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provided by Elsevier - Publisher Connector Current Biology 19, 1478–1484, September 15, 2009 ª2009 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2009.07.041 Report Key Features of the X Inactivation Process Are Conserved between Marsupials and Eutherians

Shantha K. Mahadevaiah,1 Helene Royo,1 in situ hybridization (RNA FISH) [1] and semiquantitative John L. VandeBerg,2 John R. McCarrey,3 Sarah Mackay,4 reverse transcriptase-polymerase chain reaction (RT-PCR) and James M.A. Turner1,* [2] have concluded that the X chromosome of the marsupial 1MRC National Institute for Medical Research, Mill Hill, Monodelphis domestica (opossum) remains inactive during London NW7 1AA, UK spermiogenesis. However, Cot-1 RNA FISH has since been 2Department of Genetics, Southwest Foundation for shown to primarily detect silencing of repeat rather than Biomedical Research, San Antonio, TX 78227, USA coding X-linked sequences [16, 17], and RT-PCR cannot 3Department of Biology, University of Texas at San Antonio, discriminate between steady-state RNA levels and ongoing San Antonio, TX 78249, USA transcription. We therefore sought to reexamine male germline 4Division of Integrated Biology, Faculty of Biomedical & Life X silencing and its relationship to female XCI with a gene- Sciences, University of Glasgow, Glasgow G12 8QQ, specific RNA FISH approach. Scotland, UK For our analysis of XCI in the male germline, we selected ten genes distributed across the opossum X chromosome (Fig- ure 1A). We wished to compare X gene silencing in opossum Summary with that in mouse, in which MSCI and its maintenance have been well characterized [18, 19]. For this reason, we chose In female marsupials, X chromosome inactivation (XCI) is im- genes that have X-linked orthologs in mouse (Figure 1A). printed, affecting the paternal X chromosome. One model, RNA FISH analysis showed that all ten opossum X-linked supported by recent studies [1, 2], proposes that XCI in genes and nine of the ten mouse X-linked orthologs were marsupials is achieved through inheritance of an already expressed in the testis. RNA signals were evident in multiple silent X chromosome from the father [3–6], with XCI initiated cell types, including premeiotic germ cells, in which the X chro- by meiotic sex chromosome inactivation (MSCI) [7, 8]. This mosome has not yet undergone MSCI (Figure 1B; see also model is appealing because marsupials have no Xist gene Figure S1 available online). In order to analyze meiotic inactiva- [9–12] and the marsupial inactive X chromosome is epige- tion, we combined RNA FISH for each X-linked gene with im- netically dissimilar to that of mice, apparently lacking munostaining for gH2AX, a histone modification that appears repressive histone marks such as H3K27 trimethylation on the X and Y chromosome at the onset of MSCI in both [13]. A central prediction of the meiotic inactivation model mice [20] and opossums [1]. Consistent with previous studies of XCI is that silencing of genes on the X chromosome, initi- [18, 19], we found that inactivation of all nine mouse X-linked ated during male meiosis, is stably maintained during genes was complete, as inferred by the fact that all pachytene subsequent spermiogenesis. Here we characterize XCI in cells analyzed exhibited silencing (Figures 1B and 1C; the male germline and female soma of the marsupial Mono- Figure S1; see Figure 1 legend for details on quantitation). delphis domestica. Contrary to the meiotic inactivation We also found MSCI in the opossum to be complete, with all model, we find that X genes silenced by MSCI are reactivated ten opossum X-linked genes being silenced in all pachytene after meiosis and are subsequently inactivated in the female. cells (Figures 1B and 1C; Figure S1). We conclude that MSCI A reexamination of the female somatic inactive marsupial X leads to efficient silencing of X-linked genes in both mice chromosome reveals that it does share common properties and opossums. with that of eutherians, including H3K27 trimethylation and Next, we assessed the maintenance of silencing for each targeting to the perinucleolar compartment. We conclude X-linked gene in mouse and opossum round spermatids. that aspects of the XCI process are more highly conserved Mouse X-bearing spermatids were discriminated from in therian mammals than previously thought. Y-bearing spermatids by combined RNA FISH for the Y-linked Sly gene, which we previously showed to be expressed in all Y Results and Discussion spermatids [19]. In opossums, in which no Y-linked spermatid- expressed genes have yet been identified, X- and Y-bearing Meiotic sex chromosome inactivation (MSCI) has long been spermatids were discriminated instead by carrying out DNA proposed to represent the ancestral form of X chromosome FISH with a putative Y-linked BAC probe, MAB-198O, after inactivation (XCI) in mammals [3–6]. Recent genomic studies image capture of X gene RNA FISH signals. Three observa- have found that Xist, which mediates imprinted and random tions confirmed that MAB-198O maps to the Y chromosome: XCI in eutherians [14, 15], is not conserved in marsupials [9– DNA FISH signals were seen in male but not female somatic 12], raising the possibility that MSCI has persisted as a means cells (Figure 2A), signals colocalized with the smaller of the of X dosage compensation in this mammalian lineage. The two gH2AX-positive sex chromosome domains in early pachy- meiotic inactivation model of marsupial XCI predicts that tene spermatocytes (Figure 2B), and signals were seen in half silencing of genes on the X chromosome, initiated at pachy- of all round spermatids (Figure 2C). tene, is stably maintained during the subsequent stage of Consistent with our previous findings [19], the maintenance spermiogenesis when haploid round spermatids mature into of silencing of the X chromosome was incomplete in mouse spermatozoa. Recent studies using Cot-1 RNA fluorescence round spermatids. Of the nine X-linked genes assayed, five re- tained a silent state during spermiogenesis, but the remaining four genes exhibited reactivation (Figures 2D and 2E; Figure S2 *Correspondence: [email protected] and Table S1; see Figure 2 legend for details on quantitation). Meiotic and Somatic X Inactivation in Marsupials 1479

Figure 1. Complete Silencing of Ten X-Linked Opossum Genes and Nine X-Linked Mouse Orthologs during Pachytene by Meiotic Sex Chromosome Inactivation (A) Map positions of the ten X-linked genes in opossum and mouse. Only one of the mouse genes, Gria3 (red text), could not be detected by RNA fluores- cence in situ hybridization (RNA FISH) in testis. (B) Representative RNA FISH images for six opossum X-linked genes and their mouse orthologs (images for other genes are shown in Figure S1). Pachytene cells were identified by gH2AX staining of the sex body (red). Insets show positive control testis cells with RNA signals (arrows, green). DAPI is blue. Scale bars represent 5 mm. (C) Quantitative analysis of pachytene RNA FISH data. Data are shown as means from two male mice (n = 100 pachytene cells/gene/male) and three male opossums (n = 50 pachytene cells/gene/male). aCounts for mouse Xiap and Fmr1 have been quantitated previously [19]. Current Biology Vol 19 No 17 1480

Figure 2. Reactivation of X-Linked Genes during Opossum Spermiogenesis (A) Characterization of the Y-linked MAB-198O BAC probe by DNA FISH (red) on opossum brain cells. DAPI is blue. MAB-198O DNA signals (arrowhead) were detected in 100% (n = 100) and 0% (n = 100) of male and female brain cells, respectively. (B) MAB-198O DNA signals (arrowhead) colocalize with the Y chromosome (arrow) as delineated by gH2AX staining (green) in male early pachytene cells (n = 15 cells; DNA FISH/gH2AX merge shown in inset). The larger of the two gH2AX domains is the X chromosome. (C) RNA FISH for an X-linked gene, Mbnl3 (arrow, green), followed by MAB-198O DNA FISH (arrowheads, red). This allows the percentage of X-bearing spermatids expressing Mbnl3 to be readily determined. (D) Representative RNA FISH images (arrows, green) for six opossum X-linked genes and their mouse orthologs in spermatids (images for other genes are shown in Figure S2). (E) Quantitative analysis of spermatid RNA FISH data. All ten opossum X-linked genes showed reactivation, compared with only four of the nine mouse X-linked orthologs. Data are shown as means collected from two male mice and three male opossums (see Tables S1 and S2 for individual values). Gria3 RNA could not be detected by RNA FISH in mouse. aCounts for mouse Xiap and Fmr1 have been quantitated previously [19]. Scale bars represent 5 mm. Meiotic and Somatic X Inactivation in Marsupials 1481

Figure 3. X-Linked Opossum Genes Are Subject to Somatic X Inactivation despite Reactivation during Spermiogenesis All images are from opossum brain. Scale bars represent 5 mm. (A) Comparison of female and male somatic cells reveals a female-specific H3K27me3-positive structure (arrowheads, red; DAPI is blue). (B) Combining H3K27me3 antibody staining with DNA FISH for an X-linked Gria3 probe (arrows, green) demonstrates that the female H3K27me3-enriched structure (arrowhead) is the inactive X chromosome. H3K27me3-positive inactive Xs are seen in 98% of opossum brain cells (n = 100 cells), 60% of opossum liver cells (n = 100 cells), and 98% of mouse brain cells (n = 100 cells; see Experimental Procedures for immunostaining protocol). (C) The inactive X (arrowhead) is also found close to the nucleolus, as delineated by fibrillarin staining (green) and DAPI paucity (asterisk, blue). This close proximity is found in 98% of opossum brain cells (n = 100 cells) and 99% of opossum liver cells (n = 100 cells), compared with 54% of mouse brain cells (n = 100 cells). (D) Representative RNA FISH images for three opossum X-linked genes and their mouse orthologs (images for other genes are shown in Figure S3). Most cells have a single RNA FISH signal (arrows) in both opossum and mouse tissues, indicating nonleaky X chromosome inactivation (arrowheads). (E) Quantitative analysis of somatic RNA FISH data. Expression of five of the nine mouse X-linked and seven of the ten X-linked opossum orthologs was detectable by RNA FISH. Data are shown as means collected from two female mice and two female opossums (see Table S3 for individual values and information on quantitation). Dashes represent genes not expressed in the given tissue.

In contrast, we found that all ten X-linked genes were reacti- XCI in the female opossum and therefore might not be suitable vated in round spermatids in the opossum (Figure 2D; for testing the meiotic inactivation hypothesis. Indeed, Figure S2 and Table S2), with the number of expressing X sper- previous studies with protein electrophoretic variant analysis matids varying from gene to gene (Figure 2E). In each case, [21] and restriction fragment length polymorphism analysis RNA signals were seen only in MAB-198O-negative and not [2] have suggested that somatic XCI in marsupials is imperfect. in MAB-198O-positive spermatids, thereby confirming that We therefore examined the XCI status of the same ten genes in all ten opossum genes map to the X chromosome. These find- adult female somatic cells. We used postmortem brain and ings show that MSCI does not lead to stable silencing of liver tissue (representing derivatives of ectoderm and endo- X-linked genes in opossum round spermatids and that these derm, respectively) rather than cultured cells because the genes therefore are not preinactivated by MSCI. They also latter have been shown to exhibit abnormalities in XCI [22]. show that the maintenance of X chromosome silencing is Our analysis of somatic XCI in opossums revealed a number more stable in the mouse than in the opossum for those genes of previously undocumented similarities to that in eutherians. that we studied. We found that the inactive opossum X chromosome was en- Next, we considered the possibility that the ten opossum riched in H3K27 trimethylation in both brain and liver (Figures X-linked genes that we had analyzed might go on to escape 3A and 3B; see legend for details on quantitation). This Current Biology Vol 19 No 17 1482

female somatic cells are not preinactivated by MSCI in male germ cells. A previous study using Cot-1 RNA FISH concluded that the opossum X chromosome is silent during spermiogenesis [1]. We sought to reconcile this observation with our current finding that individual X-linked genes are active in round sper- matids. To do this, we combined RNA FISH for three X-linked genes showing spermatid reactivation, Mbnl3, Mtm1, and Htatsf1, with Cot-1 RNA FISH on opossum testis cells (Fig- ure 4A). We found that in the majority of expressing sperma- tids, the X gene RNA FISH signals originated from outside or from the edge of the Cot-1-negative domain (Figures 4A and 4B). We conclude that Cot-1 RNA FISH cannot be consistently used to draw conclusions about the activity of individual coding X-linked genes on the opossum spermatid X chromo- some. In this study, we provide compelling evidence that XCI in the opossum does not result from the inheritance of an X chromo- some already inactivated by MSCI. Rather, inactivation of the genes studied herein must occur as a de novo event in the female, as has previously been demonstrated in mice [26]. Future experiments with early embryos will be needed in order to determine when XCI begins during female opossum devel- opment. In addition, we have found that several key features of the XCI process are shared between marsupials and euthe- rians, including enrichment of H3K27 trimethylation on the inactive X chromosome and association of the inactive X chro- mosome with the nucleolus. In eutherians, both of these features of XCI require the noncoding RNA Xist [25, 27]. Assembly of the PRC2 complex, which catalyzes H3K27 trime- thylation, at the HoxD cluster also requires a noncoding RNA, HOTAIR [28]. Noncoding RNAs may therefore be required for targeting the PRC2 complex in broad developmental contexts. Considering all of these similarities together, we favor a model Figure 4. X Gene RNA FISH Signals Originate from the Edge or outside of in which XCI in female marsupials is mediated by an as yet the DAPI-Dense Sex Chromosome Domain in Round Spermatids unidentified noncoding RNA that effects silencing through (A) The positions of X gene RNA FISH signals relative to the DAPI-dense (arrowheads, shown in gray in first column and green in third column), the same conserved pathways as those used by eutherians, Cot-1-negative (red, second column) region of the opossum X chromosome e.g., H3K27 trimethylation by the PRC2 complex. was determined for three X-linked genes, Mbnl3, Mtm1, and Htatsf1 (red/ Our data also uncover interesting differences in the orange, arrows in third column). In each case, the RNA FISH signal is located dynamics of XCI in the marsupial and the eutherian male either outside (Mbnl3) or on the edge (Mtm1 and Htatsf1) of the Cot-1-nega- germline. Although all of the X-linked genes that we studied tive domain, but infrequently inside it. Scale bar represents 5 mm. were silenced in both opossum and mouse pachytene cells, (B) Quantitation of gene position relative to the DAPI-dense domain from one male opossum (n = 100 cells/gene). all exhibited subsequent reactivation in opossum spermatids, whereas only half of their respective orthologs reactivated in mouse spermatids. Based on these findings, two issues repressive histone modification, which is required for the warrant further attention. First, from a mechanistic perspec- maintenance of XCI in the mouse [23, 24], was previously tive, the enhanced ability of the mouse X chromosome to thought not to be enriched on the inactive marsupial X chro- retain X gene silencing could imply that specific DNA elements mosome [13]. Furthermore, in both tissues we noticed a very showing differential abundance on the mouse X chromosome high incidence of close proximity between the inactive X chro- versus the opossum X chromosome are involved in the main- mosome and the nucleolus, as has been observed previously tenance of MSCI. Interestingly, recent sequence analysis has in mice [25] (Figure 3C; see legend for details on quantitation). found that long interspersed nuclear elements, which are Most notably, however, XCI was efficient in female opossums thought to be involved in spreading of XCI in the female for the X-linked genes and tissues that we studied. Seven of soma [29], are more abundant on the mouse than the the ten testis-expressed X-linked genes were also expressed opossum X chromosome [30]. Second, our recent work has in adult female somatic tissue, and in all seven cases, the demonstrated that the maintenance of X chromosome repres- percentage of cells exhibiting monoallelic expression was sion in mouse spermatids may be one of the driving forces 96% or greater (Figures 3D and 3E; Figure S3 and Table S3). behind the massive amplification of spermatid-expressed The percentage of cells showing monoallelic expression was genes on the mouse X chromosome [19]. If this is the case, very similar to that observed for the mouse X-linked orthologs one might predict that amplification of spermatid-expressed in brain and liver (Figures 3D and 3E; Figure S3 and Table S3). genes may not be as marked on the opossum X chromosome. We conclude that the opossum and mouse inactive X chromo- Further developments in marsupial genomics should provide somes share common epigenetic properties. Furthermore, insights into these fundamental questions of X chromosome these findings confirm that genes subject to XCI in opossum biology. Meiotic and Somatic X Inactivation in Marsupials 1483

Experimental Procedures Acknowledgments

Animals We thank T. Jenuwein for the H3K27me3 antibody and D. Page for the Material for this study was acquired from opossums and MF1 random-bred MAB-198O probe. We also thank P. Burgoyne, J. Cocquet, L. Reynard, mice maintained at the MRC National Institute for Medical Research (NIMR) H. Byers, J. Mueller, and D. Page for critical reading of the manuscript. according to UK Home Office regulations and also from opossums main- This work was supported by the Medical Research Council (UK). tained in San Antonio, TX, USA. Testis material was harvested from two 8-week-old mice and from three opossums aged 19 months, 2 years Received: May 26, 2009 (NIMR), and 1 year (San Antonio). Adult somatic tissue was harvested Revised: July 6, 2009 from two male and two female mice aged 4 months and from two male Accepted: July 7, 2009 and two female opossums aged 2 years (NIMR). Published online: August 27, 2009

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