Non-coding RNA Regulation of TNF Alpha

项目概述/摘要TNF1是一种在先天宿主防御中至关重要的细胞因子。TNFa水平的改变对宿主有害，其表达在多个水平上受到严格调控。我们认为它的调控也包括长链ncrna的作用。GCF2是一种结合染色质的转录抑制因子，定位于发现有意义和反义ncrna的TNF1启动子区域。在不表达TNF1的造血干细胞模型K562细胞中，DNA高度甲基化，染色质抑制标记明显，并且发现GCF2与启动子的-300区域结合。GCF2在表达能力细胞系中过表达，导致TNF1的表达显著降低。GCF2所在的启动子的-300区域是组蛋白修饰最大的区域，也以EZH2和Suz 12结合为标志，这两种多梳蛋白与转录抑制相关。我们在TNF1的上游区域发现了低水平的正义和反义转录本，预计这些转录本会形成高度折叠的结构。在K562细胞中，硫代寡核苷酸对反义转录物的消耗导致TNF1表达的去抑制。启动子RNA与DNA密切相关，因为识别RNA:DNA杂交结构的S9.6抗体以与GCF2相似的模式结合到TNF1上游区域。我们假设GCF2在高二级结构的反义RNA位点与染色质结合，参与RNA介导的转录抑制。支持这一假设的其他数据包括：(1)GCF2在所有报道的瞬时转染实验中都起到抑制作用，(2)GCF2结合argonautes 1和2，(3)染色质RNA- ip与GCF2回收的TNF1上游RNA的抗体结合。在Aim 1中，我们将通过转录本的定量和来自抑制、能态或主动表达TNF1的细胞的定位来更好地定义上游序列的作用。为了证明它们对TNF1 mRNA的影响，我们将用硫代寡核苷酸耗尽上游转录本。在Aim 2中，我们将定义GCF2的结合特性。我们将耗尽上游序列，观察GCF2与启动子结合的影响。为了评估GCF2的其他靶点，我们将进行GCF2 ChIP-seq分析。在Aim 3中，我们将确定GCF2与上游区域结合是否需要argonaute、EZH2或Suz 12。目的3还将定义蛋白质-蛋白质相互作用，并直接评估对转录的影响。来自这些目标的数据将定义上游转录本的特征及其在TNF1调控中的作用。这些研究将促进我们对rna介导的基因表达调控的理解，特别是对TNF1表达的复杂调控。

DESCRIPTION (provided by applicant): Project Summary/Abstract TNF1 is a cytokine critically important in innate host defense. Altered levels of TNFa are deleterious to the host and expression is tightly regulated at multiple levels. We propose that its regulation also includes effects from long ncRNAs. GCF2 is a transcriptional repressor that binds chromatin and localizes to the region of the TNF1 promoter where sense and antisense ncRNAs are found. In K562 cells, a model of hematopoietic stem cells which do not express TNF1, the DNA is highly methylated, chromatin marks of repression are evident and GCF2 is found bound to the -300 region of the promoter. Over-expression of GCF2 in a cell line competent for expression, led to significantly reduced expression of TNF1. The -300 region of the promoter, where GCF2 is found, is the region of maximal histone modifications and is also marked by binding of EZH2 and Suz 12, two polycomb proteins associated with transcriptional repression. . We identified low levels of sense and antisense transcripts throughout the upstream region of TNF1 and these transcripts were predicted to form highly folded structures. Depletion of antisense transcripts by phosphorothioate oligonucleotides led to de- repression of TNF1 expression in K562 cells. The promoter RNA was closely associated with the DNA because the S9.6 antibody, which recognizes RNA:DNA hybrid structures, bound to the TNF1 upstream region in a pattern that paralleled the binding pattern of GCF2. We hypothesize that GCF2 binds chromatin at sites of antisense RNA with high secondary structure and participates in RNA-mediated transcriptional repression. Additional data supporting this hypothesis include (1) GCF2 acts as a repressor in every transient transfection assay reported, (2) GCF2 binds argonautes 1 and 2, (3) Chromatin RNA-IP with an antibody to GCF2 recovered TNF1 upstream RNA. In Aim 1, we will better define the roles of the upstream sequences by quantitation of the transcripts and mapping from cells that are repressed, competent or actively expressing TNF1. To demonstrate their effect on TNF1 mRNA, we will deplete the upstream transcripts with phosphorothioate oligonucleotides. In Aim 2, we will define the binding characteristics of GCF2. We will deplete the upstream sequences and observe the effect on GCF2 binding to the promoter. To assess the other targets of GCF2, we will perform a GCF2 ChIP-seq analysis. In Aim 3, we will determine whether argonaute, EZH2, or Suz 12 are required for GCF2 binding to the upstream region. Aim 3 will also define protein-protein interactions and directly assess the effect on transcription. The data from these aims will define the character of the upstream transcripts and their role in the regulation of TNF1. These studies will advance our understanding of RNA-mediated regulation of gene expression and, specifically, the complex regulation of TNF1 expression. PUBLIC HEALTH RELEVANCE: Project Narrative Previous studies from my laboratory have revealed evidence of a novel form of regulation of TNF1. The studies described in this application are directed at understanding this new RNA-mediated form of transcriptional regulation. The long term goal is to be able to regulate TNF1 expression in disease states characterized by over-production such as Crohn Disease and rheumatoid arthritis.