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

热休克蛋白90 （Hsp90）是一种分子伴侣，是抗癌治疗的成功靶点。Hsp90不帮助核糖体新合成的蛋白质折叠，而是促进部分折叠的蛋白质成熟。人类的热休克蛋白90有两种同型，由两种不同的基因编码。这两种同工异构体大约90%相同。然而，基因敲除小鼠的不同表型表明，这两种同种异构体具有不重叠的功能。哺乳动物细胞中大约1- 3%的热休克蛋白90存在于细胞核中，这一比例在压力下会增加。我们之前的工作表明，Hsp90与30%的果蝇基因启动子结合，导致RNA聚合酶II暂停。Hsp90在影响基因调控的染色质上结合并伴随许多染色质修饰因子和转录因子。然而，人们对其机制仍然知之甚少。当细胞对应激（如热休克）产生转录反应时，Hsp90在基因调控中的功能变得更加突出。Hsp90亚型在正常和应激条件下如何调控转录？本提案将解决这一问题，重点关注人类细胞中染色质相关的Hsp90。我们将在ENCODE项目中的一级细胞系K562人类红白血病细胞系中进行所有实验，以便将我们的数据与已发表的染色质谱进行有意义的比较。作为应激条件，我们将采用热休克处理细胞，这引起了充分研究，稳健和高度保守的反应。使用正常和应激条件下的K562细胞，该项目将在三个目标下执行：(1)为了鉴定Hsp90的染色质特异性客户端，我们将从正常和应激条件下的K562细胞的染色质提取物中对标记的Hsp90亚型进行串联亲和纯化。通过研究这些相互作用物在Hsp90抑制作用下的稳定性和染色质定位，将对一类新型相互作用物进行验证。(2)为了找出应激前和应激时Hsp90亚型结合染色质的基因组位置，我们将进行染色质免疫沉淀，然后测序。使用计算方法，我们将以异构体特异性和应激特异性的方式识别被Hsp90靶向的基因。(3)将第一部分的基因组和蛋白质组学数据整合到目标中，以描述Hsp90亚型对应激诱导的转录反应的具体贡献。当每个亚型被敲除时，对正常和应激条件下的K562细胞进行RNA测序将提供有关Hsp90在转录中的作用的信息。与ENCODE数据对各种转录因子的染色质谱的比较将提供机制的见解。总之，该项目将首次提供Hsp90的染色质功能的详尽视图，Hsp90是一种具有巨大生物医学重要性的伴侣。

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.