Combined Chromatin and Expression Analysis Reveals Specific Regulatory Mechanisms within Cytokine Genes in the Macrophage Early Immune Response

Macrophages play a critical role in innate immunity, and the expression of early response genes orchestrate much of the initial response of the immune system. Macrophages undergo extensive transcriptional reprogramming in response to inflammatory stimuli such as Lipopolysaccharide (LPS). To identify gene transcription regulation patterns involved in early innate immune responses, we used two genome-wide approaches - gene expression profiling and chromatin immunoprecipitation-sequencing (ChIP-seq) analysis. We examined the effect of 2 hrs LPS stimulation on early gene expression and its relation to chromatin remodeling (H3 acetylation; H3Ac) and promoter binding of Sp1 and RNA polymerase II phosphorylated at serine 5 (S5P RNAPII), which is a marker for transcriptional initiation. Our results indicate novel and alternative gene regulatory mechanisms for certain proinflammatory genes. We identified two groups of up-regulated inflammatory genes with respect to chromatin modification and promoter features. One group, including highly up-regulated genes such as tumor necrosis factor (TNF), was characterized by H3Ac, high CpG content and lack of TATA boxes. The second group, containing inflammatory mediators (interleukins and CCL chemokines), was up-regulated upon LPS stimulation despite lacking H3Ac in their annotated promoters, which were low in CpG content but did contain TATA boxes. Genome-wide analysis showed that few H3Ac peaks were unique to either +/−LPS condition. However, within these, an unpacking/expansion of already existing H3Ac peaks was observed upon LPS stimulation. In contrast, a significant proportion of S5P RNAPII peaks (approx 40%) was unique to either condition. Furthermore, data indicated a large portion of previously unannotated TSSs, particularly in LPS-stimulated macrophages, where only 28% of unique S5P RNAPII peaks overlap annotated promoters. The regulation of the inflammatory response appears to occur in a very specific manner at the chromatin level for specific genes and this study highlights the level of fine-tuning that occurs in the immune response.巨噬细胞（Macrophages）在固有免疫中发挥关键作用，早期应答基因的表达调控着免疫系统的大部分初始应答反应。巨噬细胞在受到脂多糖（Lipopolysaccharide, LPS）等炎症刺激时，会发生广泛的转录重编程。为了鉴定参与早期固有免疫应答的基因转录调控模式，我们采用了两种全基因组研究方法——基因表达谱分析与染色质免疫沉淀测序（ChIP-seq）。我们探究了2小时LPS刺激对早期基因表达的影响，及其与染色质重塑（组蛋白H3乙酰化，即H3Ac）、Sp1在启动子区域的结合以及丝氨酸5位磷酸化RNA聚合酶II（S5P RNAPII）之间的关联，后者是转录起始的标志性标志物。我们的研究结果揭示了部分促炎基因全新的及非经典的基因调控机制。我们依据染色质修饰与启动子特征，将上调的炎症基因划分为两类：第一类以肿瘤坏死因子（TNF）等高度上调基因为代表，其特征为存在H3Ac修饰、高CpG含量且缺乏TATA盒；第二类包含多种炎症介质（如白细胞介素与CCL趋化因子），尽管其注释启动子区域未检测到H3Ac修饰，但仍在LPS刺激后出现上调，这类基因的CpG含量较低，但含有TATA盒。全基因组分析显示，仅有少量H3Ac峰仅存在于±LPS处理组中的某一组。然而，在这些仅存在于单一处理组的H3Ac峰中，LPS刺激会引发已存在的H3Ac峰的展开与扩增。与之相反，约40%的S5P RNAPII峰仅存在于某一处理组中，具有处理组特异性。此外，研究数据还显示存在大量此前未被注释的转录起始位点（TSSs），尤其在LPS刺激的巨噬细胞中，仅有28%的唯一S5P RNAPII峰与注释启动子区域存在重叠。炎症应答的调控似乎在染色质层面以高度特异性的方式作用于特定基因，本研究凸显了免疫应答过程中精细调控的层级与复杂程度。