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Heat Shock Transcription Factor 1 Localizes to Sex Chromatin during Meiotic Repression*

  • Malin Åkerfelt
    Footnotes
    Affiliations
    From the Department of Biosciences, Åbo Akademi University, FI-20521 Turku, Finland,

    the Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20521 Turku, Finland, and
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  • Anniina Vihervaara
    Footnotes
    Affiliations
    From the Department of Biosciences, Åbo Akademi University, FI-20521 Turku, Finland,

    the Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20521 Turku, Finland, and
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  • Asta Laiho
    Affiliations
    the Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20521 Turku, Finland, and
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  • Annie Conter
    Affiliations
    the Université Toulouse 3, UMR 5547, Centre de Biologie du Développement, Université Paul Sabatier, 31062 Toulouse, France
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  • Elisabeth S. Christians
    Affiliations
    the Université Toulouse 3, UMR 5547, Centre de Biologie du Développement, Université Paul Sabatier, 31062 Toulouse, France
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  • Lea Sistonen
    Correspondence
    To whom correspondence should be addressed: Dept. of Biosciences, Åbo Akademi University, P.O. Box 123, FI-20521 Turku, Finland. Tel.: 358-2-215-3311; Fax: 358-2-333-8000;
    Footnotes
    Affiliations
    From the Department of Biosciences, Åbo Akademi University, FI-20521 Turku, Finland,

    the Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20521 Turku, Finland, and
    Search for articles by this author
  • Eva Henriksson
    Footnotes
    Affiliations
    From the Department of Biosciences, Åbo Akademi University, FI-20521 Turku, Finland,

    the Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20521 Turku, Finland, and
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  • Author Footnotes
    * This work was supported by The Academy of Finland, The Sigrid Jusélius Foundation, The Finnish Cancer Organizations, and Åbo Akademi University (to L. S.), The Magnus Ehrnrooth Foundation and Foundation of Åbo Akademi Research Institute (to M. Å., A. V., and E. H.), and by the Turku Graduate School of Biomedical Sciences (TuBS) (to M. Å. and A. V.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S3 and Figs. S1–S3.
    1 Both authors contributed equally to this article.
    2 Both authors contributed equally to this article.
Open AccessPublished:August 27, 2010DOI:https://doi.org/10.1074/jbc.M110.157552
      Heat shock factor 1 (HSF1) is an important transcription factor in cellular stress responses, cancer, aging, and developmental processes including gametogenesis. Disruption of Hsf1, together with another HSF family member, Hsf2, causes male sterility and complete lack of mature sperm in mice, but the specific role of HSF1 in spermatogenesis has remained unclear. Here, we show that HSF1 is transiently expressed in meiotic spermatocytes and haploid round spermatids in mouse testis. The Hsf1−/− male mice displayed regions of seminiferous tubules containing only spermatogonia and increased morphological abnormalities in sperm heads. In search for HSF1 target genes, we identified 742 putative promoters in mouse testis. Among them, the sex chromosomal multicopy genes that are expressed in postmeiotic cells were occupied by HSF1. Given that the sex chromatin mostly is repressed during and after meiosis, it is remarkable that HSF1 directly regulates the transcription of sex-linked multicopy genes during postmeiotic repression. In addition, our results show that HSF1 localizes to the sex body prior to the meiotic divisions and to the sex chromocenter after completed meiosis. To the best of our knowledge, HSF1 is the first known transcription factor found at the repressed sex chromatin during meiosis.

      Introduction

      The mammalian sex chromosomes are highly heteromorphic; the X chromosome is large and gene-rich in contrast to the small, heterochromatic, and degenerate Y chromosome (
      • Ellis P.J.
      • Affara N.A.
      ). Thus, the sex chromosomes constitute a challenge to the mechanisms that ensure accurate segregation of autosomes during meiosis (
      • Hoyer-Fender S.
      ,
      • Handel M.A.
      ,
      • Turner J.M.
      ). To deal with incomplete synapsis of the sex chromosomes, the sex chromosomes are secluded into a subnuclear compartment called the sex body (
      • Solari A.J.
      ). This subnuclear domain separates the sex chromatin from the rest of the chromatin during meiosis, and it is thought that sex body formation could mask the incompletely synapsed X and Y chromosomes from meiotic surveillance mechanisms (
      • Hoyer-Fender S.
      ,
      • Handel M.A.
      ,
      • Turner J.M.
      ). The sex body is devoid of RNA synthesis, and it contains a unique repertoire of proteins and modified histone variants, which retain the meiotic sex chromosome inactivation (MSCI)
      The abbreviations used are: MSCI
      meiotic sex chromosome inactivation
      HSF
      heat shock factor
      F
      forward
      R
      reverse
      Q
      dark quencher dye
      KO
      knock-out
      ChIP-chip
      chromatin immunoprecipitation on promoter microarray analysis.
      (
      • Hoyer-Fender S.
      ,
      • Handel M.A.
      ,
      • Turner J.M.
      ). Proteins that have been shown to localize to the sex body belong predominantly to various categories of chromatin proteins, including modified histone variants and proteins associated with DNA damage repair (
      • Hoyer-Fender S.
      ,
      • Handel M.A.
      ,
      • Turner J.M.
      ). For example, the phosphorylated form of the histone variant H2AX (γH2AX) is strongly associated with the sex body as it initiates heterochromatinization of the sex chromosomes (
      • Mahadevaiah S.K.
      • Turner J.M.
      • Baudat F.
      • Rogakou E.P.
      • de Boer P.
      • Blanco-Rodríguez J.
      • Jasin M.
      • Keeney S.
      • Bonner W.M.
      • Burgoyne P.S.
      ). Due to MSCI, the X and Y chromosomes are transcriptionally silenced during meiosis at the pachytene stage of spermatogenesis (
      • Turner J.M.
      ). Most sex-linked genes remain repressed during meiosis and, throughout round spermatid development, as several repressive chromatin marks still are present (
      • Greaves I.K.
      • Rangasamy D.
      • Devoy M.
      • Marshall Graves J.A.
      • Tremethick D.J.
      ,
      • Namekawa S.H.
      • Park P.J.
      • Zhang L.F.
      • Shima J.E.
      • McCarrey J.R.
      • Griswold M.D.
      • Lee J.T.
      ,
      • Turner J.M.
      • Mahadevaiah S.K.
      • Ellis P.J.
      • Mitchell M.J.
      • Burgoyne P.S.
      ). The postmeiotic sex chromosome repression might be a direct effect of the MSCI (
      • Turner J.M.
      ).
      As a result of no recombination, the Y chromosome ensures its own survival by gene additions from other chromosomes and series of massive inverted repeats, termed palindromes (
      • Ellis P.J.
      • Affara N.A.
      ,
      • Turner J.M.
      ). The palindromes give the Y chromosome a possibility of repairing mutations via intrapalindrome, arm-to-arm recombination (
      • Ellis P.J.
      • Affara N.A.
      ,
      • Turner J.M.
      ,
      • Lange J.
      • Skaletsky H.
      • van Daalen S.K.
      • Embry S.L.
      • Korver C.M.
      • Brown L.G.
      • Oates R.D.
      • Silber S.
      • Repping S.
      • Page D.C.
      ). Repetitive gene families have been found in multiple copies in the mouse Y chromosome: Sly and Ssty1/2 (
      • Conway S.J.
      • Mahadevaiah S.K.
      • Darling S.M.
      • Capel B.
      • Rattigan A.M.
      • Burgoyne P.S.
      ,
      • Touré A.
      • Clemente E.J.
      • Ellis P.
      • Mahadevaiah S.K.
      • Ojarikre O.A.
      • Ball P.A.
      • Reynard L.
      • Loveland K.L.
      • Burgoyne P.S.
      • Affara N.A.
      ,
      • Touré A.
      • Grigoriev V.
      • Mahadevaiah S.K.
      • Rattigan A.
      • Ojarikre O.A.
      • Burgoyne P.S.
      ). Gene duplications give rise to a massive expansion in the so far unknown copy number of genes residing in the repetitive male-specific long arm of the mouse Y chromosome (MSYq) (
      • Ellis P.J.
      • Ferguson L.
      • Clemente E.J.
      • Affara N.A.
      ). The X chromosome gene content has been primarily based on the analysis of single-copy genes, but large ampliconic regions also reside in the X chromosome (
      • Mueller J.L.
      • Mahadevaiah S.K.
      • Park P.J.
      • Warburton P.E.
      • Page D.C.
      • Turner J.M.
      ). These regions contain multicopy gene families, which have not been fully characterized. Nevertheless, 33 X-chromosomal multicopy gene families have been identified in the mouse (
      • Mueller J.L.
      • Mahadevaiah S.K.
      • Park P.J.
      • Warburton P.E.
      • Page D.C.
      • Turner J.M.
      ). Some of these genes, such as Slx, have a Y-chromosomal paralogue, Sly (
      • Touré A.
      • Clemente E.J.
      • Ellis P.
      • Mahadevaiah S.K.
      • Ojarikre O.A.
      • Ball P.A.
      • Reynard L.
      • Loveland K.L.
      • Burgoyne P.S.
      • Affara N.A.
      ,
      • Reynard L.N.
      • Turner J.M.
      • Cocquet J.
      • Mahadevaiah S.K.
      • Touré A.
      • Höög C.
      • Burgoyne P.S.
      ). Interestingly, the sex chromosomal multicopy genes are expressed at high levels, predominantly in postmeiotic round spermatids (
      • Touré A.
      • Clemente E.J.
      • Ellis P.
      • Mahadevaiah S.K.
      • Ojarikre O.A.
      • Ball P.A.
      • Reynard L.
      • Loveland K.L.
      • Burgoyne P.S.
      • Affara N.A.
      ,
      • Mueller J.L.
      • Mahadevaiah S.K.
      • Park P.J.
      • Warburton P.E.
      • Page D.C.
      • Turner J.M.
      ). The mechanism by which these genes escape the postmeiotic sex chromosome repression has, however, remained obscure.
      HSF1 belongs to a family of heat shock transcription factors (HSFs) and is the principal stress-responsive regulator in mammals. HSF1 protects cells from proteotoxic stress through induction of heat shock genes encoding heat shock proteins (Hsps) (
      • Lindquist S.
      • Craig E.A.
      ). In addition to heat shock response, HSF1 is important in cancer, aging, and developmental processes like gametogenesis (
      • Khaleque M.A.
      • Bharti A.
      • Sawyer D.
      • Gong J.
      • Benjamin I.J.
      • Stevenson M.A.
      • Calderwood S.K.
      ,
      • Dai C.
      • Whitesell L.
      • Rogers A.B.
      • Lindquist S.
      ,
      • Hsu A.L.
      • Murphy C.T.
      • Kenyon C.
      ,
      • Morley J.F.
      • Morimoto R.I.
      ,
      • Westerheide S.D.
      • Anckar J.
      • Stevens Jr., S.M.
      • Sistonen L.
      • Morimoto R.I.
      ,
      • Nakai A.
      • Suzuki M.
      • Tanabe M.
      ,
      • Izu H.
      • Inouye S.
      • Fujimoto M.
      • Shiraishi K.
      • Naito K.
      • Nakai A.
      ,
      • Wang G.
      • Ying Z.
      • Jin X.
      • Tu N.
      • Zhang Y.
      • Phillips M.
      • Moskophidis D.
      • Mivechi N.F.
      ,
      • Salmand P.A.
      • Jungas T.
      • Fernandez M.
      • Conter A.
      • Christians E.S.
      ,
      • Christians E.
      • Davis A.A.
      • Thomas S.D.
      • Benjamin I.J.
      ,
      • Metchat A.
      • Åkerfelt M.
      • Bierkamp C.
      • Delsinne V.
      • Sistonen L.
      • Alexandre H.
      • Christians E.S.
      ,
      • Bierkamp C.
      • Luxey M.
      • Metchat A.
      • Audouard C.
      • Dumollard R.
      • Christians E.
      ). Mouse embryos whose mothers lack Hsf1 do not develop beyond the zygotic stage, causing female infertility, and HSF1 is thereby a maternal factor (
      • Christians E.
      • Davis A.A.
      • Thomas S.D.
      • Benjamin I.J.
      ). In males, a constitutively active form of HSF1 causes a severe disruption of spermatogenesis and death of pachytene spermatocytes (
      • Nakai A.
      • Suzuki M.
      • Tanabe M.
      ), whereas Hsf1−/− males are fertile but produce less sperm and exhibit an increase in disorganized or missing layers of germ cells in the seminiferous tubules (
      • Salmand P.A.
      • Jungas T.
      • Fernandez M.
      • Conter A.
      • Christians E.S.
      ). Interestingly, disrupting Hsf1 together with another family member Hsf2 causes a clearly potentiated phenotype associated with male infertility and a complete lack of mature spermatozoa, implying that both factors are required for normal spermatogenesis (
      • Wang G.
      • Ying Z.
      • Jin X.
      • Tu N.
      • Zhang Y.
      • Phillips M.
      • Moskophidis D.
      • Mivechi N.F.
      ). Together, these findings suggest that the activity of HSF1 is tightly intertwined with HSF2 during spermatogenesis, but the specific function of HSF1 in testis is unknown. Intriguingly, there is no correlation between HSF1 and induction of Hsps in male germ cells, highlighting the need to elucidate the HSF1 target genes during sperm maturation.
      In this study, we show that the expression of HSF1 was restricted to spermatocytes and round spermatids and that the Hsf1−/− mice displayed dispersed clusters of seminiferous tubules lacking most cell types. Utilizing a chromatin immunoprecipitation on promoter microarray analysis (ChIP-chip), we discovered 742 putative target genes for HSF1 in mouse testis. HSF1 was found to occupy sex chromosomal multicopy genes and regulate their transcription in round spermatids, where the sex chromatin mostly is repressed. Interestingly, HSF1 was localized to the sex chromatin both prior to and after the meiotic divisions in a repressed chromatin environment.

      DISCUSSION

      HSF1 is best known as the principal regulator of the heat shock response. In addition, HSF1 is a developmental factor, but its function in testis is not demonstrated conclusively. To establish the impact of HSF1 on spermatogenesis, we examined its expression and knock-out phenotype and searched for its direct target genes. The expression analyses revealed that HSF1 is localized specifically in spermatocytes and round spermatids. These results are well in line with HSF1 expression studies previously conducted with rat testis (
      • Alastalo T.P.
      • Lönnström M.
      • Leppä S.
      • Kaarniranta K.
      • Pelto-Huikko M.
      • Sistonen L.
      • Parvinen M.
      ). Moreover, a novel transient expression pattern of HSF1 was detected (Fig. 1). The HSF1 expression observed in pachytene spermatocytes prior to the meiotic divisions, in cells undergoing meiotic divisions and right after the completion of meiosis in haploid spermatids, strongly indicates that HSF1 plays a role in meiotic cell divisions and early spermatid differentiation.
      In spermatogenesis, ∼75% of the germ cells are estimated to undergo apoptotic cell death in the testis (
      • Print C.G.
      • Loveland K.L.
      ). In addition to spontaneous cell death, the developing germ cells are highly susceptible to stress (
      • Crew F.A.
      ,
      • Moore C.R.
      ). Because the process of spermatogenesis is sensitive to high temperatures, quality control mechanisms are important to eliminate injured or abnormal cells in spermatogenesis. Of all cell types in spermatogenesis, the pachytene spermatocytes are most vulnerable to elevated temperatures (
      • Almon E.
      • Goldfinger N.
      • Kapon A.
      • Schwartz D.
      • Levine A.J.
      • Rotter V.
      ,
      • Schwartz D.
      • Goldfinger N.
      • Rotter V.
      ). We found that HSF1 was expressed in discrete loci of these sensitive pachytene spermatocytes under physiological conditions (Fig. 1B, panel g). Interestingly, constitutively active HSF1 causes apoptosis of pachytene spermatocytes, and the heat-inducible apoptosis of pachytene spermatocytes is markedly inhibited in Hsf1−/− testes (
      • Nakai A.
      • Suzuki M.
      • Tanabe M.
      ,
      • Izu H.
      • Inouye S.
      • Fujimoto M.
      • Shiraishi K.
      • Naito K.
      • Nakai A.
      ). Although HSF1 is activated, the Hsps are not induced in the spermatocytes in response to heat stress. These results indicate that HSF1 promotes apoptotic cell death of pachytene spermatocytes exposed to thermal stress, thereby protecting the organism from abnormal development in the next generation (
      • Izu H.
      • Inouye S.
      • Fujimoto M.
      • Shiraishi K.
      • Naito K.
      • Nakai A.
      ).
      Recently, it was demonstrated that HSF1 and HSF2 can form heterotrimers upon stress (
      • Sandqvist A.
      • Björk J.K.
      • Åkerfelt M.
      • Chitikova Z.
      • Grichine A.
      • Vourc'h C.
      • Jolly C.
      • Salminen T.A.
      • Nymalm Y.
      • Sistonen L.
      ), which could provide an efficient mechanism to integrate the trans-activating capabilities of these factors. Moreover, HSF1 has been found to interact with HSF2 in mouse testis (
      • Sandqvist A.
      • Björk J.K.
      • Åkerfelt M.
      • Chitikova Z.
      • Grichine A.
      • Vourc'h C.
      • Jolly C.
      • Salminen T.A.
      • Nymalm Y.
      • Sistonen L.
      ). These findings, combined with our new results on their common target genes (supplemental Table S3), suggest that HSF1 and HSF2 can form heterotrimers in testis and thereby facilitate transcriptional fine-tuning of a subset of target genes. The functional relationship between HSF1 and HSF2 in spermatogenesis is intriguing, and several obvious questions, e.g. the stoichiometry in a possible heterocomplex in testis, remain to be answered. Given the slightly different DNA-binding preferences of HSF1 and HSF2 (
      • Kroeger P.E.
      • Morimoto R.I.
      ,
      • Manuel M.
      • Rallu M.
      • Loones M.T.
      • Zimarino V.
      • Mezger V.
      • Morange M.
      ), the composition of heat shock elements on the target promoters could direct the formation of a specific heterocomplex.
      In contrast to HSF1, the impact of HSF2 on spermatogenesis has earlier been examined. Hsf2−/− males display reduced size of testis, disruption of spermatogenesis at the pachytene stage, lowered number of germ cells, and increased sperm head abnormalities (
      • Kallio M.
      • Chang Y.
      • Manuel M.
      • Alastalo T.P.
      • Rallu M.
      • Gitton Y.
      • Pirkkala L.
      • Loones M.T.
      • Paslaru L.
      • Larney S.
      • Hiard S.
      • Morange M.
      • Sistonen L.
      • Mezger V.
      ,
      • Åkerfelt M.
      • Henriksson E.
      • Laiho A.
      • Vihervaara A.
      • Rautoma K.
      • Kotaja N.
      • Sistonen L.
      ,
      • Wang G.
      • Zhang J.
      • Moskophidis D.
      • Mivechi N.F.
      ). Inactivation of both Hsf1 and Hsf2 results in a more severe phenotype, manifested by arrested spermatogenesis and complete sterility (
      • Wang G.
      • Ying Z.
      • Jin X.
      • Tu N.
      • Zhang Y.
      • Phillips M.
      • Moskophidis D.
      • Mivechi N.F.
      ). This phenotype proposes that both HSFs are essential for male fertility. The Hsf1−/− testis phenotype was clearly different from that of Hsf2−/− (
      • Kallio M.
      • Chang Y.
      • Manuel M.
      • Alastalo T.P.
      • Rallu M.
      • Gitton Y.
      • Pirkkala L.
      • Loones M.T.
      • Paslaru L.
      • Larney S.
      • Hiard S.
      • Morange M.
      • Sistonen L.
      • Mezger V.
      ,
      • Wang G.
      • Zhang J.
      • Moskophidis D.
      • Mivechi N.F.
      ) because long pale zones of seminiferous tubules and regions lacking spermatocytes and spermatids were found in the Hsf1−/− testis (Fig. 2). In contrast to the distinct testis phenotype, mature sperm lacking HSF1 displayed close resemblance to Hsf2−/− sperm as abnormal head morphology and defects in the replacement of chromatin packing proteins were detected in sperm from either Hsf1−/− or Hsf2−/− male mice (
      • Åkerfelt M.
      • Henriksson E.
      • Laiho A.
      • Vihervaara A.
      • Rautoma K.
      • Kotaja N.
      • Sistonen L.
      ). The phenotypical analyses indicate that HSF1 has unique and overlapping functions with HSF2 in spermatogenesis. Our results also revealed that HSF1 and HSF2 have shared target promoters in testis (supplemental Table S3) and that both HSFs are required for the transcriptional regulation of sex chromosomal multicopy genes (Fig. 3) (
      • Åkerfelt M.
      • Henriksson E.
      • Laiho A.
      • Vihervaara A.
      • Rautoma K.
      • Kotaja N.
      • Sistonen L.
      ). It has been suggested that HSF1 requires cooperation with HSF2 in development (
      • Sandqvist A.
      • Björk J.K.
      • Åkerfelt M.
      • Chitikova Z.
      • Grichine A.
      • Vourc'h C.
      • Jolly C.
      • Salminen T.A.
      • Nymalm Y.
      • Sistonen L.
      ) and that the synergistic action of both HSFs is crucial for sperm production (
      • Wang G.
      • Ying Z.
      • Jin X.
      • Tu N.
      • Zhang Y.
      • Phillips M.
      • Moskophidis D.
      • Mivechi N.F.
      ). It is albeit possible that the different HSFs could to certain extent compensate each other.
      We discovered that the transcription of sex chromosomal multicopy genes is regulated by both HSF1 and HSF2, although they seem to have opposing actions on certain promoters (Fig. 3) (
      • Åkerfelt M.
      • Henriksson E.
      • Laiho A.
      • Vihervaara A.
      • Rautoma K.
      • Kotaja N.
      • Sistonen L.
      ), leading to either activation or repression of the target genes. Due to MSCI, the X and Y chromosomes are transcriptionally silenced in meiosis at the pachytene stage of spermatogenesis (
      • Turner J.M.
      ). The repressive state of the sex chromosomes continues throughout the development of round spermatids (
      • Greaves I.K.
      • Rangasamy D.
      • Devoy M.
      • Marshall Graves J.A.
      • Tremethick D.J.
      ,
      • Namekawa S.H.
      • Park P.J.
      • Zhang L.F.
      • Shima J.E.
      • McCarrey J.R.
      • Griswold M.D.
      • Lee J.T.
      ,
      • Turner J.M.
      • Mahadevaiah S.K.
      • Ellis P.J.
      • Mitchell M.J.
      • Burgoyne P.S.
      ). Interestingly, the sex-linked multicopy genes are expressed predominantly in round spermatids (
      • Touré A.
      • Clemente E.J.
      • Ellis P.
      • Mahadevaiah S.K.
      • Ojarikre O.A.
      • Ball P.A.
      • Reynard L.
      • Loveland K.L.
      • Burgoyne P.S.
      • Affara N.A.
      ,
      • Mueller J.L.
      • Mahadevaiah S.K.
      • Park P.J.
      • Warburton P.E.
      • Page D.C.
      • Turner J.M.
      ,
      • Touré A.
      • Szot M.
      • Mahadevaiah S.K.
      • Rattigan A.
      • Ojarikre O.A.
      • Burgoyne P.S.
      ). Specifically, the multicopy genes residing in the MSYq-region are critical for sperm differentiation and for correct packing of the chromatin in male germ cells (
      • Touré A.
      • Clemente E.J.
      • Ellis P.
      • Mahadevaiah S.K.
      • Ojarikre O.A.
      • Ball P.A.
      • Reynard L.
      • Loveland K.L.
      • Burgoyne P.S.
      • Affara N.A.
      ,
      • Touré A.
      • Szot M.
      • Mahadevaiah S.K.
      • Rattigan A.
      • Ojarikre O.A.
      • Burgoyne P.S.
      ,
      • Ellis P.J.
      • Clemente E.J.
      • Ball P.
      • Touré A.
      • Ferguson L.
      • Turner J.M.
      • Loveland K.L.
      • Affara N.A.
      • Burgoyne P.S.
      ,
      • Ward M.A.
      • Burgoyne P.S.
      ). The existence of multiple copies of the X- and Y-chromosomal genes has been proposed to counteract the repressive sex chromatin state in postmeiotic cells (
      • Mueller J.L.
      • Mahadevaiah S.K.
      • Park P.J.
      • Warburton P.E.
      • Page D.C.
      • Turner J.M.
      ). In light of these reports, we propose that the presence of both HSF1 and HSF2 is required for the transcriptional regulation of certain X- and Y-chromosomal multicopy gene promoters in postmeiotic cells.
      A hallmark of the heat shock response is the extinction of bulk transcription, whereas transcription of Hsp is activated rapidly and strongly (
      • Kugel J.F.
      • Goodrich J.A.
      ). The transcriptional activation of Hsp promoters is mainly mediated by HSF1, as HSF2 has only a modulating role upon heat stress (
      • Östling P.
      • Björk J.K.
      • Roos-Mattjus P.
      • Mezger V.
      • Sistonen L.
      ). Nevertheless, both factors can bind to the Hsp promoters in response to heat shock (
      • Östling P.
      • Björk J.K.
      • Roos-Mattjus P.
      • Mezger V.
      • Sistonen L.
      ) when most other promoters are silenced. Similarly to heat shock, transcription of all single-copy X- and Y-chromosomal genes is silenced during meiosis, and the genes remain repressed throughout spermatid development (
      • Turner J.M.
      ,
      • Namekawa S.H.
      • Park P.J.
      • Zhang L.F.
      • Shima J.E.
      • McCarrey J.R.
      • Griswold M.D.
      • Lee J.T.
      ,
      • Turner J.M.
      • Mahadevaiah S.K.
      • Ellis P.J.
      • Mitchell M.J.
      • Burgoyne P.S.
      ). Intriguingly, our results indicated that HSF1 and HSF2 occupy sex-linked multicopy genes in round spermatids (Fig. 3 and Ref.
      • Åkerfelt M.
      • Henriksson E.
      • Laiho A.
      • Vihervaara A.
      • Rautoma K.
      • Kotaja N.
      • Sistonen L.
      ), allowing the multicopy genes to escape postmeiotic sex chromosome repression. It is likely that HSF1 and HSF2, possibly with help from other factors, are capable of binding to target promoters in transcriptionally repressive environments.
      Localization studies revealed that HSF1 occupies the sex chromatin of the sex body during meiotic stages of the spermatogenesis (Fig. 4 and supplemental Fig. S1). The sex body is a specialized meiotic chromatin domain distinct from the autosomal domain, and it is characterized by a lack of RNA synthesis and by sequestration of a unique array of proteins to retain MSCI (
      • Hoyer-Fender S.
      ,
      • Handel M.A.
      ,
      • Turner J.M.
      ). Proteins that have been found to localize to the sex body are mainly chromosomal proteins, such as histone variants and proteins associated with DNA damage repair (
      • Hoyer-Fender S.
      ,
      • Handel M.A.
      ,
      • Turner J.M.
      ). To our knowledge, this is the first time a transcription factor such as HSF1 is shown to colocalize with γH2AX and the sex body in meiotic cells and with the sex chromocenter of postmeiotic cells. A challenge for forthcoming studies is to establish the specific function of HSF1 at the repressed sex chromatin in distinct cell types.

      Acknowledgments

      We are grateful to Noora Kotaja for insightful discussions. We thank Karoliina Rautoma and Johanna Manninen for expert technical assistance, Pia Roos-Mattjus and the members of our laboratory for critical comments on the manuscript.

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