Mechanisms and functions of lysine methylation of promoter-bound NFkB and STAT3

响应激活信号，NFKB 和 STATS 在特定赖氨酸残基上通过以下方式可逆甲基化： 染色质重塑酶，以深刻影响其功能的方式。我们建议可逆 当这些因子与特定的启动子结合时，甲基化与组蛋白甲基化同时发生， 改变诱导基因表达的强度和持续时间，赋予依赖的生物可塑性 反应，并有助于解释对不同促炎信号的特定反应。目标 1. 我们将 在表达 STATS 或 NFKB 的 p65 亚基的细胞中进行全局基因表达分析，其中 赖氨酸甲基化位点已发生突变，还将评估不同的 NFKB 激活剂（IL-6 与 TNF 与 TLR 相比）在 p65 上施加不同的磷酸化模式作为“条形码”，反过来 影响不同靶基因组的赖氨酸甲基化模式。目标 2. 我们将延长最初的 观察到甲基化离子发生在启动子结合的 STATS 和 NFKB 上，作为有序序列的一部分 导致转录激活的事件序列。染色质将得到广泛利用 免疫沉淀（CHIP）测定，以确定特定启动子的事件时间进程 STATS 或 p65 的赖氨酸甲基化差异调节。我们还将使用创新的质量 光谱法来识别在特定启动子上组装的未知蛋白质，作为 激活 STATS 或 NFKB 后的时间。目标 3. 对于不直接接触的赖氨酸残基 DNA 甲基化可能会促进改变功能的辅助蛋白的结合。这些蛋白质将 通过使用 GST 蛋白结构域微阵列和免疫沉淀测定来鉴定。 K218 和 K221 的 p65 确实与 DNA 接触，因此我们将通过研究纯甲基化的结合来探索这些相互作用 蛋白质直接转化为DNA。目标 4. IRFS、TIS7 和 IRAK4 是 NFKB 激活和调节的重要调节剂 功能。我们将确定每个因素如何影响 p65 及其依赖的磷酸化和甲基化 NpKB 依赖性基因表达模式。然后，激活被调节的基因的例子 如上所述进行详细研究，以确定启动子特异性机制。在项目 1、3 和 4 中，方法 NFKB 调节这些基因的激活也将在原代小鼠细胞和 小鼠肿瘤模型。这样，项目2的机理分析将直接与生物学相关 其他三个项目的分析。 相关性（参见说明）： 最近的证据表明启动子结合的转录因子被相同的酶修饰 修饰组蛋白，具有重要的功能后果。这些修饰是基因特异性的，并且是完整的 了解它们是如何发生的，以及它们如何影响对主要促炎细胞因子的反应 （IL-1、IL-6、TNF、TLR）通过激活 NFKB 和 STATS，对于充分了解如何发挥作用非常重要 免疫系统细胞与上皮细胞的相互作用导致癌症。

In response to activating signals, NFKB and STATS are reversibly methylated on specific lysine residues by chromatin remodeling enzymes, in ways that profoundly affect their functions. We propose that reversible methylation occurs, in concert with histone methylation, when the factors are bound to specific promoters, altering the strength and duration of inducible gene expression, giving plasticity to the dependent biological responses, and helping to explain specific responses to different pro-inflammatory signals. Aim 1. We will conduct global gene expression analyses in cells expressing STATS or the p65 subunit of NFKB in which the lysine methylation sites have been mutated and will also evaluate whether different activators of NFKB (IL-6 versus TNF versus TLRs) impose different patterns of phosphorylation on p65 as "bar codes" that in turn affect lysine methylation patterns at different cohorts of target genes. Aim 2. We will extend initial observations that methylafions take place on promoter-bound STATS and NFKB as part of an ordered sequence of events leading to transcriptional activation. Extensive use will be made of chromatin immunoprecipitation (CHIP) assays to determine the fime courses of events at specific promoters that are regulated differentially by lysine methylation of STATS or p65. We also will use innovative mass spectrometric approaches to identify unknown proteins that assemble on specific promoters as a function of time following activation of STATS or NFKB. Aim 3. For lysine residues that are not in direct contact with DNA, methylation may facilitate the binding of accessory proteins that alter function. These proteins will be identified by using a GST protein domain microarray and by immunopreciptation assays. K218 and K221 of p65 do contact DNA, so we will explore these interactions by studying the binding of the pure methylated protein to DNA directly. Aim 4. IRFS, TIS7 and IRAK4 are important modulators of NFKB activafion and funcfion. We will determine how each affects the phosphorylation and methylation of p65 and the dependent patterns of NpKB-dependent gene expression. Examples of genes whose activafion is modulated will then be studied in detail as above, to define promoter-specific mechanisms. In Projects 1, 3 and 4, the ways in which the activation of these genes by NFKB is modulated will also be studied in primary mouse cells and in mouse tumor models. In this way, the mechanistic analyses of Project 2 will connect directly to the biological analyses of the other three projects. RELEVANCE (See instructions): Recent evidence has shown us that promoter-bound transcription factors are modified by the same enzymes that modify histones, with important functional consequences. The modifications are gene-specific, and a full understanding of how they occur, and of how they affect the responses to major pro-inflammatory cytokines (IL-1, IL-6, TNF, TLRs) through their activation of NFKB and STATS, is important for understanding fully how the interactions of cells of the immune system with epithelial cells lead to cancer.