Coordinated transcriptional regulation of Hspa1a gene by multiple transcription factors: crucial roles for HSF-1, NF-Y, NF-κB, and CREB

Abstract

Although the transcript level of inducible heat shock protein 70.3 (Hsp70.3, also known as Hspa1a) is altered in various disease states, its transcriptional regulation remains incompletely understood. Here, we systematically analyzed the Hspa1a promoter to identify major cis elements and transcription factors that may govern the constitutive/inducible gene expression. Computational analyses coupled with extensive in vitro (promoter-reporter activity and electrophoretic mobility shift assays) and in vivo (chromatin immunoprecipitation assays) revealed interaction of several transcription factors with Hspa1a promoter motifs: HSF-1 (heat shock factor 1) at -114/-97 bp and -788/-777bp, NF-Y (nuclear transcription factor Y) at -73/-58 bp, NF-κB (nuclear factor kappa B) at -133/-124 bp, and CREB (cAMP response element binding protein) at -483/-476 bp. Consistently, siRNA (small interfering RNA)-mediated down-regulation of each of these transcription factors caused substantial reduction of endogenous Hspa1a expression. Heat-shock-induced activation of Hspa1a was coordinately regulated by HSF-1 and NF-Y/NF-κB. The Hspa1a expression was augmented by TNF-α (tumor necrosis factor-alpha) and forskolin in NF-κB and CREB-dependent manners, respectively. NF-κB and CREB also activated Hspa1a transcription in cardiac myoblasts upon exposure to ischemia-like conditions. Taken together, this study discovered previously unknown roles for NF-κB and CREB to regulate Hspa1a expression and a coordinated action by several transcription factors for Hspa1a transactivation under heat-shock/ischemia-like conditions and thereby provided new insights into the mechanism of Hspa1a regulation.

Keywords: CHO; CRE; CREB; ChIP; Chinese hamster ovary; EMSA; HEK; HS; HSE; HSF-1; Hsp; ICE; NF-Y; NF-κB; TNF-α; cAMP; cAMP response element; cAMP response element binding protein; chromatin immunoprecipitation; cyclic adenosine monophosphate; electrophoretic mobility shift assay; gene expression; heat shock; heat shock element; heat shock factor 1; heat shock protein; human embryonic kidney; inverted CCAAT element; nuclear factor kappa B; nuclear transcription factor Y; regulatory motifs; shRNA; short hairpin RNA; siRNA; small interfering RNA; transcription factors; tumor necrosis factor-alpha.