Promoter ChIP-chip analysis in mouse testis reveals Y chromosome occupancy by HSF2

Abstract

The mammalian Y chromosome is essential for spermatogenesis, which is characterized by sperm cell differentiation and chromatin condensation for acquisition of correct shape of the sperm. Deletions of the male-specific region of the mouse Y chromosome long arm (MSYq), harboring multiple copies of a few genes, lead to sperm head defects and impaired fertility. Using chromatin immunoprecipitation on promoter microarray (ChIP-chip) on mouse testis, we found a striking in vivo MSYq occupancy by heat shock factor 2 (HSF2), a transcription factor involved in spermatogenesis. HSF2 was also found to regulate the transcription of MSYq resident genes, whose transcriptional regulation has been unknown. Importantly, disruption of Hsf2 caused a similar phenotype as the 2/3 deletion of MSYq, i.e., altered expression of the multicopy genes and increased mild sperm head abnormalities. Consequently, aberrant levels of chromatin packing proteins and more frequent DNA fragmentation were detected, implying that HSF2 is required for correct chromatin organization in the sperm. Our findings define a physiological role for HSF2 in the regulation of MSYq resident genes and the quality of sperm. chromatin packing ͉ heat shock factor ͉ MSYq ͉ promoter microarray ͉ spermatogenesis

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

“…Whereas HSF1, which is the major stressactivated regulator, binds to continuous HSEs, both HSF2 and HSF4 bind preferentially to discontinuous HSEs. 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).…”

“…Given the cell-type specificity of HSF actions (Fujimoto and Nakai, 2010) it seems likely that the effects of HSFs on HIF-1a transcription observed in human breast cancer cells may differ in other cell types and may also be affected differently by heat shock and other stress conditions in different cell types (see Mathew et al, 2001;Pirkkala et al, 2001;Chi and Karliner, 2004;Xing et al, 2005;Loison et al, 2006;Akerfelt et al, 2007Akerfelt et al, , 2008Ostling et al, 2007;Sandqvist et al, 2009;Yamamoto et al, 2009). However, notwithstanding these considerations, our data show that the effects of HSF2 and HSF4 on VEGF production in at least two other cell lines-HeLa and 293T cells-are similar to those seen in MCF-7 cells.…”

“…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.…”

Transcription and post-transcriptional regulation of spermatogenesis

“…Recently, the molecular targets of HSF2 during spermatogenesis were identified at the genome-wide level by Akerfelt et al (2008). High-resolution chromatin immunoprecipitation (ChIP)-on-chip analysis using promoter microarrays to assay HSF2-binding sites in adult mouse testes revealed that HSF2 primarily interacts with sites on the mouse Y chromosome.…”

“…This study by Akerfelt et al (2008) is a landmark paper, as it is one of very few studies to perform genome-wide analysis of transcription factor-binding sites in vivo rather than in cultured cells. It remains for future studies to determine precisely how HSF2 controls male fertility and whether it directly or indirectly controls chromatin structure.…”