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

启动子结合的NFkB和STAT3赖氨酸甲基化的机制和功能

• 批准号: 8052288
• 负责人: GEORGE ROBERT STARK
• 金额: $ 51.42万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8052288
• 关键词: Affect; Affinity; Bar Codes; Binding; Biological; Biological Assay; Cell Communication; Cells; DNA; DNA Methylation; DNA-Directed RNA Polymerase; Enzymes; Epithelial Cells; Event; Gene Activation; Gene Expression; Gene Targeting; Genes; Histones; IRAK4 gene; Immune system; Individual; Inflammation; Inflammatory; Instruction; Interleukin-1; Interleukin-6; Lead; Lysine; Malignant Neoplasms; Methylation; Modeling; Modification; Mus; Mutate; NF-kappa B; Pattern; Performance; Phenotype; Phosphorylation; Play; Polymerase; Proteins; Recruitment Activity; Role; STAT3 gene; Serine/Threonine Phosphorylation; Signal Transduction; Site; Stress; TNF gene; Tertiary Protein Structure; Time; Transactivation; Transcriptional Activation; Transferase; Work; chromatin immunoprecipitation; chromatin remodeling; cohort; cytokine; histone methyltransferase; histone modification; innovation; p65; promoter; research study; response; transcription factor; tumor; tumorigenesis

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.

为了响应激活信号，NFKB和统计 染色质重塑酶，以深刻影响其功能的方式。我们提出了这种可逆的 当因子与特定启动子结合时，与组蛋白甲基化一起发生甲基化， 改变诱导基因表达的强度和持续时间，使依赖生物学可塑性 响应，并有助于解释对不同促炎信号的特定响应。目标1。我们将 在表达统计数据的细胞或NFKB的p65亚基中进行全局基因表达分析，其中 赖氨酸甲基化位点已突变，还将评估NFKB的不同激活剂（IL-6）是否（IL-6） 与TNF与TLRS）在p65上施加了不同的磷酸化模式，作为“条形码” 影响靶基因不同人群的赖氨酸甲基化模式。目标2。我们将扩展最初 观察到在启动子结合的统计数据和NFKB上进行的甲基侵蚀作为有序的一部分 导致转录激活的事件序列。大量使用将由染色质组成 免疫沉淀（芯片）测定法以确定特定启动子事件的唱片课程 通过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 光谱方法识别在特定启动子上聚集在特定启动子上的未知蛋白 激活统计或NFKB之后的时间。目标3。对于未直接接触的赖氨酸残留 DNA，甲基化可能促进改变功能的辅助蛋白的结合。这些蛋白质将是 通过使用GST蛋白质结构域微阵列和免疫沉淀测定法确定。 K218和K221 p65确实接触DNA，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 蛋白质直接蛋白质。 AIM4。IRFS，TIS7和IRAK4是NFKB Activafion和 功能。我们将确定每个人如何影响p65的磷酸化和甲基化和依赖性 NPKB依赖性基因表达的模式。然后，调节活化属性的基因的示例将 如上所述进行详细研究，以定义启动子特异性机制。在项目1、3和4中， 该基因通过NFKB的激活也将在原代小鼠细胞和 小鼠肿瘤模型。这样，项目2的机械分析将直接连接到生物学 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通过相同的酶修饰 这会改变组蛋白，并具有重要的功能后果。修饰是基因特异性的，是完整的 了解它们的发生方式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）通过其激活NFKB和统计数据，对于完全理解的方式很重要 免疫系统细胞与上皮细胞的相互作用导致癌症。