Interferon regulatory factor 1 and histone H4 acetylation in systemic lupus erythematosus

Histone acetylation modulates gene expression and has been described as increased in systemic lupus erythematosus (SLE). We investigated interferon regulatory factor 1 (IRF1) interactions that influence H4 acetylation (H4ac) in SLE. Intracellular flow cytometry for H4 acetylated lysine (K) 5, K8, K12, and K16 was performed. Histone acetylation was defined in monocytes and T cells from controls and SLE patients. RNA-Seq studies were performed on monocytes to look for an imbalance in histone acetyltransferases and histone deacetylase enzyme expression. Expression levels were validated using real-time quantitative RT-PCR. IRF1 induction of H4ac was evaluated using D54MG cells overexpressing IRF1. IRF1 protein interactions were studied using co-immunoprecipitation assays. IRF1-dependent recruitment of histone acetyltransferases to target genes was examined by ChIP assays using p300 antibody. Flow cytometry data showed significantly increased H4K5, H4K8, H4K12, and H4K16 acetylation in SLE monocytes. HDAC3 and HDAC11 gene expression were decreased in SLE monocytes. PCAF showed significantly higher gene expression in SLE than controls. IRF1-overexpressing D54MG cells were associated with significantly increased H4K5, H4K8, and H4K12 acetylation compared to vector-control D54MG cells both globally and at specific target genes. Co-immunoprecipitation studies using D54MG cells revealed IRF1 protein-protein interactions with PCAF, P300, CBP, GCN5, ATF2, and HDAC3. ChIP experiments demonstrated increased p300 recruitment to known IRF1 targets in D54MG cells overexpressing IRF1. In contrast, p300 binding to IRF1 targets decreased in D54MG cells with IRF1 knockdown. SLE appears to be associated with an imbalance in histone acetyltransferases and histone deacetylase enzymes favoring pathologic H4 acetylation. Furthermore, IRF1 directly interacts with chromatin modifying enzymes, supporting a model where recruitment to specific target genes is mediated in part by IRF1.     

Screen for DNA-damage-responsive histone modifications identifies H3K9Ac and H3K56Ac in human cells

Recognition and repair of damaged DNA occurs within the context of chromatin. The key protein components of chromatin are histones, whose post-translational modifications control diverse chromatin functions. Here, we report our findings from a large-scale screen for DNA-damage-responsive histone modifications in human cells. We have identified specific phosphorylations and acetylations on histone H3 that decrease in response to DNA damage. Significantly, we find that DNA-damage-induced changes in H3S10p, H3S28p and H3.3S31p are a consequence of cell-cycle re-positioning rather than DNA damage per se. In contrast, H3K9Ac and H3K56Ac, a mark previously uncharacterized in human cells, are rapidly and reversibly reduced in response to DNA damage. Finally, we show that the histone acetyl-transferase GCN5/KAT2A acetylates H3K56 in vitro and in vivo. Collectively, our data indicate that though most histone modifications do not change appreciably after genotoxic stress, H3K9Ac and H3K56Ac are reduced in response to DNA damage in human cells.     

Deciphering the binding mode and mechanistic insights of pentadecylidenemalonate (1b) as activator of histone acetyltransferase PCAF

Histone acetyltransferases (HATs) is one among the conspicuous posttranslational modification in eukaryotic cells. p300/CBP Associated Factor (PCAF) and CREB-binding protein (CBP) are the two highly homologous HAT family which are vastly implicated in several diseases like cancer, diabetes, etc. Pentadecylidenemalonate, a simplified analog of anacardic acid, was reported as first mixed inhibitor/activator of HATs which inhibits p300/CBP and activates PCAF. It was appointed earlier as a valuable biological tool to understand the mechanism of lysine acetyltransferases due to its powerful apoptotic effect. In this study, pentadecylidenemalonate was taken for deciphering the binding mode, key interacting residues as well as mechanistic insights on PCAF and CBP as activator and inhibitor, respectively. This study is highly believed to help in rational design on antineoplastic drugs against PCAF.

Communicated by Ramaswamy H. Sarma     

沙棘H3K9乙酰化修饰全基因组分析

为了绘制沙棘H3K9乙酰化修饰图谱,确定H3K9乙酰化修饰所调控的基因,该实验通过Western blot验证抗体与组蛋白的结合能力和ChIP-seq验证抗体富集效率,获得全基因组范围内沙棘H3K9乙酰化修饰图谱和调控基因。实验结果表明,H3K9ac抗体与复合物具有较强的结合能力。对富集到的DNA片段进行高通量测序,分别获得2.2×10~7和3.6×10~7条原始序列;唯一比对序列广泛分布于沙棘基因组中,并且在结构基因中的两端具有明显的富集。对富集区进行峰的预测结果显示,共预测出1 011个峰;对峰所处部位基因进行功能预测结果发现,H3K9ac对于沙棘细胞代谢和信号转导基因的表达具有重要调控作用。沙棘片段化DNA的富集以及高通量测序结果证明,抗体能够用于研究沙棘的组蛋白修饰类型,并且绘制了沙棘第一张H3K9乙酰化修饰遗传图谱草图,鉴定出沙棘H3K9乙酰化修饰所调控的基因,为今后研究组蛋白修饰对沙棘基因表达的调控方式奠定了基础。     

Hdac3, Setdb1, and Kap1 mark H3K9me3/H3K14ac bivalent regions in young and aged liver

Post‐translational modifications of histone tails play a crucial role in gene regulation. Here, we performed chromatin profiling by quantitative targeted mass spectrometry to assess all possible modifications of the core histones. We identified a bivalent combination, a dually marked H3K9me3/H3K14ac modification in the liver, that is significantly decreased in old hepatocytes. Subsequent sequential ChIP‐Seq identified dually marked single nucleosome regions, with reduced number of sites and decreased signal in old livers, confirming mass spectrometry results. We detected H3K9me3 and H3K14ac bulk ChIP‐Seq signal in reChIP nucleosome regions, suggesting a correlation between H3K9me3/H3K14ac bulk bivalent genomic regions and dually marked single nucleosomes. Histone H3K9 deacetylase Hdac3, as well as H3K9 methyltransferase Setdb1, found in complex Kap1, occupied both bulk and single nucleosome bivalent regions in both young and old livers, correlating to presence of H3K9me3. Expression of genes associated with bivalent regions in young liver, including those regulating cholesterol secretion and triglyceride synthesis, is upregulated in old liver once the bivalency is lost. Hence, H3K9me3/H3K14ac dually marked regions define a poised inactive state that is resolved with loss of one or both of the chromatin marks, which subsequently leads to change in gene expression.