Trypanosoma cruzi bromodomain factor 2 (BDF2) binds to acetylated histones and is accumulated after UV irradiation

Histone tail post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) regulate many cellular processes. Among these modifications, phosphorylation, methylation and acetylation have already been described in trypanosomatid histones. Bromodomains, together with chromodomains and histone-binding SANT domains, were proposed to be responsible for “histone code” reading. The Trypanosoma cruzi genome encodes four coding sequences (CDSs) that contain a bromodomain, named TcBDF1-4. Here we show that one of those, TcBDF2, is expressed in discrete regions inside the nucleus of all the parasite life cycle stages and binds H4 and H2A purified histones from T. cruzi. Immunolocalization experiments using both anti-histone H4 acetylated peptides and anti-TcBDF2 antibodies determined that TcBDF2 co-localizes with histone H4 acetylated at lysines K10 and K14. TcDBF2 and K10 acetylated H4 interaction was confirmed by co-immunoprecipitation. It is also shown that TcBDF2 was accumulated after UV irradiation of T. cruzi epimastigotes. These results suggest that TcBDF2 could be taking part in a chromatin remodelling complex in T. cruzi.     

Changes in histone modifications during in vitro maturation of porcine oocytes

Nuclear core histone modifications influence chromosome structures and functions. Recently, the involvement of histone acetylations in the cell memory of gene expression has been suggested in mouse oocyte maturation. At present, there is little available data on histone modifications in mammalian oocyte maturation. In the present study, we examined changes in the acetylation of histone H3 lysines 9 (H3K9) and 14 (H3K14), and histone H4 lysines 5 (H4K5), 8 (H4K8) and 12 (H4K12), and trimethylation of H3K9 during in vitro maturation of porcine oocytes. Immunocytochemical analyses revealed that the all of the lysines examined were highly acetylated in the germinal vesicle stage, and this level of acetylation was maintained until the first prometaphase. In the first metaphase, the lysines near the N-terminal end, H3K9 and H4K5, were completely deacetylated. The acetylation of the lysines far from the N-terminal end, H3K14, H4K8, and H4K12, was markedly decreased but still present. The acetylations were increased transiently at the first anaphase and telophase, and then decreased again at the second metaphase to the same level as the first metaphase. Since effective concentrations of trichostatin A (TSA) to inhibit the deacetylation were different in various lysine residues, multiple histone deacetylases (HDACs) were suggested to function during meiotic maturation. The trimethylation of H3K9 was maintained in a high level throughout maturation. These results suggest that the histone acetylation during porcine oocyte maturation is precisely controlled by the cell cycle.     

Isolation and characterization of acetylated histones H3 and H4 and their assembly into nucleosomes

Nucleosome and chromatin structure/function relationships of histone acetylations are not understood. To address these questions we have developed chromatographic procedures that separate subtypes of H3 and the acetylated states of histone H3 and H4 in exceptionally pure forms. The sites of acetylation of the intermediately acetylated states of H3 have been determined and show a specific pattern of acetylation. An unexpected finding was the identification of a fifth site of acetylation in H3 at lysine 27. Nucleosome particles with fully acetylated H3 and H4 have been assembled on the Lytechinus variegatus 5 S rRNA DNA phasing sequence and characterized. These defined acetylated H3 and H4 particles migrate more slowly in polyacrylamide nucleoprotein particle gels than the control particles indicating a subtle effect of acetylation in nucleosome structure. However, DNA footprinting of these particles using DNase I show only small changes when compared to control particles over the core particle DNA length. It is shown further that H3 cysteines in the particle containing fully acetylated H3 and H4 were not accessible to iodoacetamide indicating that protein factors additional to H3 and H4 acetylation are required to make H3 cysteines accessible to the label. These findings are consistent with the proposal that histones H3, H4 acetylations exert their major effects outside of the core particle 146-base pair DNA, either on the DNA segment entering and leaving the nucleosome or possibly on the internucleosome interactions that involve the amino-terminal domains of the core histones in organization and stability of higher order chromatin structures.     

Histone H3 lysine 14 acetylation is required for activation of a DNA damage checkpoint in fission yeast

Histone lysine acetylation has emerged as a key regulator of genome organization. However, with a few exceptions, the contribution of each acetylated lysine to cellular functions is not well understood because of the limited specificity of most histone acetyltransferases and histone deacetylases. Here we show that the Mst2 complex in Schizosaccharomyces pombe is a highly specific H3 lysine 14 (H3K14) acetyltransferase that functions together with Gcn5 to regulate global levels of H3K14 acetylation (H3K14ac). By analyzing the effect of H3K14ac loss through both enzymatic inactivation and histone mutations, we found that H3K14ac is critical for DNA damage checkpoint activation by directly regulating the compaction of chromatin and by recruiting chromatin remodeling protein complex RSC.     

Autoregulation of the rsc4 tandem bromodomain by gcn5 acetylation

An important issue for chromatin remodeling complexes is how their bromodomains recognize particular acetylated lysine residues in histones. The Rsc4 subunit of the yeast remodeler RSC contains an essential tandem bromodomain (TBD) that binds acetylated K14 of histone H3 (H3K14ac). We report a series of crystal structures that reveal a compact TBD that binds H3K14ac in the second bromodomain and, remarkably, binds acetylated K25 of Rsc4 itself in the first bromodomain. Endogenous Rsc4 is acetylated only at K25, and Gcn5 is identified as necessary and sufficient for Rsc4 K25 acetylation in vivo and in vitro. Rsc4 K25 acetylation inhibits binding to H3K14ac, and mutation of Rsc4 K25 results in altered growth rates. These data suggest an autoregulatory mechanism in which Gcn5 performs both the activating (H3K14ac) and inhibitory (Rsc4 K25ac) modifications, perhaps to provide temporal regulation. Additional regulatory mechanisms are indicated as H3S10 phosphorylation inhibits Rsc4 binding to H3K14ac peptides.     

Dynamic Acetylation of All Lysine 4–Methylated Histone H3 in the Mouse Nucleus: Analysis at c-fos and c-jun

A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4)–methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9–methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9–methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.     

How Epigenomics Contributes to the Understanding of Gene Regulation in Toxoplasma gondii1

ABSTRACT. How apicomplexan parasites regulate their gene expression is poorly understood. The complex life cycle of these parasites implies tight control of gene expression to orchestrate the appropriate expression pattern at the right moment. Recently, several studies have demonstrated the role of epigenetic mechanisms for control of coordinated expression of genes. In this review, we discuss the contribution of epigenomics to the understanding of gene regulation in Toxoplasma gondii. Studying the distribution of modified histones on the genome links chromatin modifications to gene expression or gene repression. In particular, coincident trimethylated lysine 4 on histone H3 (H3K4me3), acetylated lysine 9 on histone H3 (H3K9ac), and acetylated histone H4 (H4ac) mark promoters of actively transcribed genes. However, the presence of these modified histones at some non‐expressed genes and other histone modifications at only a subset of active promoters implies the presence of other layers of regulation of chromatin structure in T. gondii. Epigenomics analysis provides a powerful tool to characterize the activation state of genomic loci of T. gondii and possibly of other Apicomplexa including Plasmodium or Cryptosporidium. Further, integration of epigenetic data with expression data and other genome‐wide datasets facilitates refinement of genome annotation based upon experimental data.     

组蛋白不同乙酰化位点突变对酵母细胞生长和Ssa3、Gal1基因表达的影响

组蛋白乙酰化对基因表达和细胞生长非常重要.为揭示组蛋白H3K14和H4K8的乙酰化修饰对不同条件下细胞生长和Ssa3、Gal1基因表达的重要性及二者功能差异.构建了H3K14、H4K8分别突变为精氨酸的单突变株S14、S8及二者同时突变的双突变株D814,并对其在正常、高温、咖啡因存在等条件下生长及Ssa3、Gal1表达进行比较.结果表明,所有突变株对咖啡因敏感性增加;D814对温度敏感,且在供试条件下其生长及Ssa3和Gal1激活均明显慢于野生型和单突变株;除半乳糖和葡萄糖为单一碳源,30℃时两单突变株差别不大外,其它条件下S8生长及Ssa3和Gal1激活均慢于S14.表明H3K14、H4K8乙酰化对细胞生长和适应不利环境非常重要,而且在对不利条件的快速适应方面,H4K8的乙酰化修饰可能更为重要.组蛋白突变株的表型缺陷是因该条件下细胞生存所必需的基因激活延迟所致.     

Replication-independent nucleosome exchange is enhanced by local and specific acetylation of histone H4

We used a novel single-cell strategy to examine the fate of histones during G₂-phase. Consistent with previous results, we find that in G₂-phase, the majority of nuclear histones are assembled into chromatin, whereas a small fraction comprises an unassembled pool. Small increases in the amount of histones within the free pool affect the extent of exchange, suggesting that the free pool is in dynamic equilibrium with chromatin proteins. Unexpectedly, acetylated H4 is preferentially partitioned to the unassembled pool. Although an increase in global histone acetylation did not affect overall nucleosome dynamics, an H4 containing lysine to glutamine substitutions as mimics of acetylation significantly increased the rate of exchange, but did not affect the acetylation state of neighbouring nucleosomes. Interestingly, transcribed regions are particularly predisposed to exchange on incorporation of H4 acetylation mimics compared with surrounding regions. Our results support a model whereby histone acetylation on K8 and K16 specifically marks nucleosomes for eviction, with histones being rapidly deacetylated on reassembly.     

Dynamic acetylation of all lysine 4-methylated histone H3 in the mouse nucleus: analysis at c-fos and c-jun

A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4)–methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9–methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9–methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.