REVIEW ARTICLE The role of histones in chromatin remodelling during mammalian spermiogenesis Je ´ ro ˆ me Govin, Ce ´ cile Caron, Ce ´ cile Lestrat, Sophie Rousseaux and Saadi Khochbin Laboratoire de Biologie Mole ´ culaire et Cellulaire de la Diffe ´ renciation, INSERM U309, E ´ quipe Chromatine et Expression des ge ` nes, Institut Albert Bonniot, Faculte ´ de me ´ decine, La Tronche, France One of the most dramatic chromatin remodelling processes takes place during mammalian spermatogenesis. Indeed, during the postmeiotic maturation of male haploid germ cells, o r s permiogenesis, histones are replaced by small basic proteins, which in mammals are transition proteins and protamines. However, nothing is k nown of the mechanisms controlling the process of histone replacement. Two h ints from the literature could help to shed light on the underlying molecular events: one is the massive synthesis of histone variants, including testis-specific members, and the second is a stage specific post-translational modification of histones. A new testis-specific Ôhistone codeÕ can therefore be generated combining both histone variants and h istone post-transla- tional modifications. This review will detail these two phe- nomena and discuss possible functional significance of the global chromatin alterations occurring prior to histone replacement during spermiogenesis. Keywords: bromodomain; chromodomain; epigenetics; histone chaperone; histone structure. Introduction The basic unit of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around an octamer of core histones, including two molecules of H2A, H2B, H3 and H4 [1]. A fifth histone, H1, protects additional DNA fragments linking neighbouring nucleosomes [2]. The nucleosomes are also the building blocks of a complex organization of chromatin, which adopts different architec- tures i n r esponse to specific stimuli. These i nclude organ- ization states going from a Ôbeads-on-a-stringÕ structure to the highly condensed mitotic chromosomes. Because of the specific nature of gene expression during development and in various adult tissues, the chromatin structure also has to undergo local structural alterations. Three major strategies contributing t o l ocal and specific chromatin remodelling have so far been identified. ATP utilizing complexes act directly on nucleosomes to modify the a ccessibility of factors to limited DNA regions present in a nucleosome [3]. Histone modifying enzymes dictate combinations of post-translational modifications of histones to create specific signals defining the Ôhistone codeÕ, which in turn induces localized alterations of the c hromatin structure and function. The histone code hypothesis postu- lates that specific factors can act on chromatin by recog- nizing and binding particular histone modifications [4–6]. This hypothesis is so far supported by the discovery of chromatin interacting modules present in various factors, specifically recognizing methylated or acetylated lysines of histones [7]. Finally, variants of histones H2A, H2B, H3 and H1 have been identified. Some of these variants have a lready been shown to mediate specific functions such as DNA repair in response to genotoxic treatments [8]. In somatic c ells, these three mechanisms act together to locally induce alterations of the chromatin structure and to maintain a region-dependent differentiation of chromatin over generations of cells, although many questions remain unanswered on the molecular basis of their action. An extreme case of chromatin remodelling occurs during spermatogenesis, where histones are massively removed and replaced [9]. Although n othing is known o f the underlying me chanisms, one can e xpect a major participa- tion of the three chromatin modifying mechanisms already known to a ct in somatic cells. Indeed, disparate data from the literature suggest that histone removal during spermiogenesis is preceded by a massive incorporation of histone variants associated with the i nduction of different types of histone modifications (Fig. 1). In this review, data from the literature are analysed in order to finally discuss the functional significance of histone variants, as well a s of histone post-translational modifica- tions, during spermiogenesis. The main histone variants Histone variants are nonallelic forms of the conventional histones [8]. Conventional histones are mostly synthesized and a ssembled into nucleosomes during S phase Correspondence to S. Khochbin, Laboratoire de Biologie Mole ´ culaire et Cellulaire de la Diffe ´ renciation, INSE RM U309, E ´ quipe Chroma- tine et Expression des ge ` nes, Institut Albert Bonniot, Faculte ´ de me ´ decine, Domaine de la Merci, 38 706 La Tronche, France. Fax: +33 0474549595, Tel.: +33 0474549583, E-mail: khochbin@ujf-grenoble.fr (Received 14 May 2004, revised 16 June 2004, accepted 23 June 2004) Eur. J. Biochem. 271, 3459–3469 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04266.x progression, whereas replacement histones can be produced and incorporated throughout the cell c ycle. Testis specific variants have been described [9], but many nontissue s pecific histone variants are also e xpressed and incorporated into chromatin during spermatogenesis (Fig. 1). Linker histone variants In mammals, at least six somatic subtypes (H1.1–H1.5 and H1°), one oocyte-specific and two testis-specific linker histones (H1t and HILS1) are expressed [2,10,11]. H1t contains the usual tripartite structure of linker histones, but is highly divergent in its primary structure compared to the other five members H1.1–H1.5 (Fig. 2). Its expression has been characterized in the mouse [10] as well as in the rat [12]. In situ hybridization detects the RNA in mid-pachytene s permatocytes, a nd immunodetection indi- cates the presence of the p rotein from the stage of pachytene spermatocytes until round and elongating spermatids [10,13]. At this stage, the H1t amount constitutes up to 55% of t he total linker histones. Mice bearing invalidated H1t gene display no phenotype [14–16], but the analysis of enriched populations of pachytene s permatocytes and round spermatids in these mice has shown that its absence is partially compensated by the other H1s, still permissive to end maturation and fertilization [14,15]. Interestingly, other groups have shown that the interaction of H1t with nucleosomes leads to a less compact structure than that of other H1 subtypes [17,18], suggesting that this variant may help chromatin de-compaction, giving accessibility to other chromatin remodelling factors. Among the somatic linker histones, H1.1 (H1a) is present at a high level in spermatogonia and then decreases upon further development during mitotic and meiotic cell d ivisions [19,20]. Neverth eless H1.1 disru pted mice display no significant phenotype, and show normal spermatogenesis, fertility and testicular morphology [21]. It has been shown recently that in the absence of H1t, H1.1 is o ver-expressed to m aintain the normal ratio of H1 to core histone [22]. Interestingly, the elimination of both H1.1 and H1t led to a s ignificant d ecrease of H1/core histone ratio (75% of the normal r atio) w ith- out any defect in spermatogenesis [22]. These findings suggest that male germ cell development can normally proceed in the presence of reduced ratio of H1 to core histones. A last H 1 variant, named HILS1 (H1-like protein in spermatids 1), has been found recently in human and mouse [11,23]. Whereas H1t is essentially present until the round/ elongating spermatids stages, HILS is detected later in elongating a nd condensing spermatids nucleus, suggesting a sequential action of linker histones during chromatin remodelling. H3 variants At least five H3 variants have been described, of which one seems to be testis specific. Fig. 1. Chromatin components during spermatogenesis. The major chromatin components and their post-translational modifications are presented. Histone variants are incorpora ted during meiosis, except linker variant HILS, which shows a delayed expression. Highly basic proteins, transition proteins and protamines, replace histones during late spermiogenesis. The temporal distribution of the main post-translational histone modifi- cations is also presented (A, ac etylation; U, u biquitination ; M, m ethylation; P, phosphorylation). Spermatogenesis, the differentiation of male germinal cells, is characterized by three major stages: preme iotic, m eiotic and postmeiotic. Pre-meiotic spermagogonia divide b y mitosis. They then enter meiosis by the formation of preleptotene primary spermatocytes, which replicate DNA and subsequently go through the leptotene, zygotene, pachytene and diplotene stages of the first meiotic division prophase. Meiotic I division yields secondary spermatocytes which then rapidly go through m eiotic II division, generating haploid round spermatids. During its postmeiotic maturation, the spermatid undergoes a global remodelling of its nucleus, which e longates and compacts into the very un ique nucleus structure of the sperm atozoa. 3460 J. Govin et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Fig. 2. Sequence analysis of known histone variants expressed during spermatogenesis. The sequences of conventional histones and their sperma- togenic variants are aligned in (A), (B), (C) and (D). All sequences are murine, as most of the sequence data are available for this species, except for TH2A (rat), and hTH2B and H3t (human sequences). Con ventional histone sequences were chosen on the basis of work by M arzluff and colleagues [94]. Alignments were perf ormedwiththealgorithm CLUSTALW on the web interface o f the PBIL at http://npsa-pbil.ibcp.fr/[95] and coloured with ESPRIT at http://prodes.toulouse.inra.fr/ESPript/ES Pript/[96]. Some of the histone modifications d iscussed are indicated [67,91]. Modification cassettes (amino acids Thr/Ser-Lys or Lys-Thr/Ser) [91] were searched in conventional histones and variants, and are rep resented by small rectangles, underneath the corresponding sequenc es. Black r ectan gles underline cassettes p resent in conventional histones. Some cassettes are not conserved in variants and arrows indicate changes leading to the cassette disappearance in the variants. Open rectangles underline new cassettes specific t o a variant and absent in conventional his tones. Original crystallographic data were used for the representat ion of the secondary structu res [1,97]. Sequence accession numbers: H3.1 (P16106); H3.3 (P06351); H3t (Q16695); H2A (NP_783591); TH2A (Q00728); H2B (NP_835502); TH2B (Q00729); hTSH2B (NP_733759); H1.1 (P43275); H1t ( Q07133); HILS1 (Q9QYL0). Ó FEBS 2004 Chromatin code and spermatogenesis (Eur. J. Biochem. 271) 3461 A testis-specific H3 variant, only detected in the human, has been isolated in 1996 [24,25]. This variant, named H3t, differs from the canonical H3 by only four residues (Fig. 2A). The RNA of this variant was only detected in primary spermatocytes. T he experimental sequencing helped to identify another t estis specific variant, n amed TH3 in rat [26]. However, no gene or sequen ce information is available on this putative histone variant and no corresponding genes have b een found in known mammalian genomes [25]. More data is available on nontestis specific H3 variants. CENP-A is a centromeric specific variant, and unique by its N-terminal amino-ac id composition [27]. In somatic cells, CENP-A is deposited on newly duplicated centromeres, and is required for the recruitment of other proteins to centromeres and kinetochores. A similar function in germ cells would imply its involvement during mitotic and/or meiotic segregation. The other H3 variant is H3.3, which differs b y 4–5 amino acids from H3, depending on the allelic form considered (Fig. 2A). The two H3.3 genes, H3.3A and H3.3B,are expressed in mouse testis [28,29]. H3.3A mRNA was detected before and after meiosis while the expression of H3.3B gene was found to be restricted to cells of the meiotic prophase [29]. Interestingly, H3.3A was identified by a gene trap strategy as a gene expressed in spermatocytes, a nd of which homozygous disruption caused partial neonatal lethality and, in surviving mutants, reduced growth, neuro- muscular deficits and male subfertility [30]. The number of copulations per male, as well as the number of pregnancies per copulatory p lug, were significantly lower for H3.3A–/– mutants than for non mutants. No obvious differences in the testis, epididymis, vas deferens, or sperm numbers were reported in this study, suggesting that spermatogenesis was not quantitatively affected. Akhmanova and colleagues [31] have sho wn that Dro - sophila H3.3 is incorporated during the first meiotic prophase, then concentrated in a limited number of chromatin regions and further disappears with the other core histones during the elongation of spermatids. In somatic cells, a ctively t ranscribing regions have been shown to be enriched in H3.3 [32], suggesting that the replacement of H3 by H3.3 in spermatocytes could also be linked to the very active transcription that takes place during meiosis [9]. H2B variants Rat, mouse and human TH2B have been cloned, showing very high levels of conservation [ 33–35]. The main differ- ences between H2B and TH2B a re in the N -terminal, and to a lesser extent, the histone fold domain (Fig. 2C). Most of these differences are c onserved between the three species, suggesting a conserved role for this variant during sperma- togenesis (see below). In rat, TH2B is actively expressed in early primary spermatocytes until mid–late pachytene [19] and then remains the major form o f H2B in round and elongating spermatids. Using an antibody that, luckily, cross-reacts with TH2B, it has been shown in human testis that TH2B first appears in spermatogonia, is maximal in round spermatids, and then gradually disappears during the elongation of spermatids [36]. In contrast, the human TH2B, hTSH2B, was retained in mature sperms and presented a specific nuclear localization only in 20% of sperm populations [35]. There is also apparently a nonchromatin function for histones during spermatogenesis. Indeed, recently, in bull somatic type core histones h ave been foun d associated with the perinuclear theca, which is a layer surrounding the nucleus of mammalian sperms [37]. A h istone H2B variant, named SubH2Bv, has also been found associated with the theca in bull sperm [38]. The function of these non-nuclear histones has not been defined. H2A variants Only one testis specific H2A variant has been characterized and named TH2A, which differs from somatic H2A in several residues l ocated in its histone fold domain as well as in its N- and C-term inal tails (Fig. 2B). T H2A is actively expressed a nd incorporated in the chromatin of pachytene spermatocytes [19,39]. The expression of nontestis specific H2A variants have been studied in more detail. Mainly, t wo H2A v ariants are expressed during spermatogenesis, H2A.X, and mac- roH2A. In somatic cells, H2A.X is involved in DNA double strand b reaks (DSB) surveillance and repair [9,40]. H2A.X disruption leads to male sterility with abnormal spermatogenesis. Indeed, in the male mutants, no DNA alignment for synapsis is observed at zygotene and early pachytene stages. In the spermatocytes that progress into mid-pachytene M1h1, a mismatch repair protein, do not display t he foci characteristic of recombined DNA strands, and chromosomes X and Y are abnormally paired with autosomes, leading to apoptosis of mid pachytene spermatocytes [41]. MacroH2A is a long variant of H2A, containing a large C-terminal nonhistone region [42]. Two allelic forms, macroH2A1 and macroH2A2, are expressed. They are 80% identical [43,44]. The macroH2A1 gene encodes two proteins generated by an alternative splicing mech- anism, macroH2A1.1 and macroH2A1.2 [43]. In somatic cells of female mammals , the inactive X c hromosome has been shown marked by a high concentration of histone macroH2A [43,45,46], forming a dense structure, referred to as the macrochromatin body. MacroH2A1.2 is found at high concentrations in mice testis [47,48]. During spermatogenesis, it has been observed in the nuclei of germ cells, with a localization that is largely to the developing XY-body in early pachytene spermatocytes [49,50]. Hence, the process of X-inactivation in XX somatic cells [51] and that in XY spermatocytes show some similarities, including a heterochromatinization of the region which is densely stained (forming, respectively, the Barr Body or the S ex Vesicle) and a coating of the X with the Xist RNA, a non coding RNA specifically associated w ith the inactive X chromosome [52]. Interest- ingly, a potential relationship has been discovered between macroH2A1.2 and the mammalian HP1-like heterochromatin p rotein M31 (HP1beta or MOD1) during meiosis. The HP1-like protein M31 was found initially to colocalize with heterochromatic regions in Sertoli cells, in mid-stage pachytene spermatocytes, a s well as in round spermatids (where it localized with the 3462 J. Govin et al.(Eur. J. Biochem. 271) Ó FEBS 2004 centromeric chromocenter) [53]. Both macroH2A1.2 and M31 were found to colocalize in a time-dependent manner at specific nuclear regions, including the pseudo- autosomal region (PAR) of the sex body [50], suggesting a role for this heterochromatic region in preventing precocious desynapsis of the terminally associated X and Y chromosomes prior to a naphase I. According to the data described above, the large histone H2A variant, macroH2A1.2, along with the HP1-like protein M31, could be involved in the partial pairing of X and Y chromosomes and the formation of th e s ex vesicle, which, although of unknown function, is an indispensable f eature of a successful male meiotic division. Indeed, meiotic studies in men presenting an impaired spermatogenesis in the context of a constitutional chromosomal abn ormality have suggested that the presence of a sex vesicle is crucial for the achievement of meiosis. In one stu dy, macroH2A1.2 has also been found in murine spermatozoa, suggesting that it may be important for other functions besides meiotic recombination [49]. However, according to another stud y, macroH2A was not found among sperm nuclear proteins, not even in species fully retaining the histones in mature sperm such as catfish and bullfrog [54]. Histones and post-translational modifications The histone code hypothesis proposes that combinations of histone modifications could define specific signals, and serve as an interface languag e b etween hist ones a nd chroma tin modifying activities, to assign particular structure and function to specific chromatin domains [5,6]. In fact each histone has several sites of potential modifications including acetylation, methylation, phosphorylation, etc… Assuming that the eight core histones of each nucleosome could have different associations of modifications, their combination in a multinucleosomal microenvironment would create a tremendously complex epigenetic code. This hypothesis stands only if experimental data support the existence o f a machinery capable of specifically re cognizing and reading the histone c ode. The existe nce of cellular factors recogni- zing and binding to specifically modified histones is i n support of this hypothesis [7,55]. The histone code is probably in a ction in spermatogenic cells as stage-specific histone modifications have been reported to occur during the postmeiotic genome reorgan- ization phase. However, despite detailed descriptions of some histone modifications [9], nothing is known about their potential function in chromatin reorganization and histone replacement in elongating spermatids (Fig. 1). Histone acetylation Acetylated forms of histones have been found during spermatogenesis in various species including, trout [56], rat [57] and rooster [58]. The use of antibodies, specifically recognizing individual acetylated residues, has allowed a more precise characterization of histone acetylation pattern during spermatogenesis [59]. Spermatogonia and prelepto- tene spermatocytes contain acetylated H2A H2B and H4, whereas histones are underacetylated during meiosis and in round spermatids. The replication-dependent acetylation of H4 and H 3 [60] can partially explain the acetylation signal detected in DNA replicating cells. Interestingly, these data also showed that in elon gating spermatids, histones become hyperacetylated in the total absence of DNA replication. In the case of histone H4, this acetylation was shown to follow a stage-specific distribution [59,61]. Indeed, the H4 hyperacetylation observed in the early elongating spermatids affects the nucleus in a g lobal manner. This distribution then changes during the elonga- tion and condensation s tages and finally acetylated H4 disappears following an antero-caudal movement in con- densing spermatids. This replication and transcription-independent hist one acetylation seems to be tightly linked to histone replace- ment. Indeed, histones remain under-acetylated in species where histones remain all through spermiogenesis such as winter flounder and carp [62,63]. However, the role of acetylation of core histones in their replacement remains largely unknown. Some in vitro experiments suggest that histone acetylation could facilitate their displacement by protamines [64,65], but there is no hint in the literature on how it could affect in vivo chromatin remodelling in spermatids. The recent identification of a new bromo- domain-containing testis specific factor capable of cond en- sing acetylated chromatin suggests t hat histone acetylation could primarily be a signal for chromatin condensation [66]. Histone methylation Suv39h1 and Suv39h2 a re two histone methyltransferases (HMTs) responsible for methylating Lys9 of H3 in hetero- chromatic regions, in somatic cells [67]. Suv39h2 is over-expressed in the testis [68], where it is enriched in heterochromatic regions from leptotene spermatocytes to round spermatids stages. The H3 Lys9 methylation pattern colocalizes with Suv39h2 [69]. A disruption of heterochro- matic H MT activities (double knockout of Suv39h1 and h2) leads to c hromosomal instability, impaired homologous interactions and meiosis defects. Histone phosphorylation Ser10 and 28 of H3, both very conserved in the H3 family, are phosphorylated during mitotic chromosome formation. The mitotic-specific phosphorylation of histone H3 Ser10 has also been shown to occur during meiosis very probably associated with chromosome condensation [70]. However, no information is available a bout the phosphorylation of Ser28 during spermatogenesis. Site-specific phosphorylations of H2A [71], H2AX [40] and H2B [72] have also been reported. While nothing is known a bout the phosphorylation of H2A and H2B during spermatogenesis, that of H2AX may play a c rucial role as it is tightly linked to the function of H2AX in DNA double strands breaks repair [40]. Indeed, a transient phosphorylation of H2AX on Ser139 accom- panies double strand break damage repair, as well as DNA cleavage events such as those associated with meiotic recombination [73]. Ó FEBS 2004 Chromatin code and spermatogenesis (Eur. J. Biochem. 271) 3463 Histone ubiquitination Ubiquitination is a modification known to b e a mark for protein degradation via the proteasome pathway. How- ever, the function of protein ubiquitination is not restricted to degradation, and data from the literature suggest its involve ment in DNA repair, cell cyc le control, cellular response to stress, as well as in the histone code [74]. H1 and H 3 have been found occasionally ubiquitinated in vivo, but H2A and H2B appear to be the predominant forms of ubiquitinated histones i n e ukaryotes, encompas- sing 5–15% of H2A and 1–2% of H2B [75]. Histone ubiquitination has been described du ring sper- matogenesis in many species, including rat, mouse, trout and rooster [75]. In the mouse, a high proportion of ubiquitinated H 2A (uH2A) is detected by immunochemis- try in the specific chromatin domain formed by the sex body in pachytene spermatocytes. uH2A becomes depleted from round spermatids, but reappears in elongating spermatids [74]. In elongating spermatids H2A, H 2A.Z, H2B, H3 and TH3 were found mono and poly ubiquitinated in t he rat [74,76]. HR6, a ubiquitin-conjugating enzyme, homolo gous to the yeast RAD6 protein, ubiquitinates H2B in vivo and is strongly expressed in the testis [77]. A disruption of the HR6-encoding gene induces a spermatogenesis arrest at the round/elongating spermatids st age [78] pointing to the fundamental role of histone ubiquitination during spermio- genesis. All these data suggest that histone ubiquitination can be considered as one of the important epigenetic mark involved in chromatin remodelling in postmeiotic male germ cells. Histone variants Functional significance of sequence divergence in chromatin remodelling. One of the most distinctive characteristics of chromatin remodelling during spermatogenesis is the expression of a large number of histone variants. Indeed, in ad dition to all the somatic- type histone variants, spermatogenic cells express t estis- specific histones corresponding t o three of the four core histones. Nevertheless, understanding how each variant specifically acts on chromatin s tructure and function is a real challenge. The fundamental structu ral basis of a nucleosome is very well conserved during evolution. The incorporation of histone variants could lead to the formation of nucleosomes with altered structure and modified properties. Histone variants incorporated during spermatogenesis, although showing only small changes in their primary structures, could therefore bring major changes in the nucleosome function and stability. A detailed analysis of testis-specific histone variants shows that the histone fold is usually well conserved between variants (Fig. 2A,B,C). The N-terminal region of H3 is very similar between the variants, including H3.3 and H3t, whereas the N-terminal regions of TH2A and TH2B present several differences with their somatic c ounterparts, which m ay potentially affect residues modified by known histone post-translational modifications (Fig. 2). Interestingly, the comparison of H2A/TH2A sequence shows three amino acid changes in a region covering the end of alpha1, loop1 and the beginning of alpha2. As a structural analysi s has a lready shown that H2A Loop1 is the only area of contact between the two (H2A–H2B) dimers within the nucleosome core particle [1], the m inor sequence changes observed in TH2A could have important functional consequences, as a lready established in t he case of H2A.Z by crystallographic data [79]. The structural analysis also showed that the incorporation of two heterodimers of H2A–H2B and H2A.Z–H2B within the same nucleosome is unlikely, suggesting that the incorpor- ation of the first (H2A.Z–H2B) dimer could facilitate t he recruitment o f another H2A.Z-containing dimer [79,80]. Similarly, the i ncorporation of given testis-specific histone variants might facilitate the incorporation of other variants, creating highly specialized nucleosomes. Moreover, the H2A.Z containing nucleosomes display an altered surface, with the possible incorporation of a metal divalent ion, which could lead to changes of higher order structures or modify the recruitment of specific factors [79]. It could be assumed that similar properties associated with testis specific histones would lead to an altered chromatin structure and facilitate the recruitment of testis-specific chromatin remodelling factors. The centromere specific histone variant, CENP A, has been shown to be retained in mature spermatozoa, suggesting that it could have a role in organizing the centromeres during the final stages of spermiogenesis and/or the paternal genome during early embryogenesis [81]. A role for specific histone chaperones. Cellular machinery containing histone chaperon e HIRA, has recently been discovered that is capable of uniquely assembling histone H3 variant, H3.3, in specialized nucleosomes [82,83] enriched in transcriptionally active regions [32]. The localization of H 3.3-containing chromatin has not yet been determined in mammalian germ cells, but in Drosophila, H3.3 is incorporated in chromatin during first meiotic prophase [31]. It remains concentrated in specific regions (compared to H3, which is evenly distributed) in round and elongating spermatids, and disappears in condensed spermatids like other histones. H3.3 is therefore present in haploid male g erm cells in the total absence of transcription. One possible function of this specific H3 variant could be linked to the massive histone replacement, taking place i n elongating s permatids where HIRA, or m aybe other spermatid-specific factors, could recognize H3.3 and dismantle the nucleosomes. Histone removal by HIRA may also occur in somatic cells but to a much lesser extent than in spermatids. Therefore the identification of HIRA partners in spermatids w ould be of great interest in understanding the molecular b asis of histone replacement during spermiogenesis and furthermore in that of nucleosome disassembly in gene ral. Recently, a histone variant exchanger that specifically replaces co nventional H2A by H2A.Z has been identified in yeast [ 84,85] showing that H3 and also H2A variants c an be deposited by specific factors. Recent work showed that in yeast, a protein identified as Hif1p is a histone H3 and H4 chaperone involved in chromatin assembly [86]. Interestingly, Hif1p is the 3464 J. Govin et al.(Eur. J. Biochem. 271) Ó FEBS 2004 homologue of a H1 chaperone, known as NASP, which has a testis-specific variant expressed in different species of mammals and is present all through spermiogenesis [87]. It has been proposed that tNASP may bind and t ranslocate testicular histone variants to nucleosomes [87]. Its presence during late spermiogenesis suggests that the protein may also function as a histone remover, as no chromatin assembly occurs during these stages. It is therefore very possible that the enrichment of spermatid chromatin with different histone variants would first increase the accessibility of chromatin to various factors (such as those involved in recombination in pachytene cells or histone modifying enzymes in spermatids) and then facilitate histone replacement. Moreover, histone modifica- tions, such as acetyla tion, may mediate the action of more specialized chaperones (see below). It would therefore be important to investigate the structural characteristics of testis-specific histone v ariants, and to e xplore whether the testis-specific and somatic histone chaperones expressed in spermatogenic cells are capable of exchanging TH2A, TH2B as well as H3t with transition proteins. Histone acetylation – a signal for histone replacement? As mentioned previously, postmeiotic histone hyperacety- lation has not been observed in species where somatic histones are retained completely in spermatozoa. This specific histone modification therefore appears to be tightly associated with histone replacement. Moreover, the obser- vation that in mice spermatids, acetylated H4 disappearance follows an antero-caudal pattern similar to that of chroma- tin condensation [59], reinforces the h ypothesis of a direct link between histone acetylation, th eir replacement and nucleus condensation. The m echanisms under lying this sudden histone hyper- acetylation in early elongating spermatids are unknown. However, a recent work showed that it is associated with the degradation of the major cellular histone deacetylases [88], a phenomenon that is able to play an important role by disrupting the cellular acetylation equilibrium. According to the histone code hypothesis, histone hyperacetylation in elongating spermatids would serve as a signal for the recruitment of specific machinery acting on acetylated histones. Such machinery probably contains factors such a s bromodomain-bearing p roteins, enab ling them to bind acetylated chromatin (Fig. 3). Bromodomains are acetyl-lysine binding modules present in ATP-dependent chromatin remodelling factors as well as in some HATs and other nuclear protein s of unknown function [89]. B romodomain-containing proteins therefore appear to be excellent candidates to i nterpret the signal generated by the global histone acetylation taking place during spermiogenesis. Recently, a testis-specific double bromodomain-containing protein, named BRDT, was shown to be capable of inducin g a dramatic condensation of chromatin strictly dependent o n histone hyp eracetylation [66]. These data present a new scenario regarding the significance of histone acetylation during spermiogenesis: it could primarily act as a signal for chromatin condensation. In support of this hypothesis, nuclear domains containing condensed chromatin in elongating spermatids also corres- pond to regions enriched in acetylated histone H4 (J. Go vin, C. Caron, C. Lestrat, S. Ro usseaux and S. Khochbin, unpublished results). Bromodomain-containing factors, such as BRDT, upon their interaction with acetylated histones, could also recruit testis-specific chaperones t o mediate histone removal. In fact, a new bromodomain-interacting chap- erone, CIA-II, highly expressed in t he testis, also interacts with histon e H3 in vivo andwithhistonesH3/H4in vitro [90]. Such fac tors may establish a link between an acetylation-dependent chromatin compaction mediated by bromodomain p roteins and histone displacement. More- over, it has recently been shown in yeast that Hat1p/ Hat2p/Hif1p specifically binds acetylated histones H4 and H3 [86]. A s mentioned a bove, the testis-specific homo- logue of H if1p, tNASP, is present all through spermio- genesis, and may also provide a link between histone acetylation and histone removal (Fig. 3B). Chromatin r emodelling that o ccurs during spermiogen- esis seems to d epend simultaneously on histone variants and histone modifications (histone code). It is therefore very likely that the combination of histone variants and partic- ular histone modifications generate a testis-specific Ôchro- matin codeÕ (Fig. 3A). It is noteworthy that all the sites in histon es potentially involved in generating the histone code are conserved between histone variants expressed during spermiogenesis, with the exception of the H2B phospho-acceptor site S14, which is not conserved in TH2B. This sequence divergence signifies a modification of the TH2B related histone code in spermatogenic cells, as for H2B, Ser14 phosphorylation has been shown to play an essential role in somatic cell apoptosis [72]. In contrast, compared to H2B, hTSH2B has gained four potentially new phosphorylation sites (Fig. 2 C). The observation of a p air of neighbouring amino acids both targets of post-translational modifications has recently led to the proposal of the Ôbinary switchesÕ hypothesis modulating the readout of specific marks such as lysine methylation [91]. In fact the phophorylation of Thr/Ser in Thr/Ser-Lys or Lys-Thr/Ser pairs found in the four histones may negatively regulate the binding of chromodomains to methylated lysines. Indeed, chromo- domain-containing proteins are involved in a variety of functions, but all seem to deal with chromatin. In some of these proteins, such as heterochromatin protein 1 (HP1), the chromodomain has been shown to specifically interact with histone tails bearing methylated lysines [7]. In order to assess the potential function of these binary switches during spermatogenesis, they were searched for on the primary sequences of the different histone variants. Among the three testis-specific core histones, TH2B seems t o be the on ly variant which presents significantly divergent binary cassettes compared to its somatic counterpart. Indeed, in testis-specific H2Bs, in three cases the Thr/Ser residues occurring in somatic type H2B next to a Lys residue were replaced by nonphos- pho-acceptor residues, and three new binary cassettes were created (Fig. 2C). These analyses show that, on top of a structural role, sequence divergence in testis-specific histone variants may participate in increasing the complexity of the histone code. Ó FEBS 2004 Chromatin code and spermatogenesis (Eur. J. Biochem. 271) 3465 Concluding remarks After analysing all the available data i t clearly appears that a massive chromatin alteration occurs before histone replace- ment due to an extensive incorporation of histone variants as well as to globally specific histone modifications. Recruitment of histone variants in nucleosomes may have two general effects on chromatin structure and function. First, subtle sequence divergences can have important consequences on the stability of the nucleosome. Second, these sequence divergences may change t he potential of core histones to b e modified. A testis-specific histone code can therefore be generated directing chromatin compaction, histone removal and degradation. Very little i s known on the nature of this s pecific histone code and t he way it directs chromatin remodelling in spermatids. Recently, two factors expressed in spermatids and potentially capable of participating in chromatin remodelling have been identified [66,88,92]. One of these factors containing two bromodomains, BRDT, has been shown to have the ability to induce in vitro and in vivo an histone acetylation-dependent chromatin compaction. His- tone H4 acetylation o ccurring in elongating s permatids might primarily be a signal for chromatin c ondensation. However, more investigations are required to link this acetylation-dependent chromatin compaction to histone removal. With this regard, histone chaperones may play a crucial role. Indeed, it is very plausible that specific chaperones identified to mediate nucleosome a ssembly [93] may reverse their function and control the dismantlement of nucleosomes in spermatids. Spermatogenic cells would therefore constitute an excel- lent source for the discovery of a nucleosome disassembly machinery. The identification of such factors would not only shed light on the molecular basis of chromatin reorganization during spermiogenesis but also give valuable Fig. 3. Integrative model for chromatin remodelling during spermatogenesis. (A) Chromatin remodelling combines h istone variants (1) and the histone code ( 2, 3). In the late stages of spermiogenesis, transition proteins and protamines participate in constituting the fin al sperm chro matin structure (4). (B ) Putative factors involved in the spermatogeni c rem odelling process. Brdt is prob ably on e o f th e histo ne c ode ÔreadersÕ, binding acetylated histones, and conden sing acetylated chromatin [66]. HIRA, Hif1p (also nam ed NASP) an d tNASP are suspected to behave as histon e chaperones during t his re modelling process, with some histone specificity (see text for more det ails). 3466 J. Govin et al.(Eur. J. Biochem. 271) Ó FEBS 2004 information on the yet unkn own mechanism of nucleosome disassembly. Acknowledgements This work was supported by ‘‘Re ´ gion Rhoˆ ne-Alpes’’ emergence pro- gram. C.L. i s supported by ‘‘Re ´ gion Rhoˆ ne-Alpes’’ PhD fellowship. References 1. 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