Sex-Specific Dynamics of Global Chromatin Changes in Fetal Mouse Germ Cells Masanobu Abe1.¤, Shirley Y. Tsai1., Seung-Gi Jin2, Gerd P. Pfeifer2, Piroska E. Szabo´ 1 * 1 Department of Molecular and Cellular Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, United States of America, 2 Department of Cancer Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, United States of America Abstract Mammalian germ cells undergo global reprogramming of DNA methylation during their development. Global DNA demethylation occurs around the time when the primordial germ cells colonize the embryonic gonads and this coincides with dynamic changes in chromatin composition. Global de novo DNA methylation takes place with remarkably different dynamics between the two sexes, prospermatogonia attaining methylation during fetal stages and oocytes attaining methylation postnatally. Our hypothesis was that dynamic changes in chromatin composition may precede or accompany the wave of global DNA de novo methylation as well. We used immunocytochemistry to measure global DNA methylation and chromatin components in male and female mouse fetal germ cells compared to control somatic cells of the gonad. We found that global DNA methylation levels sharply increased in male germ cells at 17.5 days post coitum, but remained low in female germ cells at all fetal stages. Global changes in chromatin composition: i, preceded global DNA methylation in fetal germ cells; ii, sex specifically occurred in male but not in female germ cells; iii, affected active and repressive histone marks and iv, included histone tail and histone globular domain modifications. Our data suggest that dynamic changes of chromatin composition may provide a framework for the pattern of male-specific de novo DNA methylation in prospermatogonia. Citation: Abe M, Tsai SY, Jin S-G, Pfeifer GP, Szabo´ PE (2011) Sex-Specific Dynamics of Global Chromatin Changes in Fetal Mouse Germ Cells. PLoS ONE 6(8): e23848. doi:10.1371/journal.pone.0023848 Editor: Esteban Ballestar, Bellvitge Biomedical Research Institute (IDIBELL), Spain Received April 13, 2011; Accepted July 29, 2011; Published August 19, 2011 Copyright: 2011 Abe 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 work was supported by National Institutes of Health grants RO1GM064378 and RO1ES015185 to PES. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pszabo@coh.org . These authors contributed equally to this work. ¤ Current address: Department of Oral and Maxillofacial Surgery, University of Tokyo Hospital, Tokyo, Japan Introduction Global waves of epigenetic changes during development At two times during development, in the germ line and during preimplantation, the epigenome of mammals undergoes global resetting [1,2,3]. Global epigenetic reprogramming includes erasure of existing DNA methylation and establishment of new DNA methylation [4,5]. Global demethylation occurs in primor- dial germ cells (PGCs) around the time when they colonize the genital ridges. Global de novo DNA methylation occurs with different dynamics between the two sexes. Prospermatogonia attain methylation during fetal stages and oocytes attain methylation postnatally. Immunostaining experiments revealed that dynamic and orderly chromatin changes take place in PGCs at the time of their specifications and during the time when DNA methylation erasure occurs [6,7,8,9,10]. Specifically, heterochro- matin marks H3K9me2, H3K9me3 and H3K64me3 are tran- siently lost from PGCs at 8.0, 11.5 and 12.5 days post coitum (dpc), respectively [6,7]. Little is known, however, about global chromatin changes during fetal germ cell development, and the information is limited to spatial reorganization of heterochromatin components H3K9me3 and CBX5 (HP1a) [10]. The information regarding global DNA methylation establish- ment in fetal germ cells mainly comes from bisulfite sequencing experiments at differentially methylated regions (DMRs) of imprinted genes and at repeat elements. Imprinted genes are expressed from one of the parental alleles in the soma [11,12], whereas imprinted expression is neutralized in the germ line [13,14]. Gametic imprints [15] —DNA methylation marks at DMRs that originate in sperm or oocyte— are critical for allele- specific monoallelic expression of imprinted genes in the soma [16,17,18,19,20]. Parental-specific methylation is maintained at DMRs in somatic cells during the life of the individual, but is reset (erased and reestablished) in the germ line according to the sex of the individual. In the mouse, DMRs become demethylated in primordial germ cells (PGCs), between 9.5 and 12.5 dpc [4,21]. Remethylation of paternal DMRs begins at 14.5 dpc in prosper- matogonia [22,23,24,25], and of maternal DMRs after birth in growing oocytes [26,27]. The mechanism of imprint establishment is not fully understood, but non-coding RNA targeting by piRNA [28], tandem repeats [29] that serve as promoter for non-coding RNA [28], transcription-related events [30], and a chromatin modifying protein [31] play role in the process. DNA and histone methylation are structurally and functionally linked [32]. Mammalian DNA methylation depends on the methylation status of H3K4 and H3K9 residues. H3K9 methylation by SUV39H1 is required for DNA methylation at the pericentric heterochromatin in mice [33]. Significantly, the H3K4 demethylase PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23848