The XY body: a specialized meiotic chromatin domain

Experimental Cell Research 296 (2004) 57 – 63 www.elsevier.com/locate/yexcr

Review

The XY body: a specialized meiotic chromatin domain Mary Ann Handel * The Jackson Laboratory, Bar Harbor, ME 04609, USA Received 12 January 2004 Available online 12 April 2004

Abstract The sex chromosomes of mammalian spermatocytes form a specialized nuclear territory known as the XY body, where both transcription and homologous recombination are restricted. The array of proteins assembled into the XY body is typical of heterochromatin. This special subnuclear domain is in distinct contrast to the autosomal domain of the spermatocyte nucleus, where both homologous recombination and transcription occur. The special features of the XY body might reflect absence of homology between the sex chromosomes, rather than any form of dosage compensation, and may also serve to mark parental origin of the paternal X chromosome. D 2004 Elsevier Inc. All rights reserved. Keywords: X chromosome; Y chromosome; Meiosis; Transcription; Recombination; Spermatogenesis

Introduction What is the spermatocyte’s XY body and how is it related to meiosis? Meiosis is a specialized cell division process, the defining event of gametogenesis. During the extended meiotic prophase in spermatocytes, homologous chromosomes pair and undergo reciprocal recombination. Reciprocal recombination is followed by two divisions—a reductive division in which homologous chromosomes separate (segregate) from one another and an equational division in which sister chromatids segregate from each other to form haploid cells. During meiosis in mammalian males, neither sex chromosome has a homolog; nonetheless, the X and Y do pair along a spatially restricted region of each chromosome and they segregate accurately from each other in the first meiotic division. However, in contrast with the autosomal chromosomes, most of the X and Y chromatin is not paired. Recombination between these heteromorphic sex chromosomes occurs only in a small region at their distal ends, the pseudoautosomal, or pairing, region (PAR). Additionally, the sex chromosomes undergo transcriptional inactivation, MSCI (meiotic sex * The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609. Fax: +1-207-288-6073. E-mail address: [email protected]. 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.03.008

chromosome inactivation). These differences are reflected in remodeling of the XY chromatin into heterochromatin, thus forming the XY body, or sex body, as a specialized and visibly distinct domain within the nucleus of mammalian pachytene spermatocytes. This subnuclear domain is characterized and different from the autosomal domain both by lack of RNA synthesis and by sequestration of an array of proteins not found elsewhere in the spermatocyte’s nucleus. The XY chromosome territory is typically found in association with the nuclear lamina where the paired distal ends of the X and Y are anchored, but it is not membrane bound (hence a former term, the ‘‘sex vesicle,’’ is inaccurate and misleading). The first major review of the XY body appeared 30 years ago [1] and is still wonderfully comprehensive and informative. Here, I will focus on new insights and contexts for viewing chromatin remodeling and silencing of the X and Y chromosomes during male meiosis. Why is the XY body interesting? First, MSCI is a model for development of repressive chromatin structure, an important facet of eukaryotic gene regulation. Second, the lack of recombination in the XY body is a counterpoint to the active homologous recombination occurring in the autosomal domain of the spermatocyte; by understanding how reciprocal recombination is repressed in the XY body, we may learn more about how

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it is promoted elsewhere in the nucleus. Third, XY body formation is under interesting regulation, controlled intrinsically and autonomously to the germ cell (i.e., not forming in XX cells) and restricted to the testis (i.e., not forming in XY germ cells that enter meiotic prophase ectopically, outside of a testis). Finally, it is assumed, but not yet definitively demonstrated, that the formation of the XY body is critical for spermatogenic success and faithful segregation of the sex chromosomes. This review is structured on these key facets of the XY meiotic chromatin domain. The focus is on male meiotic features of the sex chromosomes in the laboratory mouse (and gene symbols are those of the mouse unless otherwise noted), but much of what we know appears to be true for all mammals studied.

The XY body: a domain of repressive chromatin The XY body is transcriptionally repressed chromatin That the X and Y chromosomes are transcriptionally silenced has been known for decades as a result of autoradiographic studies on the incorporation of RNA precursors (3H-uridine) into nuclei of pachytene spermatocytes. Universally, no grains are found over the XY body, although density of grains over the autosomal chromosomes shows that they are highly active in synthesizing RNA (studies reviewed in Refs. [1,2]). Why should the X and Y chromosomes be silenced during meiosis? Previously, it had been postulated that products of the X chromosomes were inhibitory or deleterious to spermatogenesis, but for a variety of reasons this is no longer an accepted idea [2]. In fact, since the X chromosome encodes a significant number of ‘‘housekeeping’’ proteins, the transcriptional silencing raises an interesting problem. How does the spermatocyte cope with the absence of critical gene products needed for metabolism? It appears that evolution has taken care of this with autosomal ‘‘backup’’ genes utilized only during the meiotic phase of spermatogenesis. Thus, for example, the otherwise ubiquitously expressed gene encoding phosphoglycerate kinase, Pgk1, is located on the X chromosome and silenced during male meiosis, whereas, contemporaneously, a functional retroposon variant on mouse Chr 17, Pgk2, is activated [3]. Interestingly, the spermatocytes are the only cells that transcribe Pgk2. Autosomal ‘‘backups’’ expressed only in spermatocytes exist for a number of other X-linked genes silenced during male meiotic prophase (Table 1). A possible exception to this pattern could be the Ott (ovary – testis expressed, but of unknown function) gene that is detected primarily in juvenile testes at a time when both spermatogonia and spermatocytes represent the majority of the germ-cell population [4]. However, it is not known whether Ott expression occurs in spermatogonia or spermatocytes or both, nor has the possibility been eliminated

Table 1 Autosomal variants of X-linked genes Gene

Encoded Protein

References

Pgk2 Pdha2 G6pdX Cstf2t

Variant phosphoglycerate kinase Variant E1a subunit of pyruvate dehydrogenase Variant glucose-6-phosphate dehydrogenase Tau variant of the cleavage stimulation factor polyadenylation protein CSTF2 Centrosome protein and a paralog of the X-linked Cetn2 Homologous to Zfx, a protein of unknown function

[3] [51] [52] [53]

Cetn1 Zfa

[54,55] [56,57]

that the transcript detected in juvenile testes is from a homologous autosomal gene. Furthermore, it is possible that the spermatocyte also can ‘‘cope’’ with sex-chromosome silencing by a strategy of stabilization of gene products transcribed before meiotic inactivation of the sex chromosomes [5]. These lines of evidence strongly suggest that the formation of the transcriptionally repressed XY chromatin domain is a meiotic phenomenon, unrelated to either X inactivation for dosage compensation or any possible requirement for Xor Y-encoded proteins. The XY chromatin domain is associated with a unique array of heterochromatin-associated proteins Apart from the transcriptional inactivation of the sex chromosomes, the most apparent feature of the XY body is its chromatin structure, visibly different even at the light microscope level from that of the autosomal domain of the spermatocyte nucleus, as observed by early microscopists [6]. Ever since the routine application of immunolocalization to study the protein components of meiotic prophase nuclei, it has been apparent that an aspect of this unique chromatin domain is the sequestration of an interesting array of proteins, many of which are not found elsewhere in the nucleus and are typically associated with heterochromatin, and some of which are posttranslationally modified. The proteins thus far found to localize to the XY body are primarily ones in various categories of chromosomal proteins, including modified histone variants and proteins associated with repair of DNA damage (Table 2). Other as yet unknown proteins may also localize to the XY body. But equally informative is what does not localize to the XY body; it is not associated with splicing factors [7] or RNA polymerase [8], as expected because it is transcriptionally silent. It is likely that the exact function of each of these proteins will not be known until more is known about both the meiotic role of MSCI and XY body formation. Nonetheless, a common theme among all the proteins found associated with the XY body is that they are involved with heterochromatin and/or transcriptional repression. Thus, the mammalian XY body shares common features with epigenetically heritable domains of heterochromatin in general

M.A. Handel / Experimental Cell Research 296 (2004) 57–63 Table 2 Proteins localizing to the XY body Proteins and Categories Histone variants and modified histones

Testis-specific variant of histone methyl transferase Chromobox protein orthologs

Orphan receptor germ-cell nuclear factor Recombination-related proteins Other proteins

References macroH2A1.2 gH2AX H3meK9 H2A SUV39H2

[16,58,59] [23] [10,12,15] [60] [61]

CBX1 (chromobox protein 1, formerly known as M31 and HP1h) CBX3 (formerly known as HP1g) NR6A1 (formerly known as GCNF) MRE11 and RAD51 XMR XY77 XYbp Meiosis-specific protein MW 51,000 protein ASY (asynaptin)

[16,62 – 64]

[64] [65,66] [67] [68,69] [70] [71] [72] [73] [8]

[9] and the silenced X chromosome in the germline of other species, such as Caenorhabditis elegans [10 – 12]. In an expression of the ‘‘histone code’’ [13,14], modified histones, especially histone H3meK9, play a major role in recruiting proteins for assembly of heterochromatin [9], and thus the modifications of histones H3 and H2AX that occur during XY body formation are likely to be highly significant. Nonetheless, in spermatocytes of mice doubly null for the two genes encoding the major histone methylases SUV39H1 and SUV39H2, the XY body forms, although the Y chromosome is hypocondensed and sex chromosome pairing is impaired [15]. It can be assumed from these observations that histone methylation contributes to establishment of the XY body heterochromatin, but is not solely responsible. Key to understanding the roles of these proteins and their posttranslational modifications will be sorting out those that signal MSCI, those that establish the XY body heterochromatic chromosomal territory, and those that maintain the structure. Finally, it should be considered that any of the proteins associated with the XY body could have a function not directly related to MSCI or XY body formation. As an example, CBX1 and histone macroH2A1.2 both localize in particular to the PAR and may serve to reinforce the tight association of the X and Y that is necessary to ensure their segregation from each other at the first meiotic anaphase [16].

The XY body: a domain of restricted synapsis and reciprocal recombination Unlike the rest of the spermatocyte nucleus, the XY body is a domain characterized by nonhomologous meiotic

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interactions. The X and Y chromosomes are largely differentiated and share sequence homology only in the distal ‘‘pseudoautosomal’’ pairing region, the PAR. The X and Y chromosomes pair, synapse, undergo reciprocal recombination and experience a chiasma in the PAR; this serves to ensure their disjunction during the ensuing meiosis I anaphase. Although the ‘‘differentiated’’ regions of the X and Y do not synapse, proteins of the lateral axes of the synaptonemal complex, including SYCP3 and STAG3 [17], are assembled along their axes. This suggests that these proteins may serve a role primarily in sister chromatid cohesion and segregation rather than in pairing, synapsis and recombination. Whether the differentiated regions of the X and Y chromosome are subject to the genome-wide creation of SPO11-induced recombination-related DNA double-strand breaks (DSBs) is not known. Although it has been argued that the chromatin conformation of the XY body is adopted precisely to prevent DSBs that cannot be repaired using a homolog [2], in fact, the XY body is not cytologically visible until after the time that SPO11 induces the DNA strand breaks that initiate recombination. Indeed, RAD51 protein, which recognizes DNA strand breaks and catalyzes an early step in their repair by homologous recombination, is persistently localized over the sex chromosomes [18 – 20]. Likewise, gH2AX, a phosphorylated histone that localizes to chromatin associated with DNA strand breaks [21,22], also marks the XY body [23]. Together, these observations could indicate the presence of DSBs in the X and Y. If they are present and unable to be repaired by homologous recombination in the absence of a homolog, they may serve as a signal for formation of the condensed and inactive XY body. Also, if DNA strand breaks do occur in the sex chromosomes, they must be repaired by a mechanism other than homologous recombination. It is clear is that the major portions of the X and Y chromosomes contain unpaired DNA. Interestingly, there are many observations that suggest that transcriptional silencing is the fate of meiotically unpaired DNA in many other species. This occurs in fungi, best studied in Neurospora crassa, where genetic requirements for meiotic silencing by unpaired DNA, or MSUD, are being defined [24]. In C. elegans too, unpaired sequences, both inserted tandem repeat transgenes and the single X in XO males, are subjected to silencing [11,25]. Meiotic silencing is associated with the development of repressive chromatin in these two species. In C. elegans, meiotic silencing requires the action of the MES (maternal effect sterility) proteins [26], some of which are related to the Drosophila Polycombgroup proteins, known to mediate transcriptional repression during development. In Neurospora, meiotic silencing requires an Argonaute-like protein [27]. Argonaute proteins are involved in RNA silencing [28]. Thus, this evidence provides an interesting twist: the involvement of RNAi (RNA interference) in meiotic silencing [29]. At least in

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fission yeast, RNAi mechanisms are implicated in the formation of heterochromatin and histone H3 methylation on lysine 9 [30]. Could the formation of the XY heterochromatin domain be an RNAi-mediated process? This is not known yet, but mouse orthologs (MIWI and MILI) of Drosophila Argonaute proteins are present in male germ cells and implicated in spermatogenesis [31,32], and Dicer1, another RNAi pathway member, is expressed in the testis [33].

The XY body: a domain controlled intrinsically and extrinsically It has been difficult to pinpoint the exact time of MSCI. From autoradiographic evidence, inactivation appears to occur early in meiotic prophase, probably at the same time as autosomal chromosome pairing and formation of the XY body, but perhaps as early as the premeiotic S phase (reviewed in Ref. [2]. This evidence is consistent with assays for gene-specific chromatin conformation [34]. Indeed, transcriptional silencing of the sex chromosomes may occur only transiently, during meiotic prophase when the sex chromosomes assume the unique confirmation of the XY body, as the X has been shown to be transcriptionally active after the meiotic divisions [35,36]. It is not yet known how MSCI occurs during male meiosis. Although mammalian female X inactivation for dosage compensation relies on transcription of the Xist gene, the mechanism of sex chromosome silencing in spermatocytes appears to be independent of Xist [37,38]. The only clue thus far is provided by the serendipitous finding that MSCI and formation of the XY body require a variant histone, H2AX [39]. As detailed previously, the phosphorylated form of this histone, known as gH2AX, is strongly associated with the XY body [23]. Male mice homozygous for the induced null mutation in the gene encoding H2AX are sterile, experience X –Y asynapsis and display spermatogenic arrest during meiotic prophase, at the pachytene stage [40]. Interestingly, the XY body does not form in spermatocytes of H2AX-deficient spermatocytes, the typical array of proteins does not associate with the sex chromosomes, and MSCI does not occur [39]. From these observations, it is not known if H2AX or gH2AX is a signal for MSCI or if, perhaps more likely, it is an effector, recruiting other proteins to establish heterochromatization of the sex chromosomes. Nonetheless, taken together, these observations suggest that phosphorylated histone H2AX plays a major role in the chromatin remodeling that leads to MSCI and formation of the unique domain that is the XY body. The XY body chromatin territory forms in spermatocytes, but not in oocytes. There is no such unique chromatin domain in XX or XO oocytes nor is synthesis of X-encoded transcripts inhibited; thus, meiotic XY body formation and MSCI are not properties intrinsic to the X.

Moreover, the fetal oocytes of sex-reversed XY females (XYSry-dl1Rob) have no visible XY body and may not undergo MSCI, as evidenced by failure to identify a nuclear domain that does not exhibit RNA POLII [8]. While some proteins characteristic of the spermatocyte’s XY body localize in XYSry-dl1Rob oocytes [8,16], others do not [8]. Thus, meiotic XY body formation is not a property intrinsic to an XY pair. Furthermore, an XY body forms in XSxrO spermatocytes that lack the Y chromosome [41], and formation of the XY body does not require an intact, uninterrupted X chromosome [42]. These lines of evidence suggest that both XY body formation and MSCI are functions of how male germ cells manage the X and Y chromosomes, and not necessarily intrinsic to either the X or the Y chromosome. Ectopic germ cells can also provide some insight into requirements for XY body formation and MSCI. Fetal XY germ cells found in the ovary or in extra-gonadal locations, such as the adrenal cortex, enter meiosis on a female schedule, by about 13 dpc. These ectopic XY germ cells show no evidence of forming an XY body [43,44]. However, male germ cells precociously induced to enter meiosis in fetal testes do form an XY body [45]. If these observations can be substantiated with evidence from immunolocalization of proteins characteristic of the XY body, it will suggest that a non-cell-autonomous testicular factor (perhaps from Sertoli cells?) is directly or indirectly involved in the mechanisms that lead to XY body formation. Is formation of the XY body required for survival and fertility of male germ cells? The answer to this question is not yet known definitively. Certainly, the phenotype of nullizygosity for the gene encoding histone H2AX is failure to form the XY body and sterility with arrest of spermatogenesis before completion of meiosis. This suggests that XY body formation is required for spermatogenesis and fertility, but it is not known if spermatogenesis is arrested in the absence of histone H2AX because of failure to form the XY body or for some other reason. There is no evidence of fertility in any male mammal with a translocation between the X chromosome and an autosomal chromosome; in each case, there is arrest of spermatogenesis in meiotic prophase. Yet, in mice bearing a sterility-causing X-autosome translocation, the XY body is formed [42,46], so sterility may depend on some other feature of the translocated X chromosome.

Conclusions and perspectives Clearly, the XY body is an important subnuclear chromosome territory in the mammalian spermatocyte. It is likely to reflect an important aspect of meiosis—recognition of absence of homologous pairing. Thus far, from analyses of the XY body, there has been much useful information that is nonetheless anecdotal, descriptive and serendipitous. Experimental analyses can be guided by key

M.A. Handel / Experimental Cell Research 296 (2004) 57–63

questions raised by previous work: How is absence of homology recognized, does it bring about heterochromatization, and if so, how? Which proteins signal MSCI and XY body formation, which establish chromatin remodeling, and which maintain the heterochromatic domain? Does the XY body represent an example of MSUD? Does the establishment of its heterochromatin involve RNAi mechanisms? Why does not the XY body form except in germ cells in testes, and what testicular ‘‘factor’ could be implicated? Finally, it might be useful to consider whether the inactivation of the spermatocyte’s X chromosome has a consequence beyond meiosis. In fact, in XX zygotes, the paternal X is ‘‘pre-silenced’’ [47,48]. This paternal X is imprinted; that is, it ‘‘remembers’’ its paternal origin, which predicts its destiny—to be nonrandomly inactivated in extraembryonic tissues. The evolutionary advantage of this imprinted X inactivation is not clear. However, in the context of the XY body, the ‘‘imprinting’’ of the paternal X might be understood. A key feature in its imprinting may be that the paternal X is the sex chromosome that lacks a pairing partner in the formation of the gamete. Without a pairing partner, the X is inactivated and its chromatin is remodeled into the heterochromatic domain of the XY body. In other words, does the imprint on the paternal X originate in lack of homology? And, conversely, does recognition of the imprint depend on homology? The paternal X in XX zygotes from normal fertilization is recognized and inactivated nonrandomly in extraembryonic tissues in the presence of a homolog. However, when XY male embryos are formed that inherit both their sex chromosomes from the paternal parent, there is seemingly no inactivation of the paternal X [49] since normal embryos and adults are produced. Thus, it appears that the spermatocyte’s X is silenced in the absence of homology and the zygotic paternal X recognizes itself specifically in the presence of a homology-based counting mechanism. Chromosomal homology-based phenomena are widespread [50] and may lie at the root of the processes of MSCI and formation of the XY body. Acknowledgments I am indebted to Alberto Solari whose earlier observations and review piqued my curiosity about the XY body. Thanks to Drs. John Eppig, Tim O’Brien and Beverly Richards-Smith for insightful and useful comments on the manuscript. References [1] A.J. Solari, The behavior of the XY pair in mammals, Int. Rev. Cytol. 38 (1974) 273 – 317. [2] B.D. McKee, M.A. Handel, Sex chromosomes, recombination and chromatin conformation, Chromosoma 102 (1993) 71 – 80.

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