Non-coding RNA Regulation of TNF Alpha

TNF Alpha 的非编码 RNA 调控

基本信息

  • 批准号:
    7989625
  • 负责人:
  • 金额:
    $ 25.11万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2010
  • 资助国家:
    美国
  • 起止时间:
    2010-06-01 至 2012-05-31
  • 项目状态:
    已结题

项目摘要

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.
描述(由申请人提供):项目摘要/摘要TNF 1是一种在先天宿主防御中至关重要的细胞因子。改变的TNFa水平对宿主有害,并且表达在多个水平上受到严格调控。我们认为它的调节也包括长ncRNA的影响。GCF 2是一种转录抑制因子,它与染色质结合并定位于TNF 1启动子的有义和反义ncRNA区域,在K562细胞中,一种不表达TNF 1的造血干细胞模型,DNA高度甲基化,抑制的染色质标记是明显的,并且发现GCF 2与启动子的-300区域结合。在有表达能力的细胞系中GCF 2的过表达导致TNF 1的表达显著降低。发现GCF 2的启动子的-300区域是最大组蛋白修饰的区域,并且还通过EZH 2和Suz 12(与转录抑制相关的两种多梳蛋白)的结合来标记。.我们确定了低水平的正义和反义转录整个上游区域的TNF 1和这些转录被预测形成高度折叠的结构。硫代磷酸寡核苷酸耗尽反义转录物导致K562细胞中TNF 1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GCF 2结合模式相似的模式结合到TNF 1上游区域。我们推测GCF 2在具有高级结构的反义RNA的位点结合染色质,并参与RNA介导的转录抑制。支持这一假设的其他数据包括:(1)GCF 2在报道的每个瞬时转染试验中充当阻遏物,(2)GCF 2结合argonautes 1和2,(3)染色质RNA-IP与GCF 2回收的TNF 1上游RNA的抗体。在目标1中,我们将更好地定义上游序列的作用,通过定量的转录本和映射从细胞抑制,主管或积极表达TNF 1。为了证明它们对TNF 1 mRNA的影响,我们将用硫代磷酸寡核苷酸耗尽上游转录物。在目标2中,我们将定义GCF 2的结合特性。我们将耗尽上游序列并观察对GCF 2与启动子结合的影响。为了评估GCF 2的其他靶标,我们将进行GCF 2 ChIP-seq分析。在目标3中,我们将确定argonaute、EZH 2或Suz 12是否是GCF 2与上游区域结合所需的。目标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。这些目标的数据将定义上游转录本的特征及其在TNF 1调节中的作用。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TNF 1表达的复杂调控。 公共卫生相关性:项目叙述我实验室以前的研究揭示了TNF 1调节的新形式的证据。本申请中描述的研究旨在理解这种新的RNA介导的转录调节形式。长期目标是能够在以过度产生为特征的疾病状态如克罗恩病和类风湿性关节炎中调节TNF 1表达。

项目成果

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KATHLEEN E SULLIVAN其他文献

KATHLEEN E SULLIVAN的其他文献

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{{ truncateString('KATHLEEN E SULLIVAN', 18)}}的其他基金

USIDNET: A resource for clinical immunologists
USIDNET:临床免疫学家的资源
  • 批准号:
    10410606
  • 财政年份:
    2022
  • 资助金额:
    $ 25.11万
  • 项目类别:
Non-coding RNA Regulation of TNF Alpha
TNF Alpha 的非编码 RNA 调控
  • 批准号:
    8070422
  • 财政年份:
    2010
  • 资助金额:
    $ 25.11万
  • 项目类别:
Epigenomics of SLE
SLE的表观基因组学
  • 批准号:
    8126220
  • 财政年份:
    2009
  • 资助金额:
    $ 25.11万
  • 项目类别:
Epigenomics of SLE
SLE的表观基因组学
  • 批准号:
    8521081
  • 财政年份:
    2009
  • 资助金额:
    $ 25.11万
  • 项目类别:
Epigenomics of SLE
SLE的表观基因组学
  • 批准号:
    8318805
  • 财政年份:
    2009
  • 资助金额:
    $ 25.11万
  • 项目类别:
Epigenomics of SLE
SLE的表观基因组学
  • 批准号:
    7934622
  • 财政年份:
    2009
  • 资助金额:
    $ 25.11万
  • 项目类别:
Advancing Careers of Investigators in Primary Immune Disorders
促进原发性免疫疾病研究人员的职业发展
  • 批准号:
    10250421
  • 财政年份:
    2009
  • 资助金额:
    $ 25.11万
  • 项目类别:
Advancing Careers of Investigators in Primary Immune Disorders
促进原发性免疫疾病研究人员的职业发展
  • 批准号:
    10018655
  • 财政年份:
    2009
  • 资助金额:
    $ 25.11万
  • 项目类别:
Epigenomics of SLE
SLE的表观基因组学
  • 批准号:
    7725549
  • 财政年份:
    2009
  • 资助金额:
    $ 25.11万
  • 项目类别:
Training of Clinical Investigators in Primary Immune Deficiencies
原发性免疫缺陷临床研究人员的培训
  • 批准号:
    8326287
  • 财政年份:
    2009
  • 资助金额:
    $ 25.11万
  • 项目类别:

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