DNA methylation states of ECATs expressed in GS cells. Bisulfite genomic sequencing was performed to examine the DNA methylation profiles for the individual CpG sites of ECAT genes in ES cells, somatic cells, and male germ cells. Schematic diagrams of regions analyzed are shown in the upper part of the figure. Black rectangles represent the exons, and arrows indicate transcription initiation sites. Black horizontal bars indicate the regions analyzed. All of diagrams are in scale relative to a 100-bp scale bar. In the diagram for the Rex1 gene, the shaded square and open circle indicate the binding sites for Rox1 and Oct3/4, respectively. DNA methylation states in ES cells, somatic cells, and germ cells are shown in the lower part of the figure. Open circles indicate unmethylated CpGs and closed circles indicate methylated CpGs. The numbers indicate the relative positions of CpG sites from the transcription initiation site. The CpG nearest to the transcription initiation site is described as -1 (upstream) or 1 (downstream). Positions where CpG is absent due to DNA polymorphism are indicated by hyphens. ES, undifferentiated ES cells (129 background); ES (RA), ES cells differentiated by retinoic acid treatment; MEFs, MEFs derived from E13.5 C57BL/6J embryos; GS, GS cells derived from DBA neonate testis. Brain, kidney, and testis were obtained from 8-week-old C57BL/6J mice. Sperm was isolated from the epididymis of 11-week-old C57BL/6J mice.

DNA methylation state of the Nanog locus. Bisulfite genomic sequencing was performed to examine the DNA methylation profile of individual CpG sites in the regions flanking exon 1 of Nanog in ES cells, somatic cells, and male and female germ cells. Results are shown as described in Figure 2 and 3 except for the size of the scale bar (500 bp). Oblique lines indicate regions not examined and their approximate lengths are shown. Oocytes were collected from oviducts of C57BL6 mice after superovulation. ND; not determined.

DNA methylation states of Oct3/4, Sox2, and Utf1 . Bisulfite genomic sequencing was performed to examine the DNA methylation profiles for individual CpG sites in the Oct3/4, Sox2, and Utf1 loci in ES cells, somatic cells, and male germ cells. Results are shown as described in Figure 2 and 3. Oblique lines indicate regions not examined and their approximate lengths are shown.

Expression of genes located in the ECAT cluster on chromosome 6. RT-PCR was performed to examine the expression of Aicda, Apobec1, Gdf3, Stella, Slc2a3, and Nanog. PCR amplification cycle numbers are shown on the right.

The binding of Oct3/4 and Sox2 to the Octamer/Sox elements. A. ChIP analyses was performed with anti-Oct3/4 antibody (αOct3/4) and anti-Sox2 antiserum (αSox2), or with normal mouse IgG as a negative control (IgG). The genomic region with the Octamer/Sox element in Nanog, Fgf4, Fbx15, Sox2, UTF1, and Rif1 gene was amplified with specific primers. B. Western blot analysis of Oct3/4 and Sox2 proteins in ES cells and GS cells. We used β-actin as a loading control.

Expression of ECATs in male germline stem (GS)cells and ES cells. A. RT-PCR was performed for the number of cycles shown on the right. We categorized ECATs into four groups based on their relative expression levels in GS cells compared to ES cells. To monitor amplification from genomic DNA, we amplified samples in which reverse transcriptase was omitted from the reverse transcription reaction (RT-). As negative controls, water was added instead of cDNA (H2O). NAT1 and Gapdh were used as loading controls. B. Expression of Fbx15 variants examined by RT-PCR. Our previous study using 5' RACE showed that Fbx15 is transcribed from different promoters in ES cells and testes in the mouse (unpublished data). The ES cell-specific variant was not detected in GS cells or adult testis, while the testis-specific variant was weakly detected in ES cells. The two transcripts differ only in exon 1 sequence. The common sequence of the two transcripts was also amplified. NAT1 and Gapdh were used as loading controls.C. Real-time PCR quantification of the expression of Oct3/4 and Sox2 target genes. The expression of each gene was normalized with that of Gapdh. The expression in ES cells was set as 1.0.

DNA methylation states of non-ECAT genes. Bisulfite genomic sequencing was performed to examine the DNA methylation profiles for individual CpG sites in the promoter regions of germ cell specific genes Mvh and preproacrosin, and a differentiation marker Hoxb1 in ES cells, somatic cells, and male germ cells. Results are shown as described in Figure 2. In the diagram for the Hoxb1 gene, the open square and shaded circle indicate the binding sites for Sox2 and Oct1, respectively.

DNA methylation states of Fbx15 and Fgf4 . Bisulfite genomic sequencing was performed to examine the DNA methylation profiles for individual CpG sites in the Fbx15 and Fgf4 loci in ES cells, somatic cells, and male germ cells. Results are shown as described in Figure 2 and 3. Oblique lines indicate regions not examined and their approximate lengths are shown. White rectangle in the diagram for Fgf4 gene indicates 3' UTR.

DNA methylation states of the Octamer/Sox elements of non-ECAT genes. A. Bisulfite genomic sequencing was performed to examine the DNA methylation profiles for individual CpG sites in the Octamer/Sox elements of non-ECAT genes in ES cells, somatic cells, and male germ cells. Results are shown as described in Figure 2 and 3. Oblique lines indicate regions not examined and their approximate lengths are shown. B. Results of RT-PCR showing the expression of REST/NRSF, Rif1, and Tcf3 in ES cells and GS cells.

DNA methylation states of genes in the ECAT cluster on chromosome 6. Bisulfite genomic sequencing was performed to examine the DNA methylation profile of individual CpG sites in the promoter regions of Aicda, Apobec1, Gdf3, Stella/Dppa3, LOC384480, LOC626074, Nanog, and Slc2a3. Results are shown as described in Figure 2 and 3.