Genome-wide Binding of Acetylated-STAT3 Demonstrates Negative Regulation of Dendritic Cells. Mus musculus

Dendritic cells (DC) play a vital role in the induction of activation or tolerance of immune response. STAT3 is a master transcriptional regulator of immune response in DCs by positively or negatively regulating DC function, but the mechanisms are unknown. STAT3 is post-translationally modified by acetylation or phosphorylation. While much is understood about transcriptional targets of phosphorylated STAT3, the gene targets and the functional impact of acetylated-STAT3 remain unclear. We aimed to answer the gene targets of acetylated-STAT3 and test the hypothesis that acetylation of STAT3 plays a key role in negative regulation of DCs. We performed genome-wide binding analysis of acetyl-STAT3 by ChIP-Seq coupled with gene expression microarrays. Acetylation of STAT3 induced by SAHA increased its capability to bind to target DNA sites in genome. Theses binding sites were mostly proximal but some were also distal up to over 100 kb from transcription start site. Gene expression array showed 1701 genes up-regulated and 1668 genes down-regulated. Proximal binding of acety-STAT3 showed more effective transcription function than distal binding. In top 500 binding peaks, the frequency of canonical motifs bound by acetyl-STAT3 were significantly higher than that for noncanonical motifs (p<0.00001). Functional analysis revealed that acetyl-STAT3 regulates target genes by upregulating genes that are primarily involved in negative regulation of cytokine production and IL-10 signaling, or downregulating genes that are primarily involved in immune effector process and antigen processing/presentation. Upregulation of IL-10Ra by acetyl-STAT3 contributes to the enhanced sensitivity of IL-10 signaling and negative regulation of DC function. Overall design: Bone marrow derived dendritic cells were treated with SAHA (500 nm) or diluent for 12 hours. ChIP was performed using antibodies against STAT3, H3K4me3 and matched IgG control. DNA binding profiles were generated by deep sequencing using Illumina HiSeq 2000.

树突状细胞（Dendritic cells, DC）在免疫应答的激活或耐受诱导中发挥至关重要的作用。信号转导与转录激活因子3（STAT3）是DC中免疫应答的核心转录调控因子，可通过正向或负向调控DC功能发挥作用，但其具体分子机制尚未明确。STAT3可通过乙酰化或磷酸化进行翻译后修饰。目前学界对磷酸化STAT3的转录靶标已有较为深入的认识，但乙酰化STAT3的基因靶标及其功能效应仍有待阐明。
本研究旨在明确乙酰化STAT3的基因靶标，并验证“STAT3乙酰化在DC的负向调控中发挥关键作用”这一假说。我们采用染色质免疫沉淀测序（ChIP-Seq, Chromatin Immunoprecipitation Sequencing）结合基因表达微阵列技术，开展了乙酰化STAT3的全基因组结合分析。结果显示，辛二酰苯胺异羟肟酸（SAHA）诱导的STAT3乙酰化增强了其与基因组内靶DNA位点的结合能力。此类结合位点大多位于转录起始位点的近端区域，但也有部分位点距离转录起始位点远达100 kb以上。
基因表达芯片检测结果表明，共有1701个基因表达上调，1668个基因表达下调。乙酰化STAT3的近端结合相较于远端结合，展现出更强的转录调控活性。在前500个结合峰中，乙酰化STAT3结合的经典基序频率显著高于非经典基序（p<0.00001）。
功能富集分析显示，乙酰化STAT3通过上调主要参与细胞因子产生负调控及IL-10信号通路的靶基因，同时下调主要参与免疫效应过程及抗原加工/呈递的靶基因，实现对靶基因的调控。乙酰化STAT3对IL-10受体α链（IL-10Ra）的上调作用，增强了IL-10信号通路的敏感性，进而实现DC功能的负向调控。
整体实验设计：将骨髓源树突状细胞以500 nM的SAHA或溶剂对照处理12小时。使用针对STAT3、H3K4me3的抗体及匹配的IgG阴性对照开展染色质免疫沉淀实验。通过Illumina HiSeq 2000平台进行深度测序，构建DNA结合图谱。