Fig. 6

Estrogen receptor alpha (ERα) co-binds to ten-eleven translocation 2 (TET2) and regulates the levels of mesoderm-specific transcript homologue (Mest) imprinted methylation in mouse parthenogenetic embryonic stem cells (pESCs). A, B Protein expression of TET2 and ERα following 24 h of pESCs culture with 1 nM or 100 nM was determined using western blot analysis (n = 6). C Quantification of Tet2, ERα and Mest expression levels within each group by quantitative real-time polymerase chain reaction (qRT-PCR, n = 6). D, E Chromatin immunoprecipitation (ChIP) and Co-immunoprecipitation (CoIP) assay using ERα as bait protein demonstrated the interaction between ERα and TET2 (n = 3). Anti-rabbit IgG immunoprecipitation (IP) was used as a negative control. F Jaspar online website was used to predict binding sites of ERα to Mest. G, H QRT-PCR was performed with the indicated primer pair using an anti-ERα antibody immunoprecipitated DNA fragment as template (n = 6). I PCR products were separated by agarose gel electrophoresis and subjected to quantitative analysis (n = 3). J Analysis of methylation levels at cytosine preceding a guanine base (CpG) sites within the differentially methylated region (DMR) in the Mest promoter were performed using bisulfite sequencing PCR (BSP, n = 9). Data are expressed as the means ± standard deviation (SD), *P < 0.05, **P < 0.01, ***P < 0.001. (Unpaired two-tailed t test was used to compare differences among two groups and one-way ANOVA multiple comparisons test was performed among the three groups)