Fig. 4

zPRDM14, but not sPRDM14, induces hypomethylation at pluripotency-associated genes and germline-specific genes. (A) Western blotting of DNMT3A, DNMT3B and DNMT3L in wild-type ESCs and in mPrdm14 KO ESCs expressing the empty vector, sPRDM14, zPRDM14 or mPRDM14. (B) Luciferase activity driven by Dnmt3b against each PRDM14 orthologue. Relative luciferase activities of the pGL3-basic vector are shown with the s.e.m. (biological triplicates). P values were calculated using Student's t-test. *P<0.05, **P<0.01, ***P<0.001. (C) qRT-PCR analysis of pluripotency-associated genes and germ-line specific genes in wild-type ESCs and mPrdm14 KO ESCs expressing the empty vector, sPRDM14, zPRDM14 or mPRDM14. Each Ct value was subtracted from that in wild-type ESCs and is shown with the s.e.m. (two technical replicates). (D) DNA-methylation status of pluripotency-associated genes and germline-specific genes in wild-type ESCs and mPrdm14 KO ESCs expressing the empty vector, sPRDM14, zPRDM14 or mPRDM14 were measured by bisulphite sequencing. The red bars indicate the primer positions. The white circles indicate unmethylated cytosine or 5-formylcytosine or 5-carboxylcytosine, and black circles indicate 5-methylcytosine or 5-hydroxymethylcytosine. (E) Boxplot based on the bisulphite sequencing data. The central bars indicate the medians and lower and upper limits of the boxes, marking the 25th and 75th percentiles. P values were calculated using the Mann–Whitney U-test.