Fig. 5

Platr10 coordinates DNA methylation in the Oct4 promoter by recruiting TET1 demethylase. A Differential expression of TET family genes during reprogramming. Cells were collected at different stages of reprogramming and the expression of the three TET demethylases was measured by RT-PCR. FIB, fibroblasts; non-iPSC, cells that ectopically express OSKM cocktail factors, but fail to complete reprogramming; iPSC, reprogrammed pluripotent stem cells. *p < 0.05, **p < 0.01 as compared with other two groups. B Interaction of Platr10 with TET1 DNA demethylase enzyme by RNA-chromatin immunoprecipitation (RIP). After formaldehyde crosslinking, the TET1-lncRNA chromatin complex was immunoprecipitated with a TET1-specific antibody. After de-crosslinking, the immunoprecipitated RNAs were reverse-transcribed. The TET-interacting lncRNAs were measured by PCR. IgG was used as the antibody control. Input: aliquot DNAs collected during the RIP assay. Note that lncRNA controls Palr35 and Palr34 did not interact with TET1, even though these two lncRNAs were also differentially activated in pluripotent reprogramming. C Identification of the TET1 binding fragment by RIP mapping. Top panel: Schematic diagram of RIP mapping. iPSC cells were fixed and were subject to a more stringent sonication treatment in order to break the Platr10 lncRNA regions that are not a part of the TET1 binding site. After immunoprecipitation with a TET1 antibody, the TET1 interacting Platr10 lncRNA fragments were mapped by quantitative PCR using overlapping primers (middle panel). For comparison, the value of the IgG control was set as 1. **p < 0.01 as compared with other PCR fragments. The F3 and F9 show a strong binding of TET1