Abstract
Maintaining protein homeostasis is critical for survival at the cellular and organismal level (Morimoto, R. I. (2011) Cold Spring Harb. Symp. Quant. Biol. 76, 91-99). Cells express a family of molecular chaperones, the heat shock proteins, during times of oxidative stress to protect against proteotoxicity. We have identified a second stress responsive transcription factor, dFOXO, that works alongside the heat shock transcription factor to activate transcription of both the small heat shock protein and the large heat shock protein genes. This expression likely protects cells from protein misfolding associated with oxidative stress. Here we identify the regions of the Hsp70 promoter essential for FOXO-dependent transcription using in vitro methods and find a physiological role for FOXO-dependent expression of heat shock proteins in vivo.
Highlights
The forkhead box3 superfamily of transcription factors is defined by a DNA binding domain structurally related to the forkhead protein [2]
Heat shock transcription factor (HSF) and the stress-inducible heat shock proteins (Hsps) have dFOXO bound at their promoters
HSF is solely responsible for the Hsp70 induction in response to diethyl maleate (DEM; Fig. 5C). These results show endogenous dFOXO and HSF are responsible for activating transcription of Hsp70 in response to intracellular reactive oxygen species
Summary
Heat Shock Proteins Are Targets of dFOXO—We recently identified genomic targets of constitutively active Drosophila FOXO (dFOXOCA) by chromatin immunoprecipitation (ChIP) followed by microarray analysis [21]. The stress-inducible Hsp genes are dFOXO targets in Drosophila These results identified Hsps as a direct target of dFOXO, so we looked for potential FOXO-response elements (FREs) within the promoter regions. To better characterize whether the FOXO-dependent activity requires the identified dFOXO binding sites, we carried out a promoter deletion analysis and compared these to the fulllength Hsp reporter containing all HSEs and putative FREs. Deletion 1 removes FRE 1 and 2. The construct that contains no FREs, deletion 3, has no FOXO-dependent expression (Fig. 4D) These results indicate that the putative FRE-containing sequences that dFOXO bound in vitro are necessary for FOXO-dependent activation of the Hsp promoter. Hsp expression increases in response to oxidative stress, but this
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