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DNMT3L Is a Regulator of X Chromosome Compaction and Post-Meiotic Gene Transcription

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DNMT3L Is a Regulator of X Chromosome Compaction and Post-Meiotic Gene Transcription DNMT3L Is a Regulator of X Chromosome Compaction and Post-Meiotic Gene Transcription Natasha M. Zamudio1,2, Hamish S. Scott4, Katja Wolski1, Chi-Yi Lo1, Charity Law5,6, Dillon Leong5, Sarah A. Kinkel5,7, Suyinn Chong6, Damien Jolley3, Gordon K. Smyth5, David de Kretser1, Emma Whitelaw6, Moira K. O’Bryan1,2* 1 The Department of Anatomy and Developmental Biology, Monash University, Victoria, Australia, 2 The Australian Research Council Centre of Excellence in Biotechnology and Development, Monash University, Victoria, Australia, 3 The Monash Institute of Health Services Research, Monash University, Victoria, Australia, 4 The Institute of Medical and Veterinary Science, University of Adelaide, Adelaide, Australia, 5 The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia, 6 Queensland Institute of Medical Research, Herston, Queensland, Australia, 7 Department of Medical Biology, University of Melbourne, Victoria, Australia Abstract Previous studies on the epigenetic regulator DNA methyltransferase 3-Like (DNMT3L), have demonstrated it is an essential regulator of paternal imprinting and early male meiosis. Dnmt3L is also a paternal effect gene, i.e., wild type offspring of heterozygous mutant sires display abnormal phenotypes suggesting the inheritance of aberrant epigenetic marks on the paternal chromosomes. In order to reveal the mechanisms underlying these paternal effects, we have assessed X chromosome meiotic compaction, XY chromosome aneuploidy rates and global transcription in meiotic and haploid germ cells from male mice heterozygous for Dnmt3L. XY bodies from Dnmt3L heterozygous males were significantly longer than those from wild types, and were associated with a three-fold increase in XY bearing sperm. Loss of a Dnmt3L allele resulted in deregulated expression of a large number of both X-linked and autosomal genes within meiotic cells, but more prominently in haploid germ cells. Data demonstrate that similar to embryonic stem cells, DNMT3L is involved in an auto- regulatory loop in germ cells wherein the loss of a Dnmt3L allele resulted in increased transcription from the remaining wild type allele. In contrast, however, within round spermatids, this auto-regulatory loop incorporated the alternative non- coding alternative transcripts. Consistent with the mRNA data, we have localized DNMT3L within spermatids and sperm and shown that the loss of a Dnmt3L allele results in a decreased DNMT3L content within sperm. These data demonstrate previously unrecognised roles for DNMT3L in late meiosis and in the transcriptional regulation of meiotic and post-meiotic germ cells. These data provide a potential mechanism for some cases of human Klinefelter’s and Turner’s syndromes. Citation: Zamudio NM, Scott HS, Wolski K, Lo C-Y, Law C, et al. (2011) DNMT3L Is a Regulator of X Chromosome Compaction and Post-Meiotic Gene Transcription. PLoS ONE 6(3): e18276. doi:10.1371/journal.pone.0018276 Editor: Martin Pera, University of Southern California, United States of America Received November 14, 2010; Accepted March 1, 2011; Published March 31, 2011 Copyright: ß 2011 Zamudio et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by National Health and Medical Research Council of Australia (http://www.nhmrc.gov.au/) grants and fellowships to Moira O’Bryan, Emma Whitelaw, David de Kretser, Hamish Scott, Damien Jolley; an Australian Research Council (http://www.arc.gov.au/) grant to Moira O’Bryan. Natasha Zamudio and Charity Law and Sarah Kinkel were supported by Australian Postgraduate Awards. The funders had no role in study design, data collection and analysis, decisions to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Moira.O’[email protected] Introduction isoforms is unknown, they are thought to be non-coding and to suppress translation of the full length Dnmt3L isoform [9]. The Infertility affects 1 in 20 men in the Western world. Increasingly localization of DNMT3L protein within the adult testis or sperm data is showing that in addition to genetic changes, aberrant has not been reported. epigenetic regulation during spermatogenesis can lead to human Dnmt3L was recently reported as the first example of a infertility [1–3]. Previous studies on the epigenetic regulator mammalian paternal effect gene [10]. Animals with mutations in DNMT3L, have shown that deletion of Dnmt3L in mice resulted in such genes are of particular interest as they exhibit a male hypomethylation of paternally imprinted regions and transposable heterozygous phenotype, that can affect the phenotype of their elements in gonocytes, as well as developmental arrest and death wild type progeny. Specifically, when Dnmt3L heterozygous males of early meiotic cells [4–6]. Consequently Dnmt3L knockout males were mated to wild type females, there was an increased incidence were sterile. of XO monosomy in wild type E10.5 embryos and adults Dnmt3L mRNA and protein were originally reported only in the compared to wild type matings [10]. These data suggested that primordial germ cells, where DNMT3L is needed to establish the Dnmt3L heterozygous males may have a meiotic defect related epigenetic marks required in early primary spermatocytes [4,7]. to X and Y chromosome segregation and encouraged us to study Subsequently, it was shown that Dnmt3L mRNA was present in the effects of the loss of a Dnmt3L allele in germ cells in more detail. spermatocytes and spermatids [5,8]. Furthermore, it was shown We hypothesised that decreased availability of DNMT3L in that a second promoter in intron 9 initiates three alternative Dnmt3L heterozygous males may lead to the inappropriate transcripts (Forms 1, 2 and 3) in late pachytene spermatocytes and epigenetic marking of the DNA, and a partial failure of sex round spermatids [9]. While the function of the alternative chromosomes to convert from an euchromatic state to a highly PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e18276 DNMT3L in Meiotic and Haploid Germ Cells condensed heterochromatic state leading to a meiotic instability. heterozygotes, (Fig. 1C). No significant difference was observed We hypothesised that if true, abnormal XY compaction would be between the lengths and distribution of the Dnmt3L wild type and reflected by aberrant patterns of gene expression in meiotic and the C57Bl/6 wild type XY bodies (95% CI: 22.6 to +1.1, haploid germ cells. To investigate these hypotheses we measured P = 0.42) (data not shown). These data suggest that the loss of a the lengths of the XY bodies in meiotic cells from heterozygous Dnmt3L allele resulted in a partial failure of XY body chromatin and wild type males, we defined the incidence of XY sperm compaction during meiosis. Interestingly, we observed no aneuploidies, and monitored transcriptional activity in spermato- abnormalities in autosome pairing in germ cells from Dnmt3L cytes and round spermatids. heterozygous males (data not shown). Dnmt3L heterozygous males had XY bodies which were significantly longer than those from their wild type brothers, and Dnmt3L heterozygous males had an increased risk of exhibited a significantly increased incidence of XY non-disjunc- producing XY sperm tion. We demonstrated that a loss of a Dnmt3L allele was associated To address the hypothesis that a partial failure of XY body with disregulation of a number of X-linked and autosomal genes in compaction would lead to an increase in XY bearing sperm in spermatocytes and spermatids. Consistent with these observations, Dnmt3L heterozygous males, we used chromosome paints to label we localized DNMT3L protein within spermatids and mature decondensed sperm for X and Y chromosomes (Fig. 2). The visual sperm. Furthermore, our data strongly suggested a complex assessments and counting were all conducted without knowledge Dnmt3L self-regulatory mechanism in germ cells similar to that of genotype. Sperm with no labelling were not included in the previously reported in embryos and ES cells [11], but additionally analysis in order to avoid the possibility of false negative sperm as a involving the alternative transcripts. consequence of incomplete sperm head decondensation. The number of sperm lacking a sex chromosome should however, Results equal the number carrying both an X and a Y chromosome. Dnmt3L heterozygous males displayed XY body From a total of 2,063 sperm (from 9 mice) we found 7 sperm with a XY chromosome content in wild type males, compared to abnormalities 22 sperm (from 8 mice) from a total of 2,155 sperm in Dnmt3L During male meiosis, the X and Y chromosomes form an XY heterozygous males (Supporting Information File S1). The total body and become transcriptionally silent in a process known as number of X and/or Y bearing sperm from wild type and Dnmt3L meiotic sex chromosome inactivation (MSCI) [12,13]. Unlike the heterozygous males (analysed separately) was the same, suggesting autosomes, in males sex chromosomes are heterologous and only that the FISH probes worked equally well and that the loss of a synapse at their pseudoautosomal regions. To test the hypothesis Dnmt3L allele did not result in a sex chromosome bias at the level that DNMT3L has a role in X chromosome meiotic condensation, of the sperm (data not shown). A negative binomial
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