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.

项目摘要 整体组蛋白去乙酰化与许多类型的癌症有关，并由组蛋白去乙酰化酶控制 （HDAC）。尽管HDAC抑制剂广泛用于癌症治疗，但其活性不靶向特异性靶向肿瘤。 HDAC同种型也没有特定的基因，因此对患者产生显著的副作用。由此可见，有一 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、更安全的 有效的治疗。本建议的总体目标是了解如何招募残疾咨询委员会， 基因组区域。由于HDAC不具有内在的DNA结合活性，因此认为它们被募集到细胞中。 通过与DNA结合蛋白的相互作用而与染色质结合，尽管这种机制尚未完全探索。 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 非编码RNA（lncRNA）特异性地将HDAC 3募集到未来的失活X染色体（Xi）。如此则 Xist-SHARP/SMRT/HDAC 3抑制复合物使组蛋白脱乙酰化并沉默Xi上的基因表达。 我们的实验室还证明，SHARP定位于许多核位点（超出了Xi），在RNA依赖的细胞内。 这种方式，提出了一个问题，即哪些额外的RNA招募它（沿着HDAC 3）和什么特定的基因组 地点值得注意的是，几乎所有的人HDAC都在含有SHARP的多蛋白复合物内缔合， 其他RNA结合蛋白，这表明这种RNA指导机制可能超出SHARP， Xist. 我假设HDAC复合物被RNA募集，以实现对它们的各种调节的特异性。 目标遍布整个核。首先，我将确定哪些SHARP-RNA相互作用在功能上重要， 通过遗传干扰候选RNA的SHARP结合位点。然后我将测量基因 表达以及这些作用是否需要HDAC 3活性（目的1）。第二，我会全面识别 与其他癌症相关HDAC相关的RNA结合蛋白，并定义其体内RNA结合位点。 然后，我将确定哪些蛋白结合的候选RNA从这个屏幕上调节HDAC依赖性 目标2（Aim 2）这项研究有可能改变我们对RNA和RNA的理解。 结合蛋白作为中央调节器在组织染色质修饰的HDACs在正常和 癌细胞状态。