Figure 4: H3K9me3 states at the Xm-Xist promoter region in preimplantation embryos.

(a) ChIP-qPCR analysis of Xp (sperm) at Xist 5′-regions, the H19, Gtl2, Peg1 and Peg3 promoter region, and regions in repetitive elements. n=2–4. Rabbit IgG was used as a negative control. The percentage of input for negative control DNA was >1% for all genes tested. The data were not normalized for local nucleosome occupancies. (b) Schematic representation of eChIP-qPCR analysis. H3K9me3 states at major satellite regions (c) and at Xist regions and the Gapdh promoter region (d) in PEs at the four-cell stage. Two independent experiments were performed. In each experiment, 250 embryos were used. (e) H3K9me3 states at the Xist and Gapdh promoter regions in morula-stage embryos. Three independent experiments were conducted and 40 embryos were used for each assay. H3K9me3 states in major satellite (f) and Xist regions (g) in Egfp- and Kdm4b-PEs at the four-cell stage. Three (Kdm4b-PEs) and four (Egfp-PEs) independent experiments were conducted. In each experiment, 170–250 embryos were prepared. The percentages of input for negative controls (IgG) were <0.2% (f) and 1.9% (g), respectively. Error bars indicate the mean±s.e.m. The P-values were determined using Student’s t-tests.