HSF–4 and not HSF–1 Regulates Postnatal Expression of the AlphaB–crystallin Gene in the Ocular Lens

Abstract

Abstract: : Purpose: αB–crystallin gene (αB) is highly expressed in the postnatal lens (there is a 10– fold increase in αB transcripts from fetal to day 10 postnatal stage). We have previously shown that the appearance of an active trimeric complex between heat shock factors (HSFs) and the heat shock element (HSE) in the αB promoter is developmentally regulated. Of the three known mammalian HSFs (HSF1, HSF2 and HSF4), HSF–1 is almost universally implicated in the regulation of the heat shock promoters. It is not known whether there is a gene–specific role for any of the HSFs or if they interact cooperatively to regulate genes containing stress promoters. In order to answer these questions we have investigated the nature of the canonical HSF/HSE interaction using the αB–crystallin HSE (αB–HSE) with a focus on identifying the HSF that regulates the enhanced expression of this gene in the postnatal rat lens. Methods: We have used immunoblotting, quantitative RT–PCR with a homologous internal control and real time RT–PCR, FPLC and gel–shift and super–shift binding assays to investigate the HSF/HSE interactions. Results: The 10 X increase in the expression of the αB transcripts in the postnatal lens follows the expression of HSF–4 and not HSF–1, whose expression remains largely confined to the fetal lens. Gel–shift assays indicated that the trimeric HSF/αB–HSE complex was super–shifted only by HSF–4–specific antibody, establishing that it is HSF–4 which interacts with the αB–HSE. Binding studies performed with recombinant HSF–1 and whole cell extracts containing HSF–4 demonstrate that HSF–4 binds much more efficiently to the αB–HSE. Recombinant HSF–1 shows very poor binding to the αB–HSE in comparison to its robust binding to the HSE in the HSP82 gene promoter. Mutational analysis of the αB–HSE showed that the sequences surrounding the canonical HSE play an important role in the formation of the trimeric complex between the HSF–4 and αB–HSE. Conclusion: 1. We have identified that αB–crystallin gene is downstream of HSF–4 in the postnatal ocular lens. This is important because mutations in HSF–4 gene are known to be associated with predominant postnatal cataracts in the human lens. 2. Although different heat shock promoters may share canonical HSEs, specific interactions with different HSFs seem to be dictated by promoter–specific sequences as revealed by analyses with the αB–HSE.

Keywords: gene/expression • transcription factors • crystallins