Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis

REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylation enzyme that was previously identified during a genetic screen for the silencing of both RD29A-LUC and 35S-NPTII transgenes on a T-DNA construct. Here we performed a genetic screen to identify additional mutants in which the 35S-NPTII transgene is silenced. We identified several alleles of ros1 and of the following components of the RNA-directed DNA methylation (RdDM) pathway: NRPD1 (the largest subunit of polymerase IV), RDR2, NRPE1 (the largest subunit of polymerase V), NRPD2, AGO4, and DMS3. Our results show that the silencing of 35S-NPTII in the RdDM pathway mutants is due to the reduced expression of ROS1 in the mutants. We also identified a putative histone acetyltransferase (ROS4) from the genetic screen. The acetyltransferase contains a PHD-finger domain that binds to unmethylated histone H3K4. The mutation in ROS4 led to reduction of H3K18 and H3K23 acetylation levels. We show that the silencing of 35S-NPTII and some transposable element genes was released by the ddm1 mutation but that this also required ROS4. Our study identifies a unique antisilencing factor, and reveals that the RdDM pathway has an antisilencing function due to its role in maintaining ROS1 expression.     

Global and specific transcriptional repression by the histone H3 amino terminus in yeast

The yeast CHA1 promoter is activated in the presence of serine or threonine. Activation requires the Cha4p activator, and it results in perturbation of a nucleosome that incorporates the TATA element under noninducing conditions. We show that in yeast lacking the amino terminus of histone H3, the promoter is constitutively active and the chromatin is concomitantly perturbed. This derepression occurs in the absence of elevated intracellular levels of serine or threonine and is not observed in cells lacking Rpd3p, Tup1p, or the amino terminus of histone H4. Furthermore, derepression in the absence of the H3 amino terminus requires the primary activator of this promoter, Cha4p, which we show by chromatin immunoprecipitation to be constitutively bound to the CHA1 promoter in WT yeast. Thus, the H3 amino terminus is required to prevent Cha4p from activating CHA1 in the absence of inducer. We also present results of a microarray experiment showing that the H3 amino terminus has a substantial repressive effect on a genome-wide scale.     

H3K4 demethylation by Jarid1a and Jarid1b contributes to retinoblastoma-mediated gene silencing during cellular senescence

Cellular senescence is a tumor-suppressive program that involves chromatin reorganization and specific changes in gene expression that trigger an irreversible cell-cycle arrest. Here we combine quantitative mass spectrometry, ChIP deep-sequencing, and functional studies to determine the role of histone modifications on chromatin structure and gene-expression alterations associated with senescence in primary human cells. We uncover distinct senescence-associated changes in histone-modification patterns consistent with a repressive chromatin environment and link the establishment of one of these patterns--loss of H3K4 methylation--to the retinoblastoma tumor suppressor and the H3K4 demethylases Jarid1a and Jarid1b. Our results show that Jarid1a/b-mediated H3K4 demethylation contributes to silencing of retinoblastoma target genes in senescent cells, suggesting a mechanism by which retinoblastoma triggers gene silencing. Therefore, we link the Jarid1a and Jarid1b demethylases to a tumor-suppressor network controlling cellular senescence.     

Involvement of Arabidopsis HOS15 in histone deacetylation and cold tolerance

Histone modification in chromatin is one of the key control points in gene regulation in eukaryotic cells. Protein complexes composed of histone acetyltransferase or deacetylase, WD40 repeat protein, and many other components have been implicated in this process. Here, we report the identification and functional characterization of HOS15, a WD40-repeat protein crucial for repression of genes associated with abiotic stress tolerance through histone deacetylation in Arabidopsis. HOS15 shares high sequence similarity with human transducin-beta like protein (TBL), a component of a repressor protein complex involved in histone deacetylation. Mutation of the HOS15 gene renders mutant plants hypersensitive to freezing temperatures. HOS15 is localized in the nucleus and specifically interacts with histone H4. The level of acetylated histone H4 is higher in the hos15 mutant than in WT plants. Moreover, the stress inducible RD29A promoter is hyperinduced and associated with a substantially higher level of acetylated histone H4 in the hos15 mutant under cold stress conditions. Our results suggest a critical role for gene activation/repression by histone acetylation/deacetylation in plant acclimation and tolerance to cold stress.     

DNA double-strand breaks promote methylation of histone H3 on lysine 9 and transient formation of repressive chromatin

Dynamic changes in histone modification are critical for regulating DNA double-strand break (DSB) repair. Activation of the Tip60 acetyltransferase by DSBs requires interaction of Tip60 with histone H3 methylated on lysine 9 (H3K9me3). However, how H3K9 methylation is regulated during DSB repair is not known. Here, we demonstrate that a complex containing kap-1, HP1, and the H3K9 methyltransferase suv39h1 is rapidly loaded onto the chromatin at DSBs. Suv39h1 methylates H3K9, facilitating loading of additional kap-1/HP1/suv39h1 through binding of HP1’s chromodomain to the nascent H3K9me3. This process initiates cycles of kap-1/HP1/suv39h1 loading and H3K9 methylation that facilitate spreading of H3K9me3 and kap-1/HP1/suv39h1 complexes for tens of kilobases away from the DSB. These domains of H3K9me3 function to activate the Tip60 acetyltransferase, allowing Tip60 to acetylate both ataxia telangiectasia-mutated (ATM) kinase and histone H4. Consequently, cells lacking suv39h1 display defective activation of Tip60 and ATM, decreased DSB repair, and increased radiosensitivity. Importantly, activated ATM rapidly phosphorylates kap-1, leading to release of the repressive kap-1/HP1/suv39h1 complex from the chromatin. ATM activation therefore functions as a negative feedback loop to remove repressive suv39h1 complexes at DSBs, which may limit DSB repair. Recruitment of kap-1/HP1/suv39h1 to DSBs therefore provides a mechanism for transiently increasing the levels of H3K9me3 in open chromatin domains that lack H3K9me3 and thereby promoting efficient activation of Tip60 and ATM in these regions. Further, transient formation of repressive chromatin may be critical for stabilizing the damaged chromatin and for remodeling the chromatin to create an efficient template for the DNA repair machinery.DNA double-strand breaks (DSBs) are toxic and must be repaired to maintain genomic stability. Detection of DSBs requires recruitment of the mre11–rad50–nbs1 (MRN) complex to the DNA ends (1). MRN then recruits and activates the ataxia telangiectasia-mutated (ATM) kinase (2, 3) through a mechanism that also requires the Tip60 acetyltransferase (3). Tip60 directly acetylates and activates ATM’s kinase activity (4–6) and functions, in combination with MRN, to promote ATM-dependent phosphorylation of DSB repair proteins (3), including histone H2AX. This process creates domains of phosphorylated H2AX (γH2AX) extending for hundreds of kilobases along the chromatin (7, 8). Mdc1 then binds to γH2AX, providing a landing pad for other DSB repair proteins, including the RNF8/RNF168 ubiquitin ligases (1, 3, 9, 10). Tip60 also plays a critical role in regulating chromatin structure at DSBs as part of the NuA4–Tip60 complex (11). NuA4-Tip60 catalyzes histone exchange (via the p400 ATPase subunit) and acetylation of histone H4 (by Tip60) at DSBs (12–15), leading to the formation of open, flexible chromatin domains adjacent to the break (12, 13). These open chromatin structures then facilitate histone ubiquitination, the loading of brca1 and 53BP1, and repair of the DSB (13, 16). The ordered acetylation and ubiquitination of the chromatin and loading of DNA repair proteins is therefore critical for DSB repair.Activation of Tip60’s acetyltransferase activity requires interaction between Tip60’s chromodomain and histone H3 methylated on lysine 9 (H3K9me3) on nucleosomes at the break (4, 6). This interaction, in combination with tyrosine phosphorylation of Tip60 (17), increases Tip60’s acetyltransferase activity and promotes acetylation of both the ATM kinase and histone H4 (4–6, 17). Consequently, loss of H3K9me2/3 leads to failure to activate the ATM signaling pathway, loss of H4 acetylation during DSB repair, disruption of heterochromatin, genomic instability, and defective DSB repair (4, 17–19). H3K9me3s therefore play a critical role in linking chromatin structure at DSBs to the activation of DSB signaling proteins such as Tip60 and ATM.How Tip60 gains access to H3K9me3 and how H3K9me3 levels at DSBs are regulated is not known. H3K9me3 is concentrated in heterochromatin domains, where it recruits HP1, kap-1, and H3K9 methyltransferases (20, 21) to maintain the silent, compact conformation of heterochromatin (20). This implies that Tip60’s acetyltransferase activity can only be activated at DSBs in regions of high H3K9me3 density, such as heterochromatin. Alternatively, H3K9 methylation may be actively increased at DSBs in regions of low H3K9me3 density to allow for Tip60 activation and efficient DSB repair in euchromatin. Understanding the dynamics of H3K9 methylation at DSBs is therefore critical to understanding how Tip60 activity is regulated by the local chromatin architecture. Here, we show that the suv39h1 methyltransferase is recruited to DSBs in euchromatin as part of a larger kap-1/HP1/suv39h1 complex. Suv39h1 increases H3K9me3 at DSBs, activating Tip60’s acetyltransferase activity and promoting the subsequent acetylation and activation of ATM. Further, loss of inducible H3K9me3 at DSBs leads to defective repair and increased radiosensitivity. Finally, loading of the kap-1/HP1/suv39h1 complex is transient, and the complex is rapidly released from the chromatin through a negative feedback loop driven by ATM-dependent phosphorylation of the kap-1 protein.     

Identification of JmjC domain-containing UTX and JMJD3 as histone H3 lysine 27 demethylases

Covalent modifications of histones, such as acetylation and methylation, play important roles in the regulation of gene expression. Histone lysine methylation has been implicated in both gene activation and repression, depending on the specific lysine (K) residue that becomes methylated and the state of methylation (mono-, di-, or trimethylation). Methylation on K4, K9, and K36 of histone H3 has been shown to be reversible and can be removed by site-specific demethylases. However, the enzymes that antagonize methylation on K27 of histone H3 (H3K27), an epigenetic mark important for embryonic stem cell maintenance, Polycomb-mediated gene silencing, and X chromosome inactivation have been elusive. Here we show the JmjC domain-containing protein UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome), as well as the related JMJD3 (jumonji domain containing 3), specifically removes methyl marks on H3K27 in vitro. Further, the demethylase activity of UTX requires a catalytically active JmjC domain. Finally, overexpression of UTX and JMJD3 leads to reduced di- and trimethylation on H3K27 in cells, suggesting that UTX and JMJD3 may function as H3K27 demethylases in vivo. The identification of UTX and JMJD3 as H3K27-specific demethylases provides direct evidence to indicate that similar to methylation on K4, K9, and K36 of histone H3, methylation on H3K27 is also reversible and can be dynamically regulated by site-specific histone methyltransferases and demethylases.     

The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2

Recombination activating gene (RAG) 1 and RAG2 together catalyze V(D)J gene rearrangement in lymphocytes as the first step in the assembly and maturation of antigen receptors. RAG2 contains a plant homeodomain (PHD) near its C terminus (RAG2-PHD) that recognizes histone H3 methylated at lysine 4 (H3K4me) and influences V(D)J recombination. We report here crystal structures of RAG2-PHD alone and complexed with five modified H3 peptides. Two aspects of RAG2-PHD are unique. First, in the absence of the modified peptide, a peptide N-terminal to RAG2-PHD occupies the substrate-binding site, which may reflect an autoregulatory mechanism. Second, in contrast to other H3K4me3-binding PHD domains, RAG2-PHD substitutes a carboxylate that interacts with arginine 2 (R2) with a Tyr, resulting in binding to H3K4me3 that is enhanced rather than inhibited by dimethylation of R2. Five residues involved in histone H3 recognition were found mutated in severe combined immunodeficiency (SCID) patients. Disruption of the RAG2-PHD structure appears to lead to the absence of T and B lymphocytes, whereas failure to bind H3K4me3 is linked to Omenn Syndrome. This work provides a molecular basis for chromatin-dependent gene recombination and presents a single protein domain that simultaneously recognizes two distinct histone modifications, revealing added complexity in the read-out of combinatorial histone modifications.     

C2株蓝氏贾第鞭毛虫组蛋白H2B基因的克隆与同源性分析

目的 获取C2株蓝氏贾第鞭毛虫H2B组蛋白基因序列,与WB株蓝氏贾第鞭毛虫及其他物种进行同源分析。方法 PCR扩增获取H2B组蛋白基因,连接pGM-T载体,转化E. coli DH5α感受态宿主细胞,挑选阳性克隆并进行序列分析,与美国WB株蓝氏贾第鞭毛虫及模式生物H2B组蛋白基因和蛋白序列进行同源分析。结果 测序C2株蓝氏贾第鞭毛虫H2B组蛋白基因序列,同源比对结果显示其基因序列与美国WB株完全一致,进化树分析表明贾第虫H2B组蛋白基因在进化过程中与其他物种分化较早。结论 蓝氏贾第鞭毛虫H2B组蛋白基因同源分析结果显示贾第虫与其他现存真核生物亲缘关系较为疏远,为进一步研究蓝氏贾第鞭毛虫的生物进化地位提供有价值的资料。     

系统性红斑狼疮患者外周血单个核细胞组蛋白H3赖氨酸4三甲基化水平的研究

目的 研究系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMCs)组蛋白H3赖氨酸4(H3K4)三甲基化(Me3)水平.方法 梯度密度离心法分离10例SLE活动期患者、7例SLE稳定期患者和8名健康者的PBMCs,采用染色质免疫共沉淀联合芯片技术(CHIP-chip)在全基因组范围内对SLE患者及健康者的PBMCs组蛋白H3K4me3进行高通量的筛选.染色质免疫共沉淀一实时定量聚合酶链反应(ChlP-qPCR)验证芯片结果.定量反转录一聚合酶链反应(qRT-PCR)检测H3K4me3显著差异基因的mRNA表达水平.结果 10例SLE活动期患者与健康对照相比较,鉴定出413个基因存在H3K4me3显著差异,其中137个基因显示H3K4me3程度增高,276个基因H3K4me3程度降低;7例SLE稳定期患者与健康对照相比较,发现393个基因存在H3K4me3表达差异,其中有112个基因H3K4me3程度增高,281个基因H3K4me3程度降低.ChIP-qPCR验证结果与CpG岛芯片的结果相一致.结论 SLE患者与健康者之间的PBMCs存在组蛋白H3K4me3显著改变.ChIP-chip技术有利于进一步揭示SLE分子机制,发现新的治疗靶点.