.1186/s13287-020-01742-3
  22. FASEB J. 2020 Jun 16.
    Alam C, Hoque MT, Sangha V, Bendayan R.
      Folates are important for neurodevelopment and cognitive function. Folate transport across biological membranes is mediated by three major pathways: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Brain folate transport primarily occurs at the choroid plexus through FRα and PCFT; inactivation of these transport systems results in suboptimal folate levels in the cerebrospinal fluid (CSF) causing childhood neurological disorders. Our group has reported that upregulation of RFC at the blood-brain barrier (BBB) through interactions with specific transcription factors, that is, vitamin D receptor (VDR) could increase brain folate delivery. This study investigates the role of nuclear respiratory factor 1 (NRF-1) in the regulation of RFC at the BBB. Activation of NRF-1/PGC-1α signaling through treatment with its specific ligand, pyrroloquinoline quinone (PQQ), significantly induced RFC expression and transport activity in hCMEC/D3 cells. In contrast, transfection with NRF-1 or PGC-1α targeting siRNA downregulated RFC functional expression in the same cell system. Applying chromatin immunoprecipitation (ChIP) assay, we further demonstrated that PQQ treatment increased NRF-1 binding to putative NRF-1 binding sites within the SLC19A1 promoter, which encodes for RFC. Additionally, in vivo treatment of wild type mice with PQQ-induced RFC expression in isolated mouse brain capillaries. Together, these findings demonstrate that NRF-1/PGC-1α activation by PQQ upregulates RFC functional expression at the BBB and could potentially enhance brain folate uptake.
    Keywords:  brain folate transport; folate deficiency; reduced folate carrier; transcription factor
    DOI:  https://doi.org/10.1096/fj.202000239RR