Promoter ChIP-chip analysis in mouse testis reveals Y 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 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 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 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 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 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

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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. 1B). To identify the HSF2 target population, we used R/Bioconduc- In addition, three promoters; Hsp25.1, Spata 2, and Fyn, were tor package RankProd (13) to define the significance value (P) verified as nontargets for HSF2 (Fig. 1B), based on their low for each promoter (see Materials and Methods). The data were ranking in the screen (data not shown). These results prove that filtered with P value Ͻ0.005, which resulted in identification of the screen was reliable and successfully performed. 546 target promoters for HSF2 (Table S1). In addition to To study whether the newly identified target genes were also RankProd, we used a nonarbitrary analysis method previously occupied by HSF2 in other tissues, we analyzed the HSF2 used for promoter ChIP-chip data by Squazzo et al. (14) (data binding in testis, brain, muscle, and kidney. The ChIP analyses not shown), and both methods revealed similarly ranked HSF2 showed that the target promoters were occupied by HSF2 only target gene lists. in testis (Fig. 1C), indicating that the promoter sequence alone is not sufficient to specify HSF2 binding. As supporting evidence, Validation of HSF2 Binding to Target Genes in Mouse Testis. For the same Hsp25 promoter sequence that contains a canonical verification of the target genes identified in the screen, we HSE was not an HSF2 target in testis (Fig. 1B), but has earlier selected six putative HSF2 target promoters, distributed across been shown to be bound by both HSF1 and HSF2 in mouse the ranking from 1 to 546 (Fig. 1A). Three of these genes, Ssty2, embryonic fibroblasts exposed to heat stress (17). Thus, the Sly, and Slx/Xmr (hereafter called Slx), are located on the sex ChIP-chip screen in testis made it possible to identify a multitude chromosomes, whereas Speer4a, Hsc70, and Ftmt reside on of HSF2 targets in spermatogenesis, highlighting the importance autosomal chromosomes. In Fig. 1A, the localization of the of performing a global search for target genes in the physiolog- probes is indicated on each promoter, defining the peak of ically relevant context. enrichment and thereby the putative region for HSF2 binding, in proximity to the transcription start sites. HSFs bind to heat shock The Y Chromosome Is Occupied by HSF2. A possible formation of elements (HSEs) in their target promoters, and an HSE consists HSF2-binding clusters was studied through the chromo- of inverted repeats of the NGAAN motif (15), where guanidines somal distribution of target genes by using the GeneMerge are the most conserved nucleotides (16). A potential HSE was analysis tool (18). To our surprise, a profound accumulation of indeed found at the site of HSF2 enrichment on all six promoters HSF2 targets was detected on the Y chromosome, in comparison

(Fig. 1A). The HSF2 binding to the target promoters was to the total amount of promoters per chromosome on the CELL BIOLOGY validated in a standard in vivo ChIP assay by using a different microarray (Table 1). Interestingly, 34 HSF2 target genes resid-

Åkerfelt et al. PNAS ͉ August, 2008 ͉ vol. 105 ͉ no. 32 ͉ 11225 Downloaded by guest on September 24, 2021 Table 1. The chromosomal distribution of HSF2 target genes 8 8 1v 6

A Hsf2 B rcA Hsf2 WT Chromo Population Study 66 5 Hsf2 KO -some fraction* fraction† P‡ e-score§ otno

44 4

i s

s 3

1 1,254 29 0.313003 1 22 3 erp

2 1,933 29 0.979376 1 2 xeevital 2 00 3 1,067 16 0.937303 1 8 1 4 1,300 20 0.941805 1 8 Ssty2

5 1,291 29 0.370752 1 66 eR 0 Ssty2 Sly Slx

4

6 1,182 25 0.504441 1 Gapdh 4

7 2,566 29 0.282118 1 otnoisserpxeevitaleR 22 8 1,092 14 0.983677 1 9 1,272 25 0.653106 1 00 10 1,032 12 0.991928 1 1212 Sly 11 1,652 27 0.927213 1 88 12 727 15 0.556808 1

13 915 14 0.912291 1 44 14 787 22 0.103817 1 15 830 18 0.469905 1 00

16 698 12 0.792606 1 1616 17 1,051 21 0.614983 1 Slx 1212 18 554 7 0.94583 1 19 745 12 0.856506 1 88

X 806 22 0.123984 1 44 Y 105 35 1.22E-32 2.8E-31 00 Mt 4 1 NA NA IX-XI XII-I II-VI Un 3,266 83 0.033699 0.775067 VII-VIII

NA, not applicable; Mt, mitochondrial DNA; Un, undefined chromosomal location. *Number of genes located on a certain chromosome, of the total number of genes on the array (26,129). †Number of HSF2 target genes located on a certain chromosome, of the total number of HSF2 target genes analyzed in this study (546, with a P value Ͻ0.005). ‡The probability of HSF2 occupancy on the promoters in each chromosome by random chance. I II III IV V VI VII VIII IX X XI XII §Bonferroni-corrected P value. Fig. 2. HSF2 functions as a transcriptional regulator of multicopy in spermatogenesis. (A) RT-PCR analysis of Hsf2, Ssty2, Sly, and Slx ing in the Y chromosome were multiple copies of Ssty2, Sly, and expression in the indicated stages of WT seminiferous epithelial cycle was similar to Ssty2 (LOC435023) (Table S1). The accumulation was performed. Relative quantities of mRNA were normalized to Gapdh, which was evenly expressed throughout the seminiferous epithelial cycle. A sche- evident also when the HSF2 targets were analyzed in a single matic presentation of the 12 stages (I–XII) in the mouse seminiferous epithelial copy (Table S2). The biological processes directly associated cycle is shown below. Each stage is defined by a specific collection of cell types, with the novel target genes were investigated with the DAVID which are classified by the morphology of the developing spermatids (21). (B) analysis tool (19). Target genes in a single copy were selected (P RT-PCR analysis of gene expression in whole WT (Hsf2 WT) and Hsf2 knockout value Ͻ0.0001, Table S1), including all Y-chromosomal HSF2- (Hsf2 KO) testes. Relative quantities of mRNA were normalized to Acrv1/SP-10. bound genes, and reproduction (P ϭ 0.011) was the highest All PCRs were in duplicates using samples derived from at least three biological Ϯ ranked biological process that was significantly enriched (Table repeats. Error bars denote standard deviations ( SD). The relative expression was calculated from the Hsf2 WT sample, which was arbitrarily set to 1. S3). These results strongly indicate that HSF2 regulates target genes associated with male reproduction. Ssty2, Sly, and similar to Ssty2 exist in multiple copies through- HSF2 Functions as a Transcriptional Regulator of Ssty2, Sly, and Slx in out the MSYq region (2). Sly displays substantial sequence Spermatogenesis. In mice, the cycle of the seminiferous epithe- homology to another multicopy gene on the X chromosome, Slx lium is composed of 12 stages (I–XII), and each stage contains (2, 20), which was also identified as an HSF2 target (Fig. 1B, a defined collection of cell types, which are classified by the Table S1). Accordingly, HSF2 occupancy was observed on morphology of the developing spermatids (21) (Fig. 2A). To numerous copies of the Slx promoter on the X chromosome characterize the spatiotemporal relationship between expression (Table S1). Promoter variants of each multicopy gene, Ssty2, Sly, of Hsf2 and the multicopy genes, we isolated stages IX–XI, and Slx, were defined by NimbleGen Systems, and ChIP-chip XII–I, II-VI, and VII–VIII from WT seminiferous epithelium as probes were designed for the unique promoter variants. The described by Kotaja et al. (21). Hsf2 mRNA was most abundant multiple copies of Ssty2, Sly, and Slx were differently ranked on in stages XII–I and II–VI, whereas Ssty2, Sly, and Slx mRNAs the HSF2 target list, depending on the proximity of the ChIP- were found at high levels in stages II–VI and VII–VIII (Fig. 2A). chip probes to the putative HSE (for description, see Fig. S2). These stages contain round spermatids (21), and our findings are We visualized the HSF2 binding at a chromosomal level by using in accordance with those of previous studies showing that Ssty, the SignalMap software (NimbleGen Systems) (Fig. S3). Our Sly, and Slx transcripts are present only during spermatogenesis, results demonstrate HSF2 binding to multiple copies of Ssty2, predominantly in round spermatids (2, 20, 22, 23). Importantly, Sly, and Slx, which is also supported by the ChIP assays showing the Hsf2 expression coincided with transcription of the multicopy a more intensive HSF2 enrichment on the multicopy genes than genes in stages containing round spermatids (II–VIII) (Fig. 2A). on the other targets (Fig. 1B). In addition, Hsf2 mRNA was found in earlier stages of the

11226 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800620105 Åkerfelt et al. Downloaded by guest on September 24, 2021 Hsf2 WT Hsf2 KO Hsf2 Hsf2 A A WT KO kDa B C 17 Hsf2 TNP2 WT

1212 -actin ) 32 e

viti 10 % 10

25 (stemocmreps s 8 PRM1 opfo 8

-actin 32 66 tn 25 Hsf2 KO 4

PRM2 e 4 creP 22 -actin 32 00 Hsf2 Hsf2 -tubulin 47 WT KO -actin 80 32 )%(mrepsfotn 80 B 7070 Normal 6060 Fig. 4. Altered chromatin packing protein levels and increased DNA frag- 5050 Slightly mentation in Hsf2 knockout sperm. (A) Western blot analysis of transition 4040 abnormal protein 2 (TNP2), protamine 1 (PRM1), and protamine 2 (PRM2) levels in cauda 3030 20 Grossly epididymis isolated from adult WT (Hsf2 WT) and Hsf2 knockout (Hsf2 KO)

ec 20 abnormal

r 1010 mice. The blots are representative examples of four biological repeats. Equal e

P 00 ␤ ␣ Hsf2 WT Hsf2 KO loading was assessed by -tubulin and -actin. (B) Analysis of DNA damage by comet assay. After separation of DNA fragments by electrophoresis, the sperm DNA from adult Hsf2 WT and Hsf2 KO males was stained with SYBR green. Fig. 3. HSF2-deficient mice display a significant increase in sperm head Representative examples of comets are shown with white arrows in the figure. abnormalities. (A) Analysis of hematoxylin-stained sperm smears from adult (Scale bar, 10 ␮m.) (C) The percentages of sperm positive for DNA damage WT (Hsf2 WT) and Hsf2 knockout (Hsf2 KO) males. Representative examples of after analysis by comet assay were calculated from Hsf2 WT (n ϭ 3) and Hsf2 different morphology are shown in the main figure, and a blow-up in the KO (n ϭ 3) mice. Error bars denote standard deviations (ϮSD). inset. (Scale bar, 10 ␮m.) (B) The sperm heads were classified in three catego- ries of normal, slightly abnormal, and grossly abnormal, as previously de- scribed (23). The number of sperm in each category, obtained from Hsf2 WT the chromatin condensation process, which requires replace- (n ϭ 4) and Hsf2 KO (n ϭ 4) male mice, was calculated in blind. Error bars ment of histones first with transition proteins and finally with denote standard deviations (ϮSD). protamines at the end of spermatogenesis (27). This prompted us to analyze the amounts of transition proteins and protamines incorporated in WT and Hsf2Ϫ/Ϫ sperm. We observed pro- epithelial cycle (XII–I) (Fig. 2A), which probably is a conse- foundly more transition protein 2 (TNP2), but substantially less quence of its expression in the pachytene spermatocytes (3, 4). protamine 1 and slightly less protamine 2 in the Hsf2 knockout Next, using WT and Hsf2Ϫ/Ϫ testes, we investigated whether epididymis (Fig. 4A). The difference is not due to sloughing of HSF2 is required for the transcription of the multicopy genes. Ϫ/Ϫ immature germ cells from the seminiferous epithelium of Due to enhanced apoptosis, the Hsf2 testes contain fewer Ϫ Ϫ Hsf2 / mice, as demonstrated by the morphological and cyto- spermatids (5) and the stage patterns are more diffuse than in logical analyses (Fig. S4). Thus, our data imply that the replace- the WT; therefore the mRNA expression was studied in whole ment of transition proteins with protamines is disturbed in the testis instead of stages and normalized to round spermatid- Hsf2 knockout mice. The changes in chromatin packing proteins specific Acrv1/SP-10 (24). Interestingly, Ssty2 and Sly mRNAs levels are probably an indirect effect originating from earlier were significantly reduced, whereas Slx mRNA was increased in steps of spermatogenesis, given that neither TNP2 nor prota- the Hsf2 knockout (Fig. 2B). Taken together, our results imply mines were direct targets for HSF2 (Table S1). Even small that HSF2 is a stage-specific transcriptional regulator of the changes in chromatin packing proteins have vital consequences multicopy genes in spermatogenesis. and compromise the chromatin remodeling (28), by affecting the DNA status. Therefore, we assessed the DNA fragmentation in ؊/؊ Hsf2 Mice Display Increased Sperm Head Abnormalities, Impaired Hsf2Ϫ/Ϫ sperm by performing a comet assay (29). More extensive Protamine Expression, and Prominent DNA Fragmentation. In mice, a DNA fragmentation was observed in the Hsf2Ϫ/Ϫ than in WT RIII 2/3 deletion of the MSYq region (XY qdel) causes lowered sperm (Fig. 4B). To quantify the amount of sperm with DNA transcript levels of Ssty2 and Sly in testis, but increases Slx levels damage, we calculated the percentage of positive sperm comets. (2, 25). The changes in expression of the multicopy genes are A two-fold increase in comets was found in Hsf2Ϫ/Ϫ sperm Ϫ/Ϫ strikingly similar to those detected in the Hsf2 testis (Fig. 2B). compared with WT sperm (Fig. 4C). The analyses of the Hsf2 RIII Moreover, XY qdel mutant mice display abnormalities in the knockout phenotype, i.e., sperm head morphology defect to- sperm heads (23, 25, 26), which has been interpreted to reflect gether with altered chromatin packing protein levels and in- incorrect chromatin organization in the nucleus (2, 25). We Ϫ Ϫ creased DNA damage, indicate that HSF2 is critical for sperm examined the Hsf2 / sperm morphology by assessing the degree differentiation and for correct packing of the chromatin in the of sperm abnormalities in hematoxylin-stained sperm smears male germ cells. from WT and Hsf2Ϫ/Ϫ males. Interestingly, mild but consistent defects were observed in Hsf2Ϫ/Ϫ sperm heads, manifested by Discussion flattened heads with a less hydrodynamic structure (Fig. 3A). To Our ChIP-chip screen on mouse testis revealed that a high define the severity of the head distortion, we calculated the number of the HSF2 targets are located on the Y chromosome percentage of normal, slightly abnormal and grossly abnormal (Table 1) and consist of the multicopy MSYq resident genes sperm. A marked increase in slightly abnormal heads was found Ssty2, Sly, and similar to Ssty2 (Fig. S3A and Table S1). in the Hsf2Ϫ/Ϫ sperm, in comparison to the WT, whereas gross Previous transcriptome analyses of mouse MSYq deletion sperm head defects occurred equally in both WT and knockout mutants have identified 23 down-regulated genes, of which 15 males (Fig. 3B). Our results show that in addition to the similar were recognized as copies of Ssty2 and 5 as copies of Sly (2). alterations in Ssty2, Sly, and Slx expression, the Hsf2Ϫ/Ϫ head Similarly, both Ssty2 and Sly mRNA levels were lowered in the abnormalities were equivalent to the flattened sperm head Hsf2Ϫ/Ϫ testis (Fig. 2B). We conclude that HSF2 directly binds

phenotype of the XYRIIIqdel mutant (23, 25, 26). and regulates the transcription of the Y-chromosomal multi- CELL BIOLOGY The Hsf2Ϫ/Ϫ sperm head anomaly (Fig. 3A) suggests defects in copy genes Ssty2 and Sly (Figs. 1B and 2B) in addition to the

Åkerfelt et al. PNAS ͉ August, 2008 ͉ vol. 105 ͉ no. 32 ͉ 11227 Downloaded by guest on September 24, 2021 sperm, we propose that the HSF2-mediated transcriptional regulation of MSYq multicopy genes could be crucial for proper chromatin organization and sperm quality. Our results expand the understanding of spermatogenetic anomalies and may open avenues for future research on male fertility. Materials and Methods Mice. Male hybrid mice of the B6129SF2/J strain were used in the ChIP-chip screen. Hsf2 knockout mice were obtained by matings of heterozygous mice Fig. 5. A schematic presentation of HSF2 occupancy on the male-specific Y that have been described earlier (5), and were maintained in a C57BL/6N chromosome long arm (MSYq). The majority of the Y chromosome genes are background. The pathogen-free mice were housed under controlled environ- located on the short arm (Yp) and were not found as HSF2 targets, whereas the mental conditions and fed with complete pellet chow and allowed tap water. MSYq mostly contains heterochromatin and repetitive sequences. All well The mice were killed by CO2 asphyxiation. All mice were handled in accordance annotated Y-chromosomal genes included in the ChIP-chip array are indicated with the institutional animal care policies of the Åbo Akademi University in the figure. Note that the chromosome length and the number of HSF2 (Turku, Finland). Adult (60–80 days old) mice were used for isolation of testes. molecules are only illustrative. PAR, pseudoautosomal region. ChIP. Testes were isolated and lysed in 4 ml of buffer, and the ChIP assay was performed as earlier described (8). Antibodies are described in SI Materials and X-chromosomal multicopy relative Slx (Figs. 1B and 2B). Methods. Although Sly is down-regulated in Hsf2Ϫ/Ϫ and XYRIIIqdel mutant mice, its X-linked homologue Slx is up-regulated in DNA Amplification for ChIP Experiments. PCR analysis was performed on 1/10 of each ChIP sample using puRe Taq Ready-to-Go PCR Beads (GE Healthcare). both mouse models (Fig. 2B, ref. 25). However, the mechanism For ChIP primer sequences see SI Materials and Methods. by which Slx transcription is affected in these mice might be different. Importantly, the expression of Hsf2 mRNA seems to DNA Amplification and Microarray Hybridization for ChIP-Chip Experiments. coincide with transcripts of the multicopy genes in round DNA amplification of material obtained from three biological replicates for spermatids (Fig. 2A) (2–4, 20, 22, 23), where the chromatin the microarray hybridizations was performed by using a protocol from undergoes a substantial remodeling process (27). The MSYq NimbleGen Systems. Whole ChIP samples and 20 ng of the input samples resident genes have been proposed to have a role in chromatin were used for the ligation-mediated PCR (LM-PCR). The DNA was blunted remodeling (2, 23), indicating that HSF2 could, through direct by using dNTP mix (Promega) and T4 DNA polymerase (New England regulation of Ssty2 and/or Sly, be involved in chromatin Biolabs), purified and dissolved in water. Purified DNA was ligated by using T4 DNA ligase (New England Biolabs) and annealed with linkers made from remodeling during spermatogenesis. HPLC-purified oligonucleotides: oligo 1, 5Ј-GCG GTG ACC GGG AGA TCT Deletions in the MSYq region have emerged as the most GAA TTC-3Ј and oligo 2, 5Ј-GAA TTC AGA TC-3Ј. DNA was purified again and common genetic cause for spermatogenic failures in the dissolved in water. LM-PCR was performed by using Taq DNA polymerase population worldwide, resulting in oligo- or azoospermia (30). In (New England Biolabs) and Pfu DNA polymerase (Stratagene). An aliquot of mice, deficiency in MSYq transcripts deriving from mouse- the final DNA was separated on an agarose gel for verification of the DNA specific multicopy genes causes abnormalities in the sperm fragment sizes. The experimental HSF2 amplicons were labeled with Cy5 heads, and MSYq deletion mutants display a range of teratozo- dye, and the total input amplicons were labeled with Cy3 dye (including ospermia and infertility phenotypes with severities correspond- one dye-swap) and then cohybridized to high-density oligonucleotide tiling arrays. The HSF2 ChIP signal was compared with control input signal ing to the extent of the deletion (23, 25, 26, 31–33). The increase and the data were extracted according to standard operating procedures in flat sperm heads is specific to MSYq deletions in mice, and by NimbleGen Systems (www.nimblegen.com). even minor changes in the sperm head structure can affect sperm motility and thereby fertility (26, 32, 33). Accordingly, the mild ChIP-Chip Data Analysis. The two-channel raw data were normalized between Ϫ/Ϫ but consistent head defects found in the Hsf2 sperm (Fig. 3) channels with the Lowess normalization method, and ChIP-to-input log2- could have severe consequences, although some sperm retain the ratios were produced separately from all three replicates. The target promot- fertilization capacity. The nuclear status of sperm cells is deter- ers were separately ranked in the three replicates according to the average mined by two major events that occur at the final stages of log2 ratios of all probes covering each promoter. The log2 ratios in the replicates showed positive correlation, resulting in Pearson’s correlation val- spermatogenesis: replacement of transition proteins with pro- ues between 0.34 and 0.41. The R/Bioconductor package RankProd (13) was tamines and acquisition of the correct sperm head shape. It used for determining the reliability of the bound promoters. RankProd pro- would be important to decipher whether the MSYq multicopy vides an average log2 ratio and a P value for each promoter from the sepa- genes are involved in the regulation of the replacement process. rately ranked promoters of the three replicates (Table S1). The data were The molecular and morphological changes are prerequisite for filtered with P value Ͻ0.005, which resulted in a list of 546 HSF2 bound efficient packaging of the sperm chromatin and influence the promoters (Table S1). chromatin stability, which have direct effects on fertility (28, 34). Ϫ Ϫ Altered chromatin packing protein levels were observed in the Quantitative Real-Time RT-PCR. Whole WT and Hsf2 / testes or stages IX–XI, Hsf2Ϫ/Ϫ epididymes, together with increased sperm fragmenta- XII–I, II—VI, and VII–VIII of WT seminiferous epithelial cycle were isolated as described by Kotaja et al. (21). The RT-PCR reactions were prepared and run as tion (Fig. 4), implying impaired sperm quality. Protamine defi- earlier described (17). Relative quantities of the target gene mRNAs were ciency in mature sperm from both mice and has been normalized against Acrv1/SP-10 or Gapdh, and the fold induction from WT reported as a manifestation of DNA damage and fertility samples was calculated. All reactions were in duplicates using samples derived problems, as the protamine structure protects the genetic ma- from at least three biological repeats. For primer and probe sequences, see SI terial from physical and chemical damage (35, 36). Materials and Methods. In this study, we present a promoter ChIP-chip screen focusing on the HSF2 target genes in a specific mammalian tissue, i.e., Analysis of Sperm Head Morphology and DNA Fragmentation. Adult WT and Ϫ/Ϫ mouse testis. This approach revealed a striking HSF2 occupancy Hsf2 male mice were killed, the caudae epididymes were isolated, and the on multicopy genes within the MSYq region (Fig. 5). We sperm was released into PBS by using small surgical scissors. Diluted sperm smears of Hsf2Ϫ/Ϫ (n ϭ 4) and WT (n ϭ 4) mice were spread onto microscope discovered a physiological role for HSF2 in spermatogenesis, as glass slides to dry. The dried slides were fixed in 4% paraformaldehyde, our results provide evidence for transcriptional regulation of the followed by hematoxylin staining. The glass slides were coded and random- MSYq resident genes. Based on the accumulation of HSF2 on ized, and 300–500 sperm heads from each slide were analyzed by using light Ϫ Ϫ the MSYq and the phenotypic characterization of Hsf2 / microscopy, in blind by three persons. The sperm heads were classified into

11228 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800620105 Åkerfelt et al. Downloaded by guest on September 24, 2021 three categories: normal, slightly abnormal, and grossly abnormal, as previ- smears of Hsf2Ϫ/Ϫ (n ϭ 3) and WT (n ϭ 3) mice were used. DNA from 300–500 ously described by Toure´ et al. (23). sperm from each slide was analyzed, and the percentage of positive sperm comets was calculated. Western Blot Analysis. Caudae epididymes from WT and Hsf2Ϫ/Ϫ males were lysed in 3ϫ Laemmli buffer followed by boiling, and subjected to SDS/PAGE ACKNOWLEDGMENTS. We thank Vale´rie Mezger (Ecole Normale Supe´rieure, followed by transfer to nitrocellulose membrane (Protran nitrocellulose, CNRS, Paris, France) for the Hsf2 knockout mice, the Finnish DNA Microarray Schleicher & Schuell). Proteins from four biological repeats were analyzed. Centre and the Cell Imaging Core at Turku Centre for Biotechnology for expert Antibodies are described in SI Materials and Methods. The blots were devel- technical advice, and Jukka Westermarck and all members of our laboratory oped with an enhanced chemiluminescence method (ECL kit, Amersham for stimulating discussions on the manuscript. This study was financially Biosciences). supported by The Academy of Finland, The Sigrid Juse´lius Foundation, The Finnish Cancer Organizations, and The Finnish Life and Pension Insurance Analysis of Sperm DNA Fragmentation. The single-cell gel electrophoresis, Companies, and Åbo Akademi University (L.S.). M.Å. and E.H. were supported called ‘‘comet assay,’’ was previously described by Sakkas et al. (29). Sperm was by the Magnus Ehrnrooth Foundation, and M.Å. by the Turku Graduate School isolated as for sperm analysis of the head morphology, and diluted sperm of Biomedical Sciences.

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