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

作为对激活信号的响应，NFKB和STATS在特定的赖氨酸残基上可逆地甲基化，通过 染色质重塑酶，以深刻影响其功能的方式。我们提出了可逆的 当因子与特定启动子结合时，甲基化与组蛋白甲基化同时发生， 改变可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，并帮助解释对不同促炎信号的具体反应。目标1.我们将 在表达STATS或NFKB p65亚单位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经突变，还将评估不同的NFKB(IL-6)激活剂 与肿瘤坏死因子和TLRs相比)在p65上施加了不同的磷酸化模式，这反过来又是条形码 影响不同目标基因群的赖氨酸甲基化模式。目标2.我们将延长初始 观察到在启动子结合的STAT和NFKB上发生的甲基五氟离子是有序的 导致转录激活的一系列事件。染色质将得到广泛应用 免疫沉淀(CHIP)分析确定特定启动子的FIME过程，这些启动子是 受STATS或p65赖氨酸甲基化的差异调节。我们还将用创新的大众 鉴定未知蛋白质的光谱方法，这些未知蛋白质聚集在特定启动子上 激活STATS或NFKB后的时间。目标3.对于未与之直接接触的赖氨酸残基 DNA，甲基化可能促进辅助蛋白的结合，从而改变功能。这些蛋白质将是 通过使用GST蛋白结构域芯片和免疫沉淀试验进行鉴定。K218和K221 P65确实与DNA接触，所以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。目的4.IRFS、TIS7和IRAK4是NFKB激活和释放的重要调节因子。 功能。我们将确定它们如何影响p65的磷酸化和甲基化以及依赖的 依赖于NpKB的基因表达模式。其活性受到调节的基因的例子将会 如上所述进行详细研究，以确定启动子特异的机制。在项目1、3和4中， NFKB对这些基因的激活是如何调节的，也将在原代小鼠细胞和 小鼠肿瘤模型。通过这种方式，项目2的机械分析将直接连接到生物 对其他三个项目的分析。 相关性(请参阅说明)： 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。这些修改是特定于基因的，并且完整的 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、肿瘤坏死因子、TLR)通过激活NFKB和STAT，对于充分了解 免疫系统的细胞与上皮细胞的相互作用会导致癌症。

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.