Characterizing the RNA-mediated recruitment of histone deacetylases to chromatin

项目总结 全局性组蛋白去乙酰化与多种癌症有关，并受组蛋白去乙酰化酶控制 (HDAC)。尽管HDAC抑制剂被广泛用于癌症治疗，但它们的活性并不针对特定的靶点。 HDAC异构体也不是特定的基因，因此对患者会产生显著的副作用。因此，有一个 迫切需要以高度精确的、特定于基因的方式针对HDAC活动，以开发更安全和更 有效的治疗方法。这项建议的总体目标是了解如何招募人类发展委员会到具体的 基因组区域。因为HDAC不具有固有的DNA结合活性，所以它们被认为是被招募的 通过与DNA结合蛋白相互作用到染色质，尽管这一机制还没有完全被探索。 最近，我们的实验室和其他人发现，尖锐的RNA结合蛋白直接与Xist Long相互作用 非编码RNA(LncRNA)，特异性地将HDAC3招募到未来不活跃的X染色体(Xi)。通过这种方式， Xist-Sharp/SMRT/HDAC3抑制复合体去乙酰化组蛋白，沉默XI上的基因表达。 我们的实验室还证明了在RNA依赖的情况下，夏普定位于许多核部位(不包括XI)。 方式，提出了哪些额外的RNA招募它(与HDAC3一起)以及到什么特定基因组的问题 地点。值得注意是，几乎所有的人类HDAC都结合在含有Sharp和Sharp的多蛋白复合体中 其他RNA结合蛋白，表明这种RNA引导的机制可能扩展到锐化和 希斯特。 我假设HDAC复合体是由RNA招募的，以实现对其各种调控的特异性 整个原子核的目标。首先，我将确定哪些尖锐的RNA相互作用在功能上是重要的 通过基因扰乱候选RNA的尖锐结合位点。然后我将测量对基因的影响 表达，以及是否需要HDAC3活性来实现这些效果(目标1)。第二，我将全面识别 与其他癌症相关的HDAC相关的RNA结合蛋白，并定义它们在体内的RNA结合位点。 然后我将确定这个屏幕上的哪些蛋白质结合的候选RNA在HDAC依赖的 方式(目标2)。这项拟议的研究有可能改变我们对RNA和RNA的理解。 结合蛋白在正常和高密度脂蛋白细胞染色质修饰中的中枢调节作用 癌细胞状态。

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.