The role of chromatin-associated heat-shock protein 90 in transcriptional response to stress

热休克蛋白90（Heat-shock protein 90，Hsp 90）是一种分子伴侣，是一种成功的抗癌治疗靶点。热休克蛋白90不帮助新合成的蛋白质从核糖体折叠，而是促进部分折叠的蛋白质的成熟。Hsp 90在人类中以两种亚型存在，由两种不同的基因编码。这两种亚型大约有90%相同。然而，基因敲除小鼠的不同表型表明两种亚型执行非重叠功能。在哺乳动物细胞中，总Hsp 90的约1- 3%的一小部分驻留在细胞核中，并且该比例在应激时增加。我们以前的工作表明，热休克蛋白90结合到30%的果蝇基因的启动子，并导致RNA聚合酶II暂停。热休克蛋白90结合和伴侣的染色质修饰剂和转录因子在染色质影响基因调控。然而，对这些机制仍然知之甚少。Hsp 90在基因调控中的这种功能在细胞对应激（如热休克）产生转录反应时变得更加突出。Hsp 90亚型在正常和应激条件下如何调节转录？我们将在人红白血病细胞系K562中进行所有实验，该细胞系是ENCODE项目中的一级细胞系，允许我们的数据与已发表的染色质谱之间进行有意义的比较。作为一种应激条件，我们将采用热休克处理细胞，这使得充分研究，强大和高度保守的反应。本课题以正常和应激条件下的K562细胞为研究对象，主要完成以下三个方面的工作：（1）从正常和应激条件下的K562细胞染色质提取物中，对标记的Hsp 90异构体进行串联亲和纯化，以鉴定染色质特异性的Hsp 90客户。通过研究Hsp 90抑制后这些相互作用物的稳定性和染色质定位，将验证一组新的相互作用物。(2)为了找出Hsp 90亚型在应激条件之前和期间结合染色质的基因组位置，我们将进行染色质免疫沉淀，然后进行测序。使用计算的方法，我们将确定的基因是由热休克蛋白90的亚型特异性和压力特异性的方式。(3)整合的基因组和蛋白质组学数据中产生的第一个目标，以描绘特定的贡献热休克蛋白90亚型的压力诱导的转录反应。在正常和应激条件下对K562细胞进行RNA测序，当每个亚型被敲低时，将提供关于Hsp 90在转录中的作用的信息。与ENCODE数据的染色质档案的各种转录因子的比较将提供机制的见解。总之，这个项目将提供第一个详尽的看法，染色质功能的热休克蛋白90，伴侣具有巨大的生物医学意义。

Heat-shock protein 90 (Hsp90) is a molecular chaperone that is a successful target for anti-cancer therapy. Hsp90 does not help newly synthesized proteins from the ribosome to fold, but rather facilitates maturation of partially folded proteins. Hsp90 in humans exist in two isoforms encoded by two distinct genes. The two isoforms are about 90 percent identical. However the distinct phenotypes of the knock-out mice show that the two isoforms perform non-overlapping functions. A small fraction of about 1-3 percent of total Hsp90 in a mammalian cell resides in the nucleus, and this proportion increases upon stress. Our previous work has shown that Hsp90 binds to promoters of 30% of Drosophila genes and causes RNA polymerase II to pause. Hsp90 binds and chaperones a number of chromatin modifiers and transcription factors at chromatin influencing gene regulation. However, the mechanisms remain poorly understood. This function of Hsp90 in gene regulation becomes even more prominent as cells mount a transcriptional response to stress such as heat shock. How do Hsp90 isoforms regulate transcription under normal and stress conditions? The present proposal will address this question focusing on chromatin-associated Hsp90 in human cells.We will perform all experiments in human erythroleukemia cell line K562 that is a tier 1 cell line in the ENCODE project allowing a meaningful comparison between our data and published chromatin profiles. As a stress condition, we will employ heat-shock treatment of cells, which elicits well-studied, robust and highly conserved response. Using K562 cells under normal and stress conditions, the project will be executed in three Objectives: (1) To identify chromatin-specific clients of Hsp90, we will perform tandem Affinity purification of tagged Hsp90 isoforms from chromatin extracts of K562 cells under normal and stress conditions. A subset of novel interactors will be validated by studying the stability and chromatin localization of these interactors upon Hsp90 inhibition. (2) To find out genomic locations where Hsp90 isoforms bind chromatin before and during stress conditions, we will perform chromatin immunoprecipitation followed by sequencing. Using computational approaches we will identify genes that are targeted by Hsp90 in an isoform-specific and stress-specific fashion. (3) Integration of the genomic and proteomic data generated in the first to objectives so as to delineate the specific contributions of Hsp90 isoforms to stress-induced transcriptional response. RNA sequencing of K562 cells under normal and stress conditions when each isoform has been knocked down will provide information about the role of Hsp90 in transcription. A comparison with ENCODE data on chromatin profiles of various transcription factors will provide mechanistic insights. In summary, this project will provide the first exhaustive view of chromatin functions of Hsp90, a chaperone with immense biomedical importance.