A phosphorylation switch on RbBP5 regulates histone H3 Lys4 methylation

The methyltransferase activity of the trithorax group (TrxG) protein MLL1 found within its COMPASS (complex associated with SET1)-like complex is allosterically regulated by a four-subunit complex composed of WDR5, RbBP5, Ash2L, and DPY30 (also referred to as WRAD). We report structural evidence showing that in WRAD, a concave surface of the Ash2L SPIa and ryanodine receptor (SPRY) domain binds to a cluster of acidic residues, referred to as the D/E box, in RbBP5. Mutational analysis shows that residues forming the Ash2L/RbBP5 interface are important for heterodimer formation, stimulation of MLL1 catalytic activity, and erythroid cell terminal differentiation. We also demonstrate that a phosphorylation switch on RbBP5 stimulates WRAD complex formation and significantly increases KMT2 (lysine [K] methyltransferase 2) enzyme methylation rates. Overall, our findings provide structural insights into the assembly of the WRAD complex and point to a novel regulatory mechanism controlling the activity of the KMT2/COMPASS family of lysine methyltransferases.     

Reactivation of the silenced and imprinted alleles of ARHI is associated with increased histone H3 acetylation and decreased histone H3 lysine 9 methylation

ARHI has been identified as a maternally imprinted tumor suppressor gene that maps to chromosome 1p31 and whose expression is markedly down-regulated in breast cancer. To explore possible mechanisms that could silence ARHI expression, we have tested the importance of DNA methylation, histone acetylation and histone methylation in regulating ARHI expression. We found that treatment with CpG demethylating agents and/or histone deacetylase inhibitors could reactivate both the silenced and the imprinted alleles of this tumor suppressor gene. Reactivation of ARHI expression by these reagents is related to the methylation status of the CpG islands in the ARHI promoter, especially CpG island II. Chromatin immunoprecipitation assays revealed that histone H3 lysine 9/18 acetylation levels associated with ARHI in normal cells were significantly higher than those in breast cancer cell lines that lacked ARHI expression. Treatment with a CpG demethylating agent and/or histone deacetylase inhibitor could increase ARHI expression in breast cancer cells, with a corresponding increase in histone H3 lysine 9/18 acetylation and decrease in histone H3 lysine 9 methylation.     

Impaired replication elongation in Tetrahymena mutants deficient in histone H3 Lys 27 monomethylation

Replication of nuclear DNA occurs in the context of chromatin and is influenced by histone modifications. In the ciliate Tetrahymena thermophila, we identified TXR1, encoding a histone methyltransferase. TXR1 deletion resulted in severe DNA replication stress, manifested by the accumulation of ssDNA, production of aberrant replication intermediates, and activation of robust DNA damage responses. Paired-end Illumina sequencing of ssDNA revealed intergenic regions, including replication origins, as hot spots for replication stress in ΔTXR1 cells. ΔTXR1 cells showed a deficiency in histone H3 Lys 27 monomethylation (H3K27me1), while ΔEZL2 cells, deleting a Drosophila E(z) homolog, were deficient in H3K27 di- and trimethylation, with no detectable replication stress. A point mutation in histone H3 at Lys 27 (H3 K27Q) mirrored the phenotype of ΔTXR1, corroborating H3K27me1 as a key player in DNA replication. Additionally, we demonstrated interactions between TXR1 and proliferating cell nuclear antigen (PCNA). These findings support a conserved pathway through which H3K27me1 facilitates replication elongation.     

Methylation patterns of Lys9 and Lys27 on histone H3 correlate with patient outcome and tumor progression in lung cancer

BackgroundsHistone methylation is recognized as an important component of the epigenetic mechanisms of cancer initiation and progression. Previous studies have demonstrated that aberrant alterations in histone methylation are associated with lung cancer. However, novel and specific epigenetic biomarkers for monitoring lung adenocarcinoma remain unknown.MethodsA retrospective clinicopathological analysis was performed on 71 lung adenocarcinoma (LUAD) patients who received complete ablative surgical treatment. Tissue arrays were made from the paraffin-embedded LUAD tumor tissues, and these, together with corresponding normal tissues, were examined through immunohistochemistry for several markers: histone 3 lysine 9 di-methylation (H3K9me2), histone 3 lysine 9 tri-methylation (H3K9me3), and histone 3 lysine 27 tri-methylation (H3K27me3). The expression level of each marker was analyzed according to the histological classification and clinical prognosis data.ResultsCompared with peri-cancerous tissues, cancerous tissues distinctly expressed higher proportions of H3K9me2, H3K9me3, and H3K27me3. A higher expression pattern of H3K27me3 was associated with the poorly differentiation and unfavorable prognosis in LUAD. Based on histological types, it was found that the H3K27me3 level of patients with micropapillary type is high, and it is related to worse prognosis.ConclusionsThe findings of this study show that the H3K27me3 and micropapillary type are malignant clinical factors of LUAD. H3K27me3 reduction is a novel epigenetic biomarker for defining high-risk LUAD and predicting worse prognosis. Immunohistochemical evaluation of H3K27me3 expression is an economic, easily available, and readily adaptable method.     

Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27

The chromodomain of Drosophila Polycomb protein is essential for maintaining the silencing state of homeotic genes during development. Recent studies suggest that Polycomb mediates the assembly of repressive higher-order chromatin structures in conjunction with the methylation of Lys 27 of histone H3 by a Polycomb group repressor complex. A similar mechanism in heterochromatin assembly is mediated by HP1, a chromodomain protein that binds to histone H3 methylated at Lys 9. To understand the molecular mechanism of the methyl-Lys 27 histone code recognition, we have determined a 1.4-A-resolution structure of the chromodomain of Polycomb in complex with a histone H3 peptide trimethylated at Lys 27. The structure reveals a conserved mode of methyl-lysine binding and identifies Polycomb-specific interactions with histone H3. The structure also reveals a dPC dimer in the crystal lattice that is mediated by residues specifically conserved in the Polycomb family of chromodomains. The dimerization of dPC can effectively account for the histone-binding specificity and provides new mechanistic insights into the function of Polycomb. We propose that self-association is functionally important for Polycomb.     

Abnormal methylation of imprinted genes in human sperm is associated with oligozoospermia

Genomic imprinting marks in the male germ line are already establishedin the adult germinal stem cell population. We studied the methylationpatterns of H19 and MEST imprinted genes in sperm of controland oligozoospermic patients, by bisulphite genomic sequencing.We here report that 7 out of 15 (46.7%) patients with a spermcount below 10 x 10⁶/ml display defective methylation of H19and/or MEST imprinted genes. In these cases, hypomethylationwas observed in 5.54% (1.2–8.3%) and complete unmethylationin 2.95% (0–5.9%) of H19 clones. Similarly, for the CTCF-bindingsite 6, hypomethylation occurred in 4.8% (1.2–8.9%) andcomplete unmethylation in 3.7% (0–6.9%) of the clones.Conversely, hypermethylation occurred in 8.3% (3.8–12.2%)and complete methylation in 6.1% (3.8–7.6%) of MEST clones.Of the seven patients presenting imprinting errors, two hadboth H19 hypomethylation and MEST hypermethylation, whereasfive displayed only one imprinted gene affected. The frequencyof patients with MEST hypermethylation was highest in the severeoligozoospermia group (2/5 patients), whereas H19 hypomethylationwas more frequent in the moderate oligozoospermia (2/5 patients).In all cases, global sperm genome methylation analysis (LINE1transposon) suggested that defects were specific for imprintedgenes. These findings could contribute to an explanation ofthe cause of Silver–Russell syndrome in children bornwith H19 hypomethylation after assisted reproductive technologies(ART). Additionally, unmethylation of the CTCF-binding sitecould lead to inactivation of the paternal IGF2 gene, and belinked to decreased embryo quality and birth weight, often associatedwith ART.     

SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis

The mammalian cytoplasmic protein SirT2 is a member of the Sir2 family of NAD+-dependent protein deacetylases involved in caloric restriction-dependent life span extension. We found that SirT2 and its yeast counterpart Hst2 have a strong preference for histone H4K16Ac in their deacetylation activity in vitro and in vivo. We have pinpointed the decrease in global levels of H4K16Ac during the mammalian cell cycle to the G2/M transition that coincides with SirT2 localization on chromatin. Mouse embryonic fibroblasts (MEFs) deficient for SirT2 show higher levels of H4K16Ac in mitosis, in contrast to the normal levels exhibited by SirT1-deficient MEFs. The enzymatic conversion of H4K16Ac to its deacetylated form may be pivotal to the formation of condensed chromatin. Thus, SirT2 is a major contributor to this enzymatic conversion at the time in the cell's life cycle when condensed chromatin must be generated anew.     

PR-Set7-dependent methylation of histone H4 Lys 20 functions in repression of gene expression and is essential for mitosis

The histone methyl transferase PR-Set7 mediates histone H4 Lys 20 methylation, a mark of constitutive and facultative heterochromatin. We isolated a null mutation in Drosophila PR-Set7 that suppresses position effect variegation, indicating that PR-Set7 indeed functions in silencing general gene expression. In PR-Set7 larval leg and eye discs, the number of cells is lower than normal, and the DNA content in these cells is significantly increased. These data show that PR-Set7-dependent methylation is essential for the process of mitosis. The methylation mark is highly stable and is maintained even in the absence of PR-Set7 protein.     

组蛋白H3甲基化修饰与心脏发育的研究进展

正哺乳动物心脏发育需要干细胞的精确定位、增殖以及心肌祖细胞的分化等过程~([1]),这一系列过程涉及到多个心脏目的基因的精确编程调控。心脏基因组装于致密的染色质结构中,而染色质的基本单位核小体主要是由147 bp碱基对组成的线性DNA盘绕于组蛋白八聚体外侧构成~([2])。心脏目的基因的表达除了与基因序列相关外,还与心脏关键转录因子及基因组染色质结构修饰因子等表观遗传学途径密切相关。表观遗传学修饰是在基因序列不变的情     

Context dependency of Set1/COMPASS-mediated histone H3 Lys4 trimethylation

The stimulation of trimethylation of histone H3 Lys4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied, with multiple mechanisms having been proposed for this form of histone cross-talk. Cps35/Swd2 within COMPASS (complex of proteins associated with Set1) is considered to bridge these different processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation (H3K4me3) without interacting with Cps35/Swd2, and such cross-talk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. Here, we used biochemical, structural, in vivo, and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the cross-talk. Furthermore, the apparent wild-type levels of H3K4me3 in the 762-Set1 strain are due to the rogue methylase activity of this mutant, resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to the gene bodies and intergenic regions. We also performed detailed screens and identified yeast strains lacking H2Bub but containing intact H2Bub enzymes that have normal levels of H3K4me3, suggesting that monoubiquitination may not directly stimulate COMPASS but rather works in the context of the PAF and Rad6/Bre1 complexes. Our study demonstrates that the monoubiquitination machinery and Cps35/Swd2 function to focus COMPASS''s H3K4me3 activity at promoter-proximal regions in a context-dependent manner.     

H3K27me3 and VEGF is associated with poor prognosis in patients with synovial sarcoma

Purpose

Previous studies have shown a correlation between the expression of H3K27me3 and pathological characteristics of malignant tumors. This study aimed to investigate the association of H3K27me3 and VEGF expression with clinical outcomes of synovial sarcoma patients.

Elevated histone H3 citrullination is associated with increased Beclin1 expression in HBV-related hepatocellular carcinoma

Citrullinated histone H3 (H3Cit) is the product of the conversion of peptidylarginine to citrulline in histone H3. We evaluated the H3Cit level in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) tissues and assessed its association with Beclin1 messenger RNA (mRNA) (a key autophagic regulator). The level of H3Cit was detected by a capture enzyme-linked immunosorbent assay, while Beclin1 mRNA was determined by real-time polymerase chain reaction in 80 HBV-related patients with HCC. We found that the mean level of H3Cit was 72.25 ng/mg in HCC and 44.02 ng/mg in nontumor tissues. The mean HCC/nontumor ratio of Beclin1 mRNA was higher (0.096) in tumor samples than in nontumor specimens (0.056). Specifically, Beclin1 mRNA was elevated in 51 HCC cases (63.75%) and decreased in 29 cases (36.25%). Moreover, the levels of H3Cit and Beclin1 mRNA were significantly associated with vascular invasion and serum AFP levels. A shorter survival (19 months) was associated with a high H3Cit level. We also found increased levels of Beclin1 mRNA in the H3Cit (high) group compared with the H3Cit (low) group. The results implied that elevated histone H3 citrullination is associated with increased Beclin1 expression during the development of HBV-related HCC.