Analysis of HSF4 Binding Regions Reveals Its Necessity for Gene Regulation during Development and Heat Shock Response in Mouse Lenses

Abstract

Heat shock transcription factors (HSFs) regulate gene expression in response to heat shock and in physiological conditions. In mammals, HSF1 is required for heat-mediated induction of classic heat shock genes; however, we do not know the molecular mechanisms by which HSF4 regulates gene expression or the biological consequences of its binding to chromatin. Here, we identified that HSF4 binds to various genomic regions, including the introns and distal parts of protein-coding genes in vivo in mouse lenses, and a substantial numbers of the regions were also occupied by HSF1 and HSF2. HSF4 regulated expression of some genes at a developmental stage when HSF1 and HSF2 expression decreased. Although HSF4 binding did not affect expression of many genes, it induces demethylated status of histone H3K9 on the binding regions. Unexpectedly, a lot of HSF4 targets were induced by heat shock treatment, and HSF4 is required for induction of a set of non-classic heat shock genes in response to heat shock, in part by facilitating HSF1 binding through chromatin modification. These results suggest novel mechanisms of gene regulation controlled by HSF4 in non-classic heat shock response and in lens development.

Regulation of transcription of hypoxia-inducible factor-1α (HIF-1α) by heat shock factors HSF2 and HSF4

“…Our findings are also consistent with previous evidence that different HSFs can have targets in common (Akerfelt et al, 2008;Fujimoto et al, 2008;Yamamoto et al, 2009). Whereas the DNA-binding domains of the three HSFs identified in mammalian cells are highly similar (70.5% identity between HSF1 and HSF2, 76.2% identity between HSF1 and HSF4 and 62.4% identity between HSF2 and HSF4), HSFs nevertheless show different DNA-binding preferences (Somasundaram and Bhat, 2004;Fujimoto et al, 2004Fujimoto et al, , 2008Yamamoto et al, 2009). Potentially, such preferences may have a role in determining the effects of HSF/HRE complexes in the HIF-1a promoter region.…”

“…Two alternatively spliced transcripts encoding distinct HSF4 isoforms, HSF4a and HSF4b, which possess different transcriptional activity have been described (Nakai et al, 1997;Pirkkala et al, 2001;Chi and Karliner, 2004;Min et al, 2004;Akerfelt et al, 2007;Fujimoto et al, 2004Fujimoto et al, , 2008; whereas RT-PCR results showed that both HSF4a and HSF4b mRNAs are expressed in MCF-7 cells, only the protein isoform encoded by HSF4a was detected in MCF-7 cells by western blotting. The ability of the HSF4 EST to regulate VEGF expression was shown directly, by use of an MCF-7G-derived cell clone, EK-F4 that contains the HSF4 EST inserted into the chromosome under control, in antisense orientation, of a tetracycline-repressed cytomegalovirus (CMV) promoter (Figures 1b-d).…”

“…32 P]-labeled HSE86 probe contains three perfect nGAAn pentameric units and was previously reported to bind with both HSF2 and HSF4 (Fujimoto et al, 2008). As seen in Figure 5d Heat shock factor regulation of HIF-1a transcription R Chen et al…”

“…Among these was clone C16, in which cells showed fourfold greater GFP fluorescence than the parental cell line MCF-7G, as determined by FACS analysis (Figure 1a). DNA sequencing revealed that clone C16 contains a chromosomally inserted EST (GenBank image clone 3395089) corresponding to part of the protein-coding region of heat shock factor 4 (HSF4), a transcriptional regulator previously implicated in expression of heat shock proteins (HSPs)-chaperones that assist protein folding and protect cells against proteotoxic stress (Nakai et al, 1997;Pirkkala et al, 2001;Chi and Karliner, 2004;Min et al, 2004;Fujimoto et al, 2004Fujimoto et al, , 2008Akerfelt et al, 2007). This HSF4 EST was oriented in the antisense direction relative to the lentiviral promoter.…”

“…Two alternatively spliced transcripts encoding distinct HSF2 isoforms, HSF2a and HSF2b, possessing different transcriptional activity have been described (Pirkkala et al, 2001), and RT-PCR results showed that both HSF2a and HSF2b mRNAs are expressed in MCF-7 cells (data not shown). Overproduction from a CMV promoter of either of the two HSF2 isoforms, which are similar in molecular mass and were not resolved by western blotting under the conditions employed (Figure 4c HSF4 and HSF2 bind to HSE-containing loci in the HIF-1a promoter region Earlier work has shown that HSF regulation of gene expression involves the binding of HSFs to doublestrand regions of DNA known as HSEs; HSE sequences, which are highly conserved among a broad range of eukaryotes, consist of multiple inverted repeats of the pentamer nGAAn, or of a variant of this sequence (Somasundaram and Bhat, 2004;Xing et al, 2005;Akerfelt et al, 2008;Fujimoto et al, 2008;Murphy et al, 2008). Consistent with our evidence that HSF4 and HSF2 are regulators of HIF-1a transcription, Heat shock factor regulation of HIF-1a transcription R Chen et al analysis of the 2-kb DNA region upstream from the HIF-1a transcriptional start site by the Vector NTI motif program revealed two discontinuous regions rich of 'GAA/TTC' repeats.…”