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Promoter Chip-Chip Analysis in Mouse Testis Reveals Y Chromosome Occupancy by HSF2

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Promoter Chip-Chip Analysis in Mouse Testis Reveals Y Chromosome Occupancy by HSF2 Promoter ChIP-chip analysis in mouse testis reveals Y chromosome occupancy by HSF2 Malin Åkerfelt*†‡, Eva Henriksson*†‡, Asta Laiho*, Anniina Vihervaara*†, Karoliina Rautoma*†, Noora Kotaja§ and Lea Sistonen*†¶ *Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FIN-20520 Turku, Finland; †Department of Biology, Åbo Akademi University, FIN-20520 Turku, Finland; and §Department of Physiology, University of Turku, FIN-20520, Turku, Finland Edited by David C. Page, Massachusetts Institute of Technology, Cambridge, MA, and approved May 21, 2008 (received for review January 21, 2008) The mammalian Y chromosome is essential for spermatogenesis, High-resolution chromatin immunoprecipitation on microarray which is characterized by sperm cell differentiation and chromatin (ChIP-chip) screens have successfully been used for identifying condensation for acquisition of correct shape of the sperm. Dele- direct target genes for many transcription factors (10). For example, tions of the male-specific region of the mouse Y chromosome long Ϸ3% of the genomic loci were found to be targets for HSF in arm (MSYq), harboring multiple copies of a few genes, lead to Saccharomyces cerevisiae and Drosophila exposed to heat stress (11, sperm head defects and impaired fertility. Using chromatin immu- 12). In mammals, however, the existence of three differently noprecipitation on promoter microarray (ChIP-chip) on mouse expressed HSFs (HSF1, HSF2, and HSF4) requires a strategy testis, we found a striking in vivo MSYq occupancy by heat shock to investigate each HSF in a tissue-specific manner. Here, we factor 2 (HSF2), a transcription factor involved in spermatogenesis. chose to dissect the specific role for HSF2 in spermato- HSF2 was also found to regulate the transcription of MSYq resident genesis and to map the in vivo targets for HSF2, by using mouse genes, whose transcriptional regulation has been unknown. Im- testis in a promoter ChIP-chip screen. We identified a multitude of portantly, disruption of Hsf2 caused a similar phenotype as the 2/3 target genes, and analysis of their chromosomal distribution led to deletion of MSYq, i.e., altered expression of the multicopy genes an interesting discovery that the Y chromosome is predominantly and increased mild sperm head abnormalities. Consequently, ab- occupied by HSF2 in testis. Accordingly, HSF2 regulates the errant levels of chromatin packing proteins and more frequent transcription of the Y-chromosomal genes critical for sperm dif- DNA fragmentation were detected, implying that HSF2 is required ferentiation. Functional analyses of HSF2-deficient mice revealed for correct chromatin organization in the sperm. Our findings increased sperm head anomalies, showing striking similarity to define a physiological role for HSF2 in the regulation of MSYq those in MSYq deletion mutants. Moreover, sperm lacking HSF2 resident genes and the quality of sperm. displayed altered chromatin packing protein levels and more fre- quent DNA fragmentation, implying that HSF2 is required for chromatin packing ͉ heat shock factor ͉ MSYq ͉ promoter microarray ͉ correct chromatin organization during spermatogenesis. spermatogenesis Results he mammalian Y chromosome is essential for spermatogen- Global Mapping of Target Genes for HSF2 in Spermatogenesis. To Tesis and sex determination, and contains mainly heterochro- identify novel target genes for HSF2 in spermatogenesis, we matin and only a few genes (1). It is the smallest of all cross-linked chromatin from three wild-type (WT) mouse testes chromosomes and consists mostly of a male-specific region, in and sonicated it into fragments of 100–500 bp. The quality of addition to short pseudoautosomal regions (PAR), which are DNA was controlled before the immunoprecipitation and homologous to the regions of the X chromosome required for sex showed no signs of degradation [supporting information (SI) Fig. chromosome pairing (1). In the male-specific region of the S1]. The DNA amplified from the HSF2 immunoprecipitation mouse Y chromosome long arm (MSYq), a few genes exist in samples was labeled and hybridized against the total input DNA samples, on a first-generation 1.5-kb promoter tiling array from hundreds of copies (2). These genes are expressed predomi- Ϸ nantly in testis, and their multicopy nature is suggested to be a NimbleGen Systems, covering 26,000 promoters of the mouse defense mechanism against degeneration in a non-recombining genome. After hybridization and scanning, HSF2 hybridization environment (2). To date, the transcriptional regulation of the signals were divided by the input signals to provide a value for multicopy MSYq resident genes is unknown. enrichment for each oligonucleotide probe covering the pro- Heat shock factor 2 (HSF2) is a transcription factor involved moters, on the three replicate arrays. Further, the target pro- in mammalian spermatogenesis (3–6). HSF2 belongs to a tran- moters were separately ranked in the replicates according to the scription factor family, the members of which were originally average log2-ratios of all probes covering each promoter. The found to regulate the heat shock response and later also revealed complete data set is available at the Gene Expression Omnibus to orchestrate development (7). In addition to spermatogenesis, the only other developmental process where HSF2 is known to Author contributions: M.Å., E.H., A.V., N.K., and L.S. designed research; M.Å., E.H., A.V., and be active is corticogenesis (5, 6, 8). Although HSF2 exists in K.R. performed research; M.Å., E.H., and A.L. contributed new reagents/analytic tools; many tissues, it is most abundantly expressed in testis (9). During M.Å., E.H., A.L., A.V., N.K., and L.S. analyzed data; and M.Å., E.H., and L.S. wrote the paper. spermatogenesis, HSF2 is expressed in a stage-specific manner The authors declare no conflict of interest. in the nuclei of early pachytene spermatocytes and postmeioti- This article is a PNAS Direct Submission. cally in round spermatids (3, 4). Disruption of Hsf2 causes Freely available online through the PNAS open access option. reduced size of testis and epididymis, altered morphology of the Data deposition: The data reported in this paper have been deposited in the Gene seminiferous tubules displaying extensive vacuolization, and a Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE9289). low number of differentiating spermatids due to elevated apo- ‡M.Å and E.H. contributed equally to this work. ptosis at the pachytene stage (5, 6). In addition, the synaptone- ¶To whom correspondence should be addressed at: Department of Biology, Åbo Akademi Ϫ/Ϫ mal complex is disorganized in Hsf2 pachytene spermatocytes University, Tykisto¨katu 6, FIN-20520 Turku, Finland. E-mail: lea.sistonen@btk.fi. (5). However, no correlation between HSF2 and expression of This article contains supporting information online at www.pnas.org/cgi/content/full/ the classical HSF targets, Hsps, has been found (3–6), and the 0800620105/DCSupplemental. HSF2 target genes in spermatogenesis have remained obscure. © 2008 by The National Academy of Sciences of the USA 11224–11229 ͉ PNAS ͉ August, 2008 ͉ vol. 105 ͉ no. 32 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800620105 Downloaded by guest on September 24, 2021 4HcA 2FSH A Ssty2 B 1100 - Input +400 +300 +200 +100 +1 -100 -200 -300 -400 -500 -600 -700 -800 -900 -1000 -1200 NS Log2 2.7 Ssty2 0.0 Chr. Y TTCtaGCGccTCCaaGTG Sly ** * * Slx Sly Speer4a -1400 -1300 -1200 -1000 -900 -800 -700 -600 -500 -400 -300 -200 -100 +1 +100 +200 +300 +400 Hsc70 Log2 2.7 0.0 Ftmt Chr. Y GAGgaGCTaaGCAcaGAA **** Hsp25 Slx Fyn +180 +150 +120 +90 +60 +30 +1 -30 -60 -90 -120 -150 -180 -210 -240 -270 -300 -330 -360 -390 Log2 2.6 Spata2 0.0 Chr. X TTCctGGActGAGgaTTC ** * * 2FS 2 2FSH C FSH SN Speer4a S NS Input Input N Input H +600 +500 +400 +300 +200 +100 +1 -100 -200 -300 -400 -500 -600 -700 -800 -900 -1000 Testis Log2 3.2 0.0 Chr. 5 GAGcaTTCacTTC Brain *** Muscle Hsc70 Kidney -1400 -1300 -1200 -1100 -1000 -900 -800 -700 -600 -500 -400 -300 -200 -100 +1 +100 +200 +300 Log2 3.6 Ssty2 Sly Slx 0.0 Chr. 9 TTCtgGAAggTTC Testis ** * Brain Ftmt -1400 -1300 -1200 -1100 -1000 -900 -800 -700 -600 -500 -400 -300 -200 -100 +1 +100 +200 +300 +400 Muscle Log2 3.2 Kidney 0.0 Chr. 18 GAAgaGAAggGCCgtGTGacGCA Speer4a Hsc70 Ftmt **** * Fig. 1. In vivo HSF2 binding to novel target genes in testis. (A) Visualization of the HSF2-binding profile on six selected target promoters, using the SignalMap software (NimbleGen Systems): Ssty2, Sly, Slx, Speer4a, Hsc70, and Ftmt. The localization of 15 probes per promoter is indicated as bars above the promoters, determining the HSF2 enrichment. One representative promoter is displayed for the multicopy genes. A putative heat shock element (HSE) is indicated below each promoter. Asterisk indicates key nucleotides required for HSF binding; arrows indicate primers used in the ChIP assay. Log2, log2 ratio of HSF2 enrichment, indicated as difference in positions of the bar in the log2 scale; Chr., chromosome; ϩ1, transcription start site (note that the genes are transcribed in different directions). (B) Verification of the ChIP-chip screen using a standard ChIP assay of WT testis extracts. Analysis of HSF2-binding (HSF2) on the six selected promoters, in addition to three nontarget promoters for HSF2: Hsp25.1, Fyn, and Spata 2.(C) Target genes are occupied by HSF2 only in testis. ChIP analysis of HSF2-binding (HSF2) on six target promoters in WT testis, brain, muscle, and kidney. Nonspecific antibody (NS) was used as a negative control, and acetylated histone 4 antibody (AcH4) was used as an indicator of transcriptionally active promoters. Input represents 1% of the total material used in the ChIP assay. (GEO) database (http://www.ncbi.nlm.nih.gov/geo/, GSE9289). HSF2 antibody than in the original ChIP-chip screen (Fig.
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