Characterizing the RNA-mediated recruitment of histone deacetylases to chromatin

表征 RNA 介导的组蛋白脱乙酰酶向染色质的募集

• 批准号: 10680276
• 负责人: James Kang Guo
• 金额: $ 5.27万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680276
• 关键词: Affinity Chromatography; Antisense RNA; Binding; Binding Proteins; Binding Sites; Cancerous; Cell Cycle; Cell Nucleus; Cells; Chromatin; Complex; DNA Binding; DNA-Binding Proteins; Data; Enzymes; Future; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genetic; Genome; Guide RNA; HDAC1 gene; HDAC3 gene; Health; Histone Deacetylase; Histone Deacetylase Inhibitor; Histone Deacetylation; Human; Immunoprecipitation; Knock-out; Link; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Methods; Mission; Molecular; Multiprotein Complexes; Mutate; Nuclear; Pathology; Patients; Pharmaceutical Preparations; Protein Isoforms; Proteins; RNA; RNA Binding; RNA-Binding Proteins; Repression; Research; Research Project Grants; Role; Site; Specificity; Suppressor-Effector T-Lymphocytes; Testing; Therapeutic; Time; Toxic effect; Tumor Suppressor Genes; United States National Institutes of Health; Untranslated RNA; Work; X Chromosome; cancer therapy; cancer type; chromatin modification; crosslink; effective therapy; experimental study; gene repression; genetic manipulation; genetic regulatory protein; genomic locus; improved; in vivo; inhibitor; link protein; mRNA Precursor; novel; pharmacologic; protein complex; public database; recruit; side effect; therapeutic target; transcription factor; tumor

PROJECT SUMMARY Global histone deacetylation is linked to many types of cancer and is controlled by histone deacetylases (HDACs). Although HDAC inhibitors are widely used in cancer treatment, their activity does not target specific HDAC isoforms nor specific genes and therefore results in significant side effects for patients. Thus, there is a pressing need to target HDAC activity in a highly precise, gene-specific manner to develop safer and more effective treatments. The overall objective of this proposal is to understand how HDACs are recruited to specific genome regions. Because HDACs do not possess intrinsic DNA binding activity, they are thought to be recruited to chromatin through interactions with DNA binding proteins, though this mechanism has not been fully explored. Recently, our lab and others identified that the SHARP RNA-binding protein directly interacts with the Xist long noncoding RNA (lncRNA) to specifically recruit HDAC3 to the future inactive X chromosome (Xi). In this way, the Xist-SHARP/SMRT/HDAC3 repressive complex deacetylates histones and silences gene expression on the Xi. Our lab has also demonstrated that SHARP localizes to many nuclear sites (beyond the Xi) in an RNA-dependent manner, raising the question of which additional RNAs recruit it (along with HDAC3) and to what specific genomic locations. Notably, nearly all human HDACs associate within multi-protein complexes containing SHARP and other RNA-binding proteins, suggesting that this mechanism of RNA-guidance may extend beyond SHARP and Xist. I hypothesize that HDAC complexes are recruited by RNAs to achieve specificity to their various regulatory targets throughout the nucleus. First, I will determine which SHARP-RNA interactions are functionally important by genetically perturbing the SHARP binding sites of candidate RNAs. I will then measure effects on gene expression and if HDAC3 activity is required for these effects (Aim 1). Second, I will comprehensively identify RNA-binding proteins associated with other cancer-associated HDACs and define their in vivo RNA binding sites. I will then determine which protein-bound candidate RNAs from this screen are regulated in an HDAC-dependent manner (Aim 2). The proposed research has the potential to transform our understanding of RNAs and RNA- binding proteins as central regulators in organizing chromatin modifications by HDACs in both normal and cancerous cell states.

项目概要 整体组蛋白脱乙酰化与多种癌症有关，并由组蛋白脱乙酰酶控制 （HDAC）。尽管 HDAC 抑制剂广泛用于癌症治疗，但其活性并不针对特定目标 HDAC 亚型也不是特定基因，因此会给患者带来显着的副作用。因此，有一个 迫切需要以高度精确、基因特异性的方式靶向 HDAC 活性，以开发更安全、更高效的产品 有效的治疗。该提案的总体目标是了解 HDAC 如何被招募到特定的领域 基因组区域。由于 HDAC 不具有内在的 DNA 结合活性，因此它们被认为是被招募的 通过与 DNA 结合蛋白的相互作用而形成染色质，尽管这一机制尚未得到充分探索。 最近，我们的实验室和其他人发现 SHARP RNA 结合蛋白直接与 Xist 长相互作用 非编码 RNA (lncRNA) 特异性地将 HDAC3 招募到未来失活的 X 染色体 (Xi)。这样， Xist-SHARP/SMRT/HDAC3 抑制复合物使组蛋白去乙酰化并沉默 Xi 上的基因表达。 我们的实验室还证明，SHARP 以 RNA 依赖性方式定位于许多核位点（Xi 之外） 方式，提出了哪些额外的 RNA 招募它（以及 HDAC3）以及针对什么特定基因组的问题 地点。值得注意的是，几乎所有人类 HDAC 都与含有 SHARP 和 其他 RNA 结合蛋白，表明这种 RNA 引导机制可能超出了 SHARP 和 存在。 我假设 HDAC 复合物由 RNA 招募以实现其各种调节的特异性 目标遍及细胞核。首先，我将确定哪些 SHARP-RNA 相互作用在功能上很重要 通过基因干扰候选 RNA 的 SHARP 结合位点。然后我将测量对基因的影响 表达以及这些效应是否需要 HDAC3 活性（目标 1）。二、我会全面识别 RNA 结合蛋白与其他癌症相关的 HDAC 相关，并定义了它们的体内 RNA 结合位点。 然后我将确定该筛选中哪些与蛋白质结合的候选 RNA 在 HDAC 依赖性中受到调节 方式（目标 2）。拟议的研究有可能改变我们对 RNA 和 RNA 的理解 结合蛋白作为 HDAC 组织染色质修饰的中心调节因子，在正常和 癌细胞状态。