Enhancement of hsp70 gene expression by redox factor-1 is mediated through CCAAT elements

The production of heat shock proteins, which is the hallmark of the heat shock response, can be induced in response to many stress signals including environmental stress, pathophysiological state and even non-stress conditions. Since many of these conditions are redox (reduction/oxidation)-related, redox-based mechanism was studied to elucidate how redox signal is sensed and monitored and transduced to have elevated expression of heat shock genes. To this end, cellular reducing activity, Redox Factor-1 (Ref-1) was investigated for its possible involvement in heat shock gene expression.;With the use of reporter gene construct in which hsp70 promoter drives the gene expression, it was shown that transient overexpression of Ref-1 enhanced the reporter gene expression indicating the enhancement of hsp70 promoter activity by Ref-1. The enhancement of hsp70 promoter activity by Ref-1 was also reflected in endogenous hsp gene expression.;Since the enhancement in promoter activity and endogenous heat shock gene expression by Ref-1 suggests involvement of transcriptional regulation in these processes, the responsible cis- and trans -elements that mediate Ref-1's effect were identified. Mapping analysis of hsp70 promoter revealed the importance of CCAAT motif. Mutation of CCAAT motif in hsp70 promoter caused severe reduction in promoter activity indicating the importance of this site for the enhancement of heat shock gene expression by Ref-1. In addition to CCAAT motif, its flanking sequences were also found to be necessary for optimal enhancement by Ref-1.;The identification of transcription factor that mediates the enhancing effect of Ref-1 in hsp gene expression by binding to CCAAT motif of hsp70 promoter was facilitated by use of dominant negative mutant construct of NF-Y transcription factor. In vivo demonstration of the involvement of NF-Y with the use of dominant negative mutant construct corroborates the notion that NF-Y transcription factor mediates transcriptional enhancing activity of Ref-1 in hsp70 promoter.;A major implication of this work is that It may provide a paradigm in which a reduction/oxidation stress signal in heat shock gene expression is sensed and transduced to NF-Y transcription factor by Ref-1 and the activated NF-Y enhances the gene expression. Further analysis of the target genes of Ref-1 should provide insights into the regulation mechanism of gene expression by redox condition and NF-Y.