Dimethylation of histone H3 lysine 9 （H3K9me2） is an important epigenetic mark associated with transcription repression. Here, we identified PHF8, a JmjC-domain-containing protein, as a histone demethylase specific ...Dimethylation of histone H3 lysine 9 （H3K9me2） is an important epigenetic mark associated with transcription repression. Here, we identified PHF8, a JmjC-domain-containing protein, as a histone demethylase specific for this repressing mark. Recombinant full-length wild type protein could remove methylation from H3K9me2, but mutation of a conserved histidine to alanine H247A abolished the demethylase activity. Overexpressed exogenous PHF8 was colocalized with B23 staining. Endogenous PHF8 was also colocalized with B23 and fibrillarin, two well-established nucleolus proteins, suggesting that PHF8 is localized in the nucleolus and may regulate rRNA transcription. Indeed, PHF8 bound to the promoter region of the rDNA gene. Knockdown of PHF8 reduced the expression of rRNA, and overexpression of the gene resulted in upregulation of rRNA transcript. Concomitantly, H3K9me2 level was elevated in the promoter region of the rDNA gene in PHF8 knockdown cells and reduced significantly when the wild type but not the catalytically inactive H247A mutant PHF8 was overexpressed. Thus, our study identified a histone demethylase for H3K9me2 that regulates rRNA transcription.展开更多
Histone H3 lysine 27 trimethylation(H3K27me3) is a histone modification associated with transcriptional repression. However, insights into the genome-wide pattern of H3K27me3 in grapevines are limited. Here, anti-H3K2...Histone H3 lysine 27 trimethylation(H3K27me3) is a histone modification associated with transcriptional repression. However, insights into the genome-wide pattern of H3K27me3 in grapevines are limited. Here, anti-H3K27 chromatin immunoprecipitation(ChIP), high-throughput sequencing, and transcriptome analysis were performed using leaves of Vitis amurensis. The leaves were treated at 4°C for 2 h and 24 h and used to investigate changes in H3K27me3 under chilling treatment. The results show that H3K27me3 is well-distributed both in gene regions(-50%) and in the intergenic region(-50%) in the grapevine genome(Vitis vinifera ‘Pinot Noir PN40024'). H3K27me3 was found to be localized in8 368 annotated gene regions in all detected samples(leaves at normal temperature and under chilling treatments) and mainly enriched in gene bodies with the adjacent promoter and downstream areas. The short-term chilling treatments(4°C for 2 h) induced 2 793 gains and 305losses in H3K27me3 modification. Subsequently, 97.3% of the alterations were restored to original levels after 24 h treatment. The ChIP-qPCR for five differential peaks showed similar results to the data for ChIP-seq, indicating that the chilling-induced H3K27me3 modification is reliable.Integrative analysis of transcriptome and ChIP-seq results showed that the expression of H3K27me3 target genes was significantly lower than those of non-target genes, indicating transcriptional repression of H3K27me3 in grapevine leaves. Furthermore, histone methylation alterations were detected in 82 genes and were related to either repression or activation of their expression during chilling stress. The findings provide the genome-wide H3K27me3 patterns in grapevines and shed light on uncovering its regulation in chilling stress responses.展开更多
Histone methylation is an important epigenetic phenomenon that participates in a diverse array of cellular processes and has been found to be associated with cancer. Recent identification of several histone demethylas...Histone methylation is an important epigenetic phenomenon that participates in a diverse array of cellular processes and has been found to be associated with cancer. Recent identification of several histone demethylases has proved that histone methylation is a reversible process. Through a candidate approach, we have biochemically identified JMJD3 as an H3K27 demethylase. Transfection of JMJD3 into HeLa cells caused a specific reduction oftrimethyl H3K27, but had no effect on di-and monomethyl H3K27, or histone lysine methylations on H3K4 and H3K9. The enzymatic activity requires the JmjC domain and the conserved histidine that has been suggested to be important for a cofactor binding. In vitro biochemical experiments demonstrated that JMJD3 directly catalyzes the demethylation. In addition, we found that JMJD3 is upregulated in prostate cancer, and its expression is higher in metastatic prostate cancer. Thus, we identified JMJD3 as a demethylase capable of removing the trimethyl group from histone H3 lysine 27 and upregulated in prostate cancer.展开更多
Dynamic regulation of histone methylation/demethylation plays an important role during development. Mutations and truncations in human plant homeodomain （PHD） finger protein 8 （PHF8） are associated with X-linked m...Dynamic regulation of histone methylation/demethylation plays an important role during development. Mutations and truncations in human plant homeodomain （PHD） finger protein 8 （PHF8） are associated with X-linked mental retardation and facial anomalies, such as a long face, broad nasal tip, cleft lip/cleft palate and large hands, yet its molecular function and structural basis remain unclear. Here, we report the crystal structures of the catalytic core of PHF8 with or without α-ketoglutarate （α-KG） at high resolution. Biochemical and structural studies reveal that PHF8 is a novel histone demethylase specific for di- and mono-methylated histone H3 lysine 9 （H3K9me2/1）, but not for H3K9me3. Our analyses also reveal how human PHF8 discriminates between methylation states and achieves sequence specificity for methylated H3K9. The in vitro demethylation assay also showed that the F279S mutant observed in clinical patients possesses no demethylation activity, suggesting that loss of enzymatic activity is crucial for pathogenesis of PHF8 patients. Taken together, these results will shed light on the molecular mechanism underlying PHF8-associated developmental and neurological diseases.展开更多
LSD1 （KDM1 under the new nomenclature） was the first identified lysine-specific histone demethylase belonging to the flavin-dependent amine oxidase family. Here, we report that AOF1 （KDM1B under the new nomenclatur...LSD1 （KDM1 under the new nomenclature） was the first identified lysine-specific histone demethylase belonging to the flavin-dependent amine oxidase family. Here, we report that AOF1 （KDM1B under the new nomenclature）, a mammalian protein related to LSD1, also possesses histone demethylase activity with specificity for H3K4mel and H3K4me2. Like LSD1, the highly conserved SWIRM domain is required for its enzymatic activity. However, AOF1 differs from LSD1 in several aspects. First, AOF1 does not appear to form stable protein complexes containing histone deacetylases. Second, AOF1 is found to localize to chromosomes during the mitotic phase of the cell cycle, whereas LSD1 does not. Third, AOF1 represses transcription when tethered to DNA and this repression activity is independent of its demethylase activity. Structural and functional analyses identified its unique N-terminal Zf-CW domain as essential for the demethylase activity-independent repression function. Collectively, our study identifies AOF1 as the second histone demethylase in the family of flavin-dependent amine oxidases and reveals a demethylase-independent repression function of AOF1.展开更多
H3K9me2 and H3K27me2 are important epigenetic marks associated with transcription repression, while H3K4me3 is associated with transcription activation. It has been shown that active and repressive histone methylation...H3K9me2 and H3K27me2 are important epigenetic marks associated with transcription repression, while H3K4me3 is associated with transcription activation. It has been shown that active and repressive histone methylations distribute in a mutually exclusive manner, but the underlying mechanism was poorly understood. Here we identified ceKDM7A, a PHD （plant homeodomain）- and JmjC domain-containing protein, as a histone demethylase specific for H3K9me2 and H3K27me2. We further demonstrated that the PHD domain of ceKDM7A bound H3K4me3 and H3K4me3 co-localized with ceKDM7A at the genome-wide level. Disruption of the PHD domain binding to H3K4me3 reduced the demethylase activity in vivo, and loss of ceKDM7A reduced the expression of its associated target genes. These results indicate that ceKDM7A is recruited to the promoter to demethylate H3K9me2 and H3K27me2 and activate gene expression through the binding of the PHD domain to H3K4me3. Thus, our study identifies a dual-specificity histone demethylase and provides novel insights into the regulation of histone methylation.展开更多
Background: The histone code is an established epigenetic regulator of early embryonic development in mammals.The lysine residue K9 of histone H3(H3 K9) is a prime target of SIRT1, a member of NAD+-dependent histone d...Background: The histone code is an established epigenetic regulator of early embryonic development in mammals.The lysine residue K9 of histone H3(H3 K9) is a prime target of SIRT1, a member of NAD+-dependent histone deacetylase family of enzymes targeting both histone and non-histone substrates. At present, little is known about SIRT1-modulation of H3 K9 in zygotic pronuclei and its association with the success of preimplantation embryo development. Therefore, we evaluated the effect of SIRT1 activity on H3 K9 methylation and acetylation in porcine zygotes and the significance of H3 K9 modifications for early embryonic development.Results: Our results show that SIRT1 activators resveratrol and BML-278 increased H3 K9 methylation and suppressed H3 K9 acetylation in both the paternal and maternal pronucleus. Inversely, SIRT1 inhibitors nicotinamide and sirtinol suppressed methylation and increased acetylation of pronuclear H3 K9. Evaluation of early embryonic development confirmed positive effect of selective SIRT1 activation on blastocyst formation rate(5.2 ± 2.9% versus 32.9 ± 8.1% in vehicle control and BML-278 group, respectively; P ≤ 0.05). Stimulation of SIRT1 activity coincided with fluorometric signal intensity of ooplasmic ubiquitin ligase MDM2, a known substrate of SIRT1 and known limiting factor of epigenome remodeling.Conclusions: We conclude that SIRT1 modulates zygotic histone code, obviously through direct deacetylation and via non-histone targets resulting in increased H3 K9 me3. These changes in zygotes lead to more successful pre-implantation embryonic development and, indeed, the specific SIRT1 activation due to BML-278 is beneficial for in vitro embryo production and blastocyst achievement.展开更多
Histone H3K79 modifications are essential to regulate chromatin structure and gene transcription,but understanding of the molecular mechanisms is limited.Because H3K79 is at globular domain,short histone peptide canno...Histone H3K79 modifications are essential to regulate chromatin structure and gene transcription,but understanding of the molecular mechanisms is limited.Because H3K79 is at globular domain,short histone peptide cannot mimic H3K79 in chromatin.Instead,reconstituted nucleosome-based chemical tools are ideally used to investigate H3K79 modifications.In consequence,H3K79-modified histone H3 with additional chemical handles are required,but such synthesis is challenging and laborious.Here we report a facile semisynthesis method that enables multifunctional histone H3 readily available.H3K79-containing fragment is short for straight peptide synthesis that was later ligated to recombinant expressed H3 fragments for full-length product in large scale.As a result,nucleosomes with H3K79 modifications as well as photo-reactive group and affinity tag were obtained to investigate potential binding proteins.We believe this method that enhances synthetic accessibility of nucleosome probes will accelerate understanding of the underexplored H3K79 modifications.展开更多
Histone H3 Lys36(H3K36)methylation and its associated modifiers are crucial for DNA double-strand break(DSB)repair,but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways i...Histone H3 Lys36(H3K36)methylation and its associated modifiers are crucial for DNA double-strand break(DSB)repair,but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear.Here,we unveil the distinct roles of H3K36 dimethylation(H3K36me2)and H3K36 trimethylation(H3K36me3)in DSB repair via non-homologous end joining(NHEJ)or homologous recombination(HR).Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency.y Ku70 and Rfa1 bind H3K36me2-or H3K36me3-modified peptides and chromatin,respectively.Disrupting these interactions impairs y Ku70 and Rfa1 recruitment to damaged H3K36me2-or H3K36me3-rich loci,increasing DNA damage sensitivity and decreasing repair efficiency.Conversely,H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit y Ku70 or Rfa1 under DSB stress.Importantly,human KU70 and RPA1,the homologs of y Ku70 and Rfa1,exclusively associate with H3K36me2 and H3K36me3 in a conserved manner.These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways,highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.展开更多
RING finger 187(RNF187),a ubiquitin-ligating(E3)enzyme,plays a crucial role in the proliferation of cancer cells.However,it remains unclear whether RNF187 exhibits comparable functionality in the development of germli...RING finger 187(RNF187),a ubiquitin-ligating(E3)enzyme,plays a crucial role in the proliferation of cancer cells.However,it remains unclear whether RNF187 exhibits comparable functionality in the development of germline cells.To investigate thepotential involvement of RNF187 in germ cell development,we conducted interference and overexpression assays using GC-2 cells,a mouse spermatocyte-derived cell line.Our findings reveal that the interaction between RNF187 and histone H3 increases theviability,proliferation,and migratory capacity of GC-2 cells.Moreover,we provide evidence demonstrating that RNF187 interactswith H3 and mediates the ubiquitination of H3 at lysine 57(K57)or lysine 80(K80),directly or indirectly resulting in increasedcellular transcription.This is a study to report the role of RNF187 in maintaining the development of GC-2 cells by mediatinghistone H3 ubiquitination,thus highlighting the involvement of the K57 and K80 residues of H3 in the epistatic regulation of genetranscription.These discoveries provide a new theoretical foundation for further comprehensive investigations into the functionof RNF187 in the reproductive system.展开更多
Histone modifications play crucial roles in the pathogenesis of myocardial ischaemia/reperfusion(I/R)injury.However,a genome-wide map of histone modifications and the underlying epigenetic signatures in myocardial I/R...Histone modifications play crucial roles in the pathogenesis of myocardial ischaemia/reperfusion(I/R)injury.However,a genome-wide map of histone modifications and the underlying epigenetic signatures in myocardial I/R injury have not been established.Here,we integrated transcriptome and epigenome of histone modifications to characterize epigenetic signatures after I/R injury.Disease-specific histone mark alterations were mainly found in H3K27me3-,H3K27ac-,and H3K4me1-marked regions 24 and 48 h after I/R.Genes differentially modified by H3K27ac,H3K4me1 and H3K27me3 were involved in immune response,heart conduction or contraction,cytoskeleton,and angiogenesis.H3K27me3 and its methyltransferase polycomb repressor complex 2(PRC2)were upregulated in myocardial tissues after I/R.Upon selective inhibition of EZH2(the catalytic core of PRC2),the mice manifest improved cardiac function,enhanced angiogenesis,and reduced fibrosis.Further investigations confirmed that EZH2 inhibition regulated H3K27me3 modification of multiple pro-angiogenic genes and ultimately enhanced angiogenic properties in vivo and in vitro.This study delineates a landscape of histone modifications in myocardial I/R injury,and identifies H3K27me3 as a key epigenetic modifier in I/R process.The inhibition of H3K27me3 and its methyltransferase might be a potential strategy for myocardial I/R injury intervention.展开更多
Histone H2A monoubiquitination is associated with transcriptional repression and needs to be removed by deubiquitinases to facilitate gene transcription in eukaryotes.However,the deubiquitinase responsible for genome-...Histone H2A monoubiquitination is associated with transcriptional repression and needs to be removed by deubiquitinases to facilitate gene transcription in eukaryotes.However,the deubiquitinase responsible for genome-wide H2A deubiquitination in plants has yet to be identified.In this study,we found that the previously identified PWWP-EPCR-ARID-TRB(PEAT)complex components interact with both the ubiquitin-specific protease UBP5 and the redundant histone acetyltransferases HAM1 and HAM2(HAM1/2)to form a larger version of PEAT complex in Arabidopsis thaliana.UBP5 functions as an H2A deubiquitinase in a nucleosome substrate-dependent manner in vitro and mediates H2A deubiquitination at the whole-genome level in vivo.HAM1/2 are shared subunits of the PEAT complex and the conserved NuA4 histone acetyltransferase com-plex,and are responsible for histone H4K5 acetylation.Within the PEAT complex,the PWWP components(PWWP1,PWWP2,and PWWP3)directly interact with UBP5 and are necessary for UBP5-mediated H2A deu-biquitination,while the EPCR components(EPCR1 and EPCR2)directly interact with HAM1/2 and are required for HAM1/2-mediated H4K5acetylation.Collectively,our study not onlyidentifies dual roles of thePEAT com-plex in H2A deubiquitination and H4K5 acetylation but also illustrates how these processes collaborate at the whole-genome level to regulate the transcription and development in plants.展开更多
Tens of thousands of long non-coding RNAs have been uncovered in plants,but few of them have been comprehensively studied for their biological function and molecular mechanism of their mode of action.Here,we show that...Tens of thousands of long non-coding RNAs have been uncovered in plants,but few of them have been comprehensively studied for their biological function and molecular mechanism of their mode of action.Here,we show that the Arabidopsis long non-coding RNA DANA2 interacts with an AP2/ERF transcription factor ERF84 in the cell nucleus and then affects the transcription of JMJ29 that encodes a Jumonji C domain-containing histone H3K9 demethylase.Both RNA sequencing(RNA-seq)and genetic analyses demonstrate that DANA2 positively regulates drought stress responses through JMJ29.JMJ29 positively regulates the expression of ERF15 and GOLS2 by modulation of H3K9me2 demethylation.Accordingly,mutation of JMJ29 causes decreased ERF15 and GOLS2 expression,resulting in impaired drought tolerance,in agreement with drought-sensitive phenotypes of dana2 and erf84 mutants.Taken together,these results demonstrate that DANA2 is a positive regulator of drought response and works jointly with the transcriptional activator ERF84 to modulate JMJ29 expression in plant response to drought.展开更多
Histone methylation is believed to provide binding sites for specific reader proteins, which translate histone code into biological function. Here we show that a family of acidic domain-containing proteins including n...Histone methylation is believed to provide binding sites for specific reader proteins, which translate histone code into biological function. Here we show that a family of acidic domain-containing proteins including nucleophosmin （NPM 1）, pp32, SET/TAF 113, nucleolin （NCL） and upstream binding factor （UBF） are novel H3K4me2-binding proteins. These proteins exhibit a unique pattern of interaction with methylated H3K4, as their binding is stimulated by H3K4me2 and inhibited by H3K4mel and H3K4me3. These proteins contain one or more acidic domains consisting mainly of aspartic and/or glutamic residues that are necessary for preferential binding of H3K4me2. Furthermore, we demonstrate that the acidic domain with sufficient length alone is capable of binding H3K4me2 in vitro and in vivo. NPM1, NCL and UBF require their acidic domains for association with and transcriptional activation ofrDNA genes. Interestingly, by defining acidic domain as a sequence with at least 20 acidic residues in 50 continuous amino acids, we identified 655 acidic domain-containing protein coding genes in the human genome and Gene Ontology （GO） analysis showed that many of the acidic domain proteins have chromatin-related functions. Our data suggest that acidic domain is a novel histone binding motif that can differentially read the status of H3K4 methylation and is broadly present in chromatin-associated proteins.展开更多
Interleukin(IL)17-producing T helper(Th17)cells play critical roles in the clearance of extracellular bacteria and fungi as well as the pathogenesis of various autoimmune diseases,such as multiple sclerosis,psoriasis,...Interleukin(IL)17-producing T helper(Th17)cells play critical roles in the clearance of extracellular bacteria and fungi as well as the pathogenesis of various autoimmune diseases,such as multiple sclerosis,psoriasis,and ulcerative colitis.Although a global transcriptional regulatory network of Th17 cell differentiation has been mapped recently,the participation of epigenetic modifications in the differentiation process has yet to be elucidated.We demonstrated here that histone H3 lysine-27(H3K27)demethylation,predominantly mediated by the H3K27 demethylase Jmjd3,crucially regulated Th17 cell differentiation.Activation of naı¨ve CD41 T cells immediately induced high expression of Jmjd3.Genetic depletion of Jmjd3 in CD41 T cells specifically impaired Th17 cell differentiation both in vitro and in vivo.Ectopic expression of Jmjd3 largely rescued the impaired differentiation of Th17 cells in vitro in Jmjd3-deficientCD41 T cells.Importantly,Jmjd3-deficient mice were resistant to the induction of experimental autoimmune encephalomyelitis(EAE).Furthermore,inhibition of the H3K27 demethylase activity with the specific inhibitor GSK-J4 dramatically suppressed Th17 cell differentiation in vitro.At the molecular level,Jmjd3 directly bound to and reduced the level of H3K27 trimethylation(me3)at the genomic sites ofRorc,which encodes the masterTh17 transcription factorRorgt,and Th17 cytokine genes such as Il17,Il17f,and Il22.Therefore,our studies established acritical role of Jmjd3-mediatedH3K27demethylation inTh17 cell differentiation andsuggest that Jmjd3 can be a novel therapeutic target for suppressing autoimmune responses.展开更多
Trimethylated histone H3 lysine 27(H3 K27 me3)is a repressive histone marker that regulates a variety of developmental processes,including those that determine flowering time.However,relatively little is known about t...Trimethylated histone H3 lysine 27(H3 K27 me3)is a repressive histone marker that regulates a variety of developmental processes,including those that determine flowering time.However,relatively little is known about the mechanism of how H3 K27 me3 is recognized to regulate transcription.Here,we identified BAH domain-containing transcriptional regulator 1(BDT1)as an H3 K27 me3 reader.BDT1 is responsible for preventing flowering by suppressing the expression of flowering genes.Mutation of the H3 K27 me3 recognition sites in the BAH domain disrupted the binding of BDT1 to H3 K27 me3,leading to de-repression of H3 K27 me3-enriched flowering genes and an earlyflowering phenotype.We also found that BDT1 interacts with a family of PHD finger-containing proteins,which we named PHD1–6,and with CPL2,a Pol II carboxyl terminal domain(CTD)phosphatase responsible for transcriptional repression.Pull-down assays showed that the PHD finger-containing proteins can enhance the binding of BDT1 to the H3 K27 me3 peptide.Mutations in all of the PHD genes caused increased expression of flowering genes and an earlyflowering phenotype.This study suggests that the binding of BDT1 to the H3 K27 me3 peptide,which is enhanced by PHD proteins,is critical for preventing early flowering.展开更多
基金Acknowledgments We thank the cell biology core facility for confocal study. The PHF8 antibody was kindly provided by Dr Jiemin Wong （East China Normal University）. This work was supported by the National Basic Research Program of China （2007CB947900, 2010CB529705, 2007CB947100）, the Chinese Academy of Sci- ences （KSCX2-YW-R-04, KSCX2-YW-R-I 11）, the National Natural Science Foundation of China （30870538, 90919026）, Postdoctoral fellowship （20090460670）, and the Council of Shanghai Municipal Government for Science and Technology.
文摘Dimethylation of histone H3 lysine 9 （H3K9me2） is an important epigenetic mark associated with transcription repression. Here, we identified PHF8, a JmjC-domain-containing protein, as a histone demethylase specific for this repressing mark. Recombinant full-length wild type protein could remove methylation from H3K9me2, but mutation of a conserved histidine to alanine H247A abolished the demethylase activity. Overexpressed exogenous PHF8 was colocalized with B23 staining. Endogenous PHF8 was also colocalized with B23 and fibrillarin, two well-established nucleolus proteins, suggesting that PHF8 is localized in the nucleolus and may regulate rRNA transcription. Indeed, PHF8 bound to the promoter region of the rDNA gene. Knockdown of PHF8 reduced the expression of rRNA, and overexpression of the gene resulted in upregulation of rRNA transcript. Concomitantly, H3K9me2 level was elevated in the promoter region of the rDNA gene in PHF8 knockdown cells and reduced significantly when the wild type but not the catalytically inactive H247A mutant PHF8 was overexpressed. Thus, our study identified a histone demethylase for H3K9me2 that regulates rRNA transcription.
基金supported by the National Key Research and Development Program of China (Grant No. 2018YFD1000300)the National Natural Science Foundation of China (Grant No. 32025032)+1 种基金the Grape Breeding Project of Ningxia (Grant No. NXNYYZ202101-04)Major Program of Technological Innovation in Hubei Province (Grant No. 2019ABA093).
文摘Histone H3 lysine 27 trimethylation(H3K27me3) is a histone modification associated with transcriptional repression. However, insights into the genome-wide pattern of H3K27me3 in grapevines are limited. Here, anti-H3K27 chromatin immunoprecipitation(ChIP), high-throughput sequencing, and transcriptome analysis were performed using leaves of Vitis amurensis. The leaves were treated at 4°C for 2 h and 24 h and used to investigate changes in H3K27me3 under chilling treatment. The results show that H3K27me3 is well-distributed both in gene regions(-50%) and in the intergenic region(-50%) in the grapevine genome(Vitis vinifera ‘Pinot Noir PN40024'). H3K27me3 was found to be localized in8 368 annotated gene regions in all detected samples(leaves at normal temperature and under chilling treatments) and mainly enriched in gene bodies with the adjacent promoter and downstream areas. The short-term chilling treatments(4°C for 2 h) induced 2 793 gains and 305losses in H3K27me3 modification. Subsequently, 97.3% of the alterations were restored to original levels after 24 h treatment. The ChIP-qPCR for five differential peaks showed similar results to the data for ChIP-seq, indicating that the chilling-induced H3K27me3 modification is reliable.Integrative analysis of transcriptome and ChIP-seq results showed that the expression of H3K27me3 target genes was significantly lower than those of non-target genes, indicating transcriptional repression of H3K27me3 in grapevine leaves. Furthermore, histone methylation alterations were detected in 82 genes and were related to either repression or activation of their expression during chilling stress. The findings provide the genome-wide H3K27me3 patterns in grapevines and shed light on uncovering its regulation in chilling stress responses.
文摘Histone methylation is an important epigenetic phenomenon that participates in a diverse array of cellular processes and has been found to be associated with cancer. Recent identification of several histone demethylases has proved that histone methylation is a reversible process. Through a candidate approach, we have biochemically identified JMJD3 as an H3K27 demethylase. Transfection of JMJD3 into HeLa cells caused a specific reduction oftrimethyl H3K27, but had no effect on di-and monomethyl H3K27, or histone lysine methylations on H3K4 and H3K9. The enzymatic activity requires the JmjC domain and the conserved histidine that has been suggested to be important for a cofactor binding. In vitro biochemical experiments demonstrated that JMJD3 directly catalyzes the demethylation. In addition, we found that JMJD3 is upregulated in prostate cancer, and its expression is higher in metastatic prostate cancer. Thus, we identified JMJD3 as a demethylase capable of removing the trimethyl group from histone H3 lysine 27 and upregulated in prostate cancer.
基金Supplementary information is linked to the online version of the paper on the Cell Research website.Acknowledgments We thank Dr Dawei Li （China Agricultural University） for generously providing us with the experimental conditions during the early stages of this project. We thank Dr Ruiming Xu （Institute of Biophysics, Chinese Academy of Sciences） for critical reading of this manuscript and advice. We thank Dr Pinchao Mei （Chinese Academy of Medical Sciences and Peking Union Medical College）, Xinqi Liu （Nankai University） and Jiemin Wong （East China Normal University） for discussions and advice. The synchrotronradiation experiments were performed at Shanghai Synchrotron Radiation Facility （SSRF） and NE3A in the Photon Factory. Z.C. is supported by the National Basic Research Program of China （973 Program, 2009CB825501）, the National Natural Science Foundation of China （30870494 and 90919043）, the New Century Excellent Talents in University （NCET-07-0808） and the Innovative Project of SKLAB. S. H. is supported by the National Key Laboratory Special Fund 2060204. Z. D. is supported by the National Natural Science Foundation of China （J0730639）.
文摘Dynamic regulation of histone methylation/demethylation plays an important role during development. Mutations and truncations in human plant homeodomain （PHD） finger protein 8 （PHF8） are associated with X-linked mental retardation and facial anomalies, such as a long face, broad nasal tip, cleft lip/cleft palate and large hands, yet its molecular function and structural basis remain unclear. Here, we report the crystal structures of the catalytic core of PHF8 with or without α-ketoglutarate （α-KG） at high resolution. Biochemical and structural studies reveal that PHF8 is a novel histone demethylase specific for di- and mono-methylated histone H3 lysine 9 （H3K9me2/1）, but not for H3K9me3. Our analyses also reveal how human PHF8 discriminates between methylation states and achieves sequence specificity for methylated H3K9. The in vitro demethylation assay also showed that the F279S mutant observed in clinical patients possesses no demethylation activity, suggesting that loss of enzymatic activity is crucial for pathogenesis of PHF8 patients. Taken together, these results will shed light on the molecular mechanism underlying PHF8-associated developmental and neurological diseases.
基金We thank Dr Ramin Shiekhattar （Wistar Institute, USA） for the baculoviruses expressing Flag-LSD1 and Drs Jianguo Song and Degui Chen （Shanghai Institute of Biochemistry and Cell Biol- ogy, China） for anti-HDAC1 antibody and H3K36me2 antibody, respectively. This study was partially supported by grants from the National Natural Science Foundation of China （90919025, 30871381）, the Ministry of Science and Technology of China （2009CB918402, 2009CB825601） and the Research Platform for Cell Signaling Networks from the Science and Technology Com- mission of Shanghai Municipality （06DZ22923）.
文摘LSD1 （KDM1 under the new nomenclature） was the first identified lysine-specific histone demethylase belonging to the flavin-dependent amine oxidase family. Here, we report that AOF1 （KDM1B under the new nomenclature）, a mammalian protein related to LSD1, also possesses histone demethylase activity with specificity for H3K4mel and H3K4me2. Like LSD1, the highly conserved SWIRM domain is required for its enzymatic activity. However, AOF1 differs from LSD1 in several aspects. First, AOF1 does not appear to form stable protein complexes containing histone deacetylases. Second, AOF1 is found to localize to chromosomes during the mitotic phase of the cell cycle, whereas LSD1 does not. Third, AOF1 represses transcription when tethered to DNA and this repression activity is independent of its demethylase activity. Structural and functional analyses identified its unique N-terminal Zf-CW domain as essential for the demethylase activity-independent repression function. Collectively, our study identifies AOF1 as the second histone demethylase in the family of flavin-dependent amine oxidases and reveals a demethylase-independent repression function of AOF1.
文摘H3K9me2 and H3K27me2 are important epigenetic marks associated with transcription repression, while H3K4me3 is associated with transcription activation. It has been shown that active and repressive histone methylations distribute in a mutually exclusive manner, but the underlying mechanism was poorly understood. Here we identified ceKDM7A, a PHD （plant homeodomain）- and JmjC domain-containing protein, as a histone demethylase specific for H3K9me2 and H3K27me2. We further demonstrated that the PHD domain of ceKDM7A bound H3K4me3 and H3K4me3 co-localized with ceKDM7A at the genome-wide level. Disruption of the PHD domain binding to H3K4me3 reduced the demethylase activity in vivo, and loss of ceKDM7A reduced the expression of its associated target genes. These results indicate that ceKDM7A is recruited to the promoter to demethylate H3K9me2 and H3K27me2 and activate gene expression through the binding of the PHD domain to H3K4me3. Thus, our study identifies a dual-specificity histone demethylase and provides novel insights into the regulation of histone methylation.
基金supported by Agriculture and Food Research Initiative Competitive(Grant no.2015–67,015-23,231)from the USDA National Institute of Food and Agricultureseed funding from the Food for the twenty-first Century program of the University of Missouri to P.S.Work in Y-J.Y.+6 种基金supported by Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education(NRF-2013R1A6A3A04063769)M.K.and J.N.was supported by Charles University(PROGRES Q-39)the National Sustainability Program I(NPU I)NrLO1503 provided by the Ministry of Education,Youth and Sports of the Czech RepublicK.A.,T.Z.,K.H.,J.P.and J.N.were supported by the National Agency of Agriculture Sciences(NAZV QJ1510138)the Czech Ministry of Agriculture(MZe RO 0714)
文摘Background: The histone code is an established epigenetic regulator of early embryonic development in mammals.The lysine residue K9 of histone H3(H3 K9) is a prime target of SIRT1, a member of NAD+-dependent histone deacetylase family of enzymes targeting both histone and non-histone substrates. At present, little is known about SIRT1-modulation of H3 K9 in zygotic pronuclei and its association with the success of preimplantation embryo development. Therefore, we evaluated the effect of SIRT1 activity on H3 K9 methylation and acetylation in porcine zygotes and the significance of H3 K9 modifications for early embryonic development.Results: Our results show that SIRT1 activators resveratrol and BML-278 increased H3 K9 methylation and suppressed H3 K9 acetylation in both the paternal and maternal pronucleus. Inversely, SIRT1 inhibitors nicotinamide and sirtinol suppressed methylation and increased acetylation of pronuclear H3 K9. Evaluation of early embryonic development confirmed positive effect of selective SIRT1 activation on blastocyst formation rate(5.2 ± 2.9% versus 32.9 ± 8.1% in vehicle control and BML-278 group, respectively; P ≤ 0.05). Stimulation of SIRT1 activity coincided with fluorometric signal intensity of ooplasmic ubiquitin ligase MDM2, a known substrate of SIRT1 and known limiting factor of epigenome remodeling.Conclusions: We conclude that SIRT1 modulates zygotic histone code, obviously through direct deacetylation and via non-histone targets resulting in increased H3 K9 me3. These changes in zygotes lead to more successful pre-implantation embryonic development and, indeed, the specific SIRT1 activation due to BML-278 is beneficial for in vitro embryo production and blastocyst achievement.
基金support from National Natural Science Foundation of China(Nos.22077103 and 22161132006)Westlake University startup。
文摘Histone H3K79 modifications are essential to regulate chromatin structure and gene transcription,but understanding of the molecular mechanisms is limited.Because H3K79 is at globular domain,short histone peptide cannot mimic H3K79 in chromatin.Instead,reconstituted nucleosome-based chemical tools are ideally used to investigate H3K79 modifications.In consequence,H3K79-modified histone H3 with additional chemical handles are required,but such synthesis is challenging and laborious.Here we report a facile semisynthesis method that enables multifunctional histone H3 readily available.H3K79-containing fragment is short for straight peptide synthesis that was later ligated to recombinant expressed H3 fragments for full-length product in large scale.As a result,nucleosomes with H3K79 modifications as well as photo-reactive group and affinity tag were obtained to investigate potential binding proteins.We believe this method that enhances synthetic accessibility of nucleosome probes will accelerate understanding of the underexplored H3K79 modifications.
基金supported by the National Key Research and Development Program of China(2019YFA0802501)the National Natural Science Foundation of China(32270617,31971231)+1 种基金the Fundamental Research Funds for the Central Universities(2042022dx0003)the Application Fundamental Frontier Foundation of Wuhan(2020020601012225)。
文摘Histone H3 Lys36(H3K36)methylation and its associated modifiers are crucial for DNA double-strand break(DSB)repair,but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear.Here,we unveil the distinct roles of H3K36 dimethylation(H3K36me2)and H3K36 trimethylation(H3K36me3)in DSB repair via non-homologous end joining(NHEJ)or homologous recombination(HR).Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency.y Ku70 and Rfa1 bind H3K36me2-or H3K36me3-modified peptides and chromatin,respectively.Disrupting these interactions impairs y Ku70 and Rfa1 recruitment to damaged H3K36me2-or H3K36me3-rich loci,increasing DNA damage sensitivity and decreasing repair efficiency.Conversely,H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit y Ku70 or Rfa1 under DSB stress.Importantly,human KU70 and RPA1,the homologs of y Ku70 and Rfa1,exclusively associate with H3K36me2 and H3K36me3 in a conserved manner.These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways,highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.
基金supported by the National Natural Science Foundation ofChina(82271633 to BZ and 82201762 to TTG)the Gusu Health Talent Programof Suzhou(GSWS2020068 to BZ)+2 种基金the Top Talent Support Program for Youngand Middle-aged People of Wuxi Health Committee(BJ2020047 to YBW)theScience and Technology Project of Changzhou(CJ20220143 to TTG)the Changzhou Health Committee Funded Young Investigator Training Project(CZQM2020099 to TTG)。
文摘RING finger 187(RNF187),a ubiquitin-ligating(E3)enzyme,plays a crucial role in the proliferation of cancer cells.However,it remains unclear whether RNF187 exhibits comparable functionality in the development of germline cells.To investigate thepotential involvement of RNF187 in germ cell development,we conducted interference and overexpression assays using GC-2 cells,a mouse spermatocyte-derived cell line.Our findings reveal that the interaction between RNF187 and histone H3 increases theviability,proliferation,and migratory capacity of GC-2 cells.Moreover,we provide evidence demonstrating that RNF187 interactswith H3 and mediates the ubiquitination of H3 at lysine 57(K57)or lysine 80(K80),directly or indirectly resulting in increasedcellular transcription.This is a study to report the role of RNF187 in maintaining the development of GC-2 cells by mediatinghistone H3 ubiquitination,thus highlighting the involvement of the K57 and K80 residues of H3 in the epistatic regulation of genetranscription.These discoveries provide a new theoretical foundation for further comprehensive investigations into the functionof RNF187 in the reproductive system.
基金supported by the National Natural Science Foundation of China (82088101,81930013,82000377,31871491)the National Key Research and Development Plan (2019YFA0801501)+5 种基金Key Research Center Construction Project of Shanghai (2022ZZ01008)Shanghai Key clinical specialty Project (shslczdzk06202)Key Disciplines Group Construction Project of Pudong Health Bureau of Shanghai (PWZxq2017-05)Top-level Clinical Discipline Project of Shanghai Pudong District (PWYgf2021-01)Program for the Research Unit of Origin and Regulation of Heart Rhythm,Chinese Academy of Medical Sciences (2019RU045)Innovative research team of high-level local universities in Shanghai and a key laboratory program of the Education Commission of Shanghai Municipality (ZDSYS14005)。
文摘Histone modifications play crucial roles in the pathogenesis of myocardial ischaemia/reperfusion(I/R)injury.However,a genome-wide map of histone modifications and the underlying epigenetic signatures in myocardial I/R injury have not been established.Here,we integrated transcriptome and epigenome of histone modifications to characterize epigenetic signatures after I/R injury.Disease-specific histone mark alterations were mainly found in H3K27me3-,H3K27ac-,and H3K4me1-marked regions 24 and 48 h after I/R.Genes differentially modified by H3K27ac,H3K4me1 and H3K27me3 were involved in immune response,heart conduction or contraction,cytoskeleton,and angiogenesis.H3K27me3 and its methyltransferase polycomb repressor complex 2(PRC2)were upregulated in myocardial tissues after I/R.Upon selective inhibition of EZH2(the catalytic core of PRC2),the mice manifest improved cardiac function,enhanced angiogenesis,and reduced fibrosis.Further investigations confirmed that EZH2 inhibition regulated H3K27me3 modification of multiple pro-angiogenic genes and ultimately enhanced angiogenic properties in vivo and in vitro.This study delineates a landscape of histone modifications in myocardial I/R injury,and identifies H3K27me3 as a key epigenetic modifier in I/R process.The inhibition of H3K27me3 and its methyltransferase might be a potential strategy for myocardial I/R injury intervention.
基金supported by the National Natural Science Foundation of China(grant number:32025003).
文摘Histone H2A monoubiquitination is associated with transcriptional repression and needs to be removed by deubiquitinases to facilitate gene transcription in eukaryotes.However,the deubiquitinase responsible for genome-wide H2A deubiquitination in plants has yet to be identified.In this study,we found that the previously identified PWWP-EPCR-ARID-TRB(PEAT)complex components interact with both the ubiquitin-specific protease UBP5 and the redundant histone acetyltransferases HAM1 and HAM2(HAM1/2)to form a larger version of PEAT complex in Arabidopsis thaliana.UBP5 functions as an H2A deubiquitinase in a nucleosome substrate-dependent manner in vitro and mediates H2A deubiquitination at the whole-genome level in vivo.HAM1/2 are shared subunits of the PEAT complex and the conserved NuA4 histone acetyltransferase com-plex,and are responsible for histone H4K5 acetylation.Within the PEAT complex,the PWWP components(PWWP1,PWWP2,and PWWP3)directly interact with UBP5 and are necessary for UBP5-mediated H2A deu-biquitination,while the EPCR components(EPCR1 and EPCR2)directly interact with HAM1/2 and are required for HAM1/2-mediated H4K5acetylation.Collectively,our study not onlyidentifies dual roles of thePEAT com-plex in H2A deubiquitination and H4K5 acetylation but also illustrates how these processes collaborate at the whole-genome level to regulate the transcription and development in plants.
基金supported by the National Natural Science Foundation of China(grants 32070627 and 31960138 to D.W.,31800224 and 32160070 to R.H.,and 31788103 to X.C.)the Chinese Academy of Sciences(Strategic Priority Research Program XDB27030201 to X.C.)the Natural Science Foundation of Jiangxi Province(grant 20171ACB20001 to D.W.).
文摘Tens of thousands of long non-coding RNAs have been uncovered in plants,but few of them have been comprehensively studied for their biological function and molecular mechanism of their mode of action.Here,we show that the Arabidopsis long non-coding RNA DANA2 interacts with an AP2/ERF transcription factor ERF84 in the cell nucleus and then affects the transcription of JMJ29 that encodes a Jumonji C domain-containing histone H3K9 demethylase.Both RNA sequencing(RNA-seq)and genetic analyses demonstrate that DANA2 positively regulates drought stress responses through JMJ29.JMJ29 positively regulates the expression of ERF15 and GOLS2 by modulation of H3K9me2 demethylation.Accordingly,mutation of JMJ29 causes decreased ERF15 and GOLS2 expression,resulting in impaired drought tolerance,in agreement with drought-sensitive phenotypes of dana2 and erf84 mutants.Taken together,these results demonstrate that DANA2 is a positive regulator of drought response and works jointly with the transcriptional activator ERF84 to modulate JMJ29 expression in plant response to drought.
基金supported by the Ministry of Science and Technology of China(2015CB910402)to Jiemin Wongthe National Natural Science Foundation of China(91419303)+1 种基金The Science and Technology Commission of Shanghai Municipality(14XD1401700,11DZ2260300)the National Science&Technology Major Project“Key New Drug Creation and Manufacturing Program”of China(2014ZX09507002-002)
文摘Histone methylation is believed to provide binding sites for specific reader proteins, which translate histone code into biological function. Here we show that a family of acidic domain-containing proteins including nucleophosmin （NPM 1）, pp32, SET/TAF 113, nucleolin （NCL） and upstream binding factor （UBF） are novel H3K4me2-binding proteins. These proteins exhibit a unique pattern of interaction with methylated H3K4, as their binding is stimulated by H3K4me2 and inhibited by H3K4mel and H3K4me3. These proteins contain one or more acidic domains consisting mainly of aspartic and/or glutamic residues that are necessary for preferential binding of H3K4me2. Furthermore, we demonstrate that the acidic domain with sufficient length alone is capable of binding H3K4me2 in vitro and in vivo. NPM1, NCL and UBF require their acidic domains for association with and transcriptional activation ofrDNA genes. Interestingly, by defining acidic domain as a sequence with at least 20 acidic residues in 50 continuous amino acids, we identified 655 acidic domain-containing protein coding genes in the human genome and Gene Ontology （GO） analysis showed that many of the acidic domain proteins have chromatin-related functions. Our data suggest that acidic domain is a novel histone binding motif that can differentially read the status of H3K4 methylation and is broadly present in chromatin-associated proteins.
基金supported by grants from the National Basic Research Program(2014CB541904,2011CB946102,and 2014CB943600)the National Natural Science Foundation of China(31370881,90919017,and 30972695)+1 种基金the Knowledge Innovation Project of Chinese Academy of Sciences(KSCX1-YW-22)the CAS-CSIRO Cooperative Research Program(GJHZ1409).
文摘Interleukin(IL)17-producing T helper(Th17)cells play critical roles in the clearance of extracellular bacteria and fungi as well as the pathogenesis of various autoimmune diseases,such as multiple sclerosis,psoriasis,and ulcerative colitis.Although a global transcriptional regulatory network of Th17 cell differentiation has been mapped recently,the participation of epigenetic modifications in the differentiation process has yet to be elucidated.We demonstrated here that histone H3 lysine-27(H3K27)demethylation,predominantly mediated by the H3K27 demethylase Jmjd3,crucially regulated Th17 cell differentiation.Activation of naı¨ve CD41 T cells immediately induced high expression of Jmjd3.Genetic depletion of Jmjd3 in CD41 T cells specifically impaired Th17 cell differentiation both in vitro and in vivo.Ectopic expression of Jmjd3 largely rescued the impaired differentiation of Th17 cells in vitro in Jmjd3-deficientCD41 T cells.Importantly,Jmjd3-deficient mice were resistant to the induction of experimental autoimmune encephalomyelitis(EAE).Furthermore,inhibition of the H3K27 demethylase activity with the specific inhibitor GSK-J4 dramatically suppressed Th17 cell differentiation in vitro.At the molecular level,Jmjd3 directly bound to and reduced the level of H3K27 trimethylation(me3)at the genomic sites ofRorc,which encodes the masterTh17 transcription factorRorgt,and Th17 cytokine genes such as Il17,Il17f,and Il22.Therefore,our studies established acritical role of Jmjd3-mediatedH3K27demethylation inTh17 cell differentiation andsuggest that Jmjd3 can be a novel therapeutic target for suppressing autoimmune responses.
基金supported by the National Natural Science Foundation of China(32025003)by the National Key Research and Development Program of China(2016YFA0500801)from the Chinese Ministry of Science and Technology。
文摘Trimethylated histone H3 lysine 27(H3 K27 me3)is a repressive histone marker that regulates a variety of developmental processes,including those that determine flowering time.However,relatively little is known about the mechanism of how H3 K27 me3 is recognized to regulate transcription.Here,we identified BAH domain-containing transcriptional regulator 1(BDT1)as an H3 K27 me3 reader.BDT1 is responsible for preventing flowering by suppressing the expression of flowering genes.Mutation of the H3 K27 me3 recognition sites in the BAH domain disrupted the binding of BDT1 to H3 K27 me3,leading to de-repression of H3 K27 me3-enriched flowering genes and an earlyflowering phenotype.We also found that BDT1 interacts with a family of PHD finger-containing proteins,which we named PHD1–6,and with CPL2,a Pol II carboxyl terminal domain(CTD)phosphatase responsible for transcriptional repression.Pull-down assays showed that the PHD finger-containing proteins can enhance the binding of BDT1 to the H3 K27 me3 peptide.Mutations in all of the PHD genes caused increased expression of flowering genes and an earlyflowering phenotype.This study suggests that the binding of BDT1 to the H3 K27 me3 peptide,which is enhanced by PHD proteins,is critical for preventing early flowering.
