Fig. 6

Mutation of the Oct4 binding element and TET1 binding elements abolishes the function of Platr10 lncRNA. A Diagram of TET1 binding element (TBE) mapping. The biotin-labeled Platr10 full-length lncRNA was incubated with TET1 recombinant protein. After binding, RNase A was used to remove the free Platr10 lncRNA fragments. After treatment with proteinase K, the TET1 protected Platr10 lncRNA fragment was purified and was used for RNA library cloning. DNA sequencing was then performed to map the TET1 binding elements. B Identification of the TET1 binding element. Left panel: Western blot detection of the Oscrl8-TET1 interaction. After binding, the Platr10-Tet1 complex was pulled-down by streptavidin beads and was subject to Western blot analysis. Platr10, biotin-labeled Platr10 full-length lncRNA; CTL, biotin-labeled Platr10 antisense lncRNA control; TET1, recombinant protein. Right panel: read sequences of the TET1 binding library. TBE58: consensus TET1 binding element. C Requirement of the OBE and TBE elements in Platr10 lncRNA. Fibroblasts (BMW2) were transfected with lentiviruses carrying full-length Platr10 or Platr10 mutants that lack either the OBE or TBE. After puromycin selection, mixed cells were collected for qPCR analyses of the endogenous core stem cell factor genes. *p < 0.01 as compared with controls; **p < 0.01 as compared with the full length Platr10 lncRNA. The function of Platr10 was abrogated in the OBE50D, TBE58D, and TBE58M groups. D Putative model of Platr10 in maintaining pluripotency. Open circle under the promoter: unmethylated CpG islands; TET1: DNA demethylases. In un-reprogrammed cells, such as fibroblasts, the Oct4 promoter is fully methylated and is transcriptionally inactive. During reprogramming, Platr10 becomes activated. By binding to the core stem cell gene network, Platr10 orchestrates an intrachromosomal loop, juxtaposing the enhancers close to the promoter. In addition, Platr10 also recruits TET1 and induces DNA demethylation in the promoter. By activating pluripotency-associated genes, the lncRNA promotes reprogramming and keeps stem cells from differentiation.