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

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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.