Global Chromatin Reprogramming in Fetal Germ Cells Figure 9. Nuclear distribution of H3K79me2 and H3K79me3 in fetal germ cells. Female and male germ cells (FGC and MGC, respectively) and female somatic cells (SC) at 15.5 dpc were stained with the H3K79me2 and H3K79me3 antibodies as indicated and are shown in the different channels separately and in red-blue composite. (A) DAPI-stained chromocenters are excluded from H3K79me2 signals (open arrowheads). (B) DAPI-rich chromocenters colocalize with H3K79me3 signals (closed arrowheads). Other details are as in legend to Figure 1. doi:10.1371/journal.pone.0023848.g009 indicated the presence of Y chromosome and male sex, whereas amplification of the Snrpn gene served as a positive control. Primer sequences were as follows: Sry forward: 59-ATGGAGGGC- CATGTCAAGCG-39; Sry reverse: 59-TGCCACTCCTCTGT- GACACTTTAG-39; Snrpn forward: 59-CTTTTGGTAGCTGC- CTTTTGG-39 and Snrpn reverse: 59-CTAGTCTTGCCGCA- ATGGCTC-39. Embryo tails were boiled at 95uC for 10 min in distilled water and the genomic DNA was used for template in PCR. The PCR condition was as follows; 95uC for 2 min, 95uC for 30 s, 58uC for 45 s, 72uC for 40 s (31 cycles), followed by 72uC for 5 min. Antibodies The following antibodies were used for immunostaining (dilution and source indicated in parentheses): rabbit anti- H3K9me2 (1/1000, Upstate 07-441); rabbit anti-H3K9me3 (1/ 1000, Upstate 17-625); rabbit anti-H3K9ac (1/2000, Upstate 07- 352); rabbit anti-H3K4me3 (1/2000, Upstate 07-473); rabbit anti- H3K27me3 (1/2000, Upstate 07-449); rat anti-Oct3/4 (1/1000, RD systems MAB1759); mouse anti-5-MeC (1/500, Eurogentec Figure 10. Summary of global epigenetic changes in fetal germ cells. The relative staining intensities of germ cells versus somatic germ cells (GC/SC) were combined from Figures 1–8. The data points at different fetal stages (in dpc) were connected to show the general trends in global chromatin changes. Male fetal germ cells (blue lines) exhibit strong increase in both active and repressive histone marks at 15.5–17.5 dpc, preceding the peak of global de novo DNA methylation that occurs at 17.5 dpc. Female germ cells (red lines) do not undergo global de novo methylation and do not exhibit global chromatin changes during fetal development. doi:10.1371/journal.pone.0023848.g010 900 P-A); mouse anti-DNA (1/200, QED 12404); rabbit anti-Ddx4/ Mvh (1/2000, Abcam ab13840-100); rabbit anti-H3K79me2 (1/ 2000, Upstate 07-366) and rabbit anti-H3K79me3 (1/2000, Abcam ab2621). The following secondary antibodies from Molecular Probes were used at a 1/1000 dilution: Alexa Fluor 488 goat anti-rat IgG; Alexa Fluor 488 goat anti-rabbit IgG; Alexa Fluor 568 goat anti- rabbit IgG; Alexa Fluor 568 goat anti-mouse IgG; Alexa Fluor 350 goat anti-mouse IgG. Immunofluorescence staining of chromatin marks The genital ridges or gonads of the relevant developmental stages were trypsinised and the single cells suspension was settled on the poly-L-lysine (Sigma Aldrich, St. Louis, MO) treated slides PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e23848