Coordination of Engineered Factors with TET1/2 Promotes Early-Stage Epigenetic Modification during Somatic Cell Reprogramming

Abstract

SummarySomatic cell reprogramming toward induced pluripotent stem cells (iPSCs) holds great promise in future regenerative medicine. However, the reprogramming process mediated by the traditional defined factors (OSMK) is slow and extremely inefficient. Here, we develop a combination of modified reprogramming factors (OySyNyK) in which the transactivation domain of the Yes-associated protein is fused to defined factors and establish a highly efficient and rapid reprogramming system. We show that the efficiency of OySyNyK-induced iPSCs is up to 100-fold higher than the OSNK and the reprogramming by OySyNyK is very rapid and is initiated in 24 hr. We find that OySyNyK factors significantly increase Tet1 expression at the early stage and interact with Tet1/2 to promote reprogramming. Our studies not only establish a rapid and highly efficient iPSC reprogramming system but also uncover a mechanism by which engineered factors coordinate with TETs to regulate 5hmC-mediated epigenetic control.

Distinctive Klf4 mutants determine preference for DNA methylation status

“…E446D exhibited 2–2.5X greater activities in transactivation on the 2C reporter than with 2T. The same trend is observed when Klf4 was transfected together with native Oct4, Sox2 and Nanog (OSN in Figure 3C; the red lines between panels B, C and D indicate the changed scales of the luciferase activity along y axes), and with modified factors fused to the murine Yap transcription activation domain (28) (OySyNy in Figure 3D and Supplementary Figure S2). While overall transactivation activities are lower than those of the D446 variant, both E446 (WT) and the P446 variant seem to have higher activity with 2T than with 2C under the CMV driven expression (Figure 3C and D).…”

“…We first modified a pGL4.2-Basic-6XCR4 reporter plasmid (28) to have two copies of CR4, which contains at different positions the binding sites of Klf4, Oct4 and Sox2/Nanog (Figure 3A). We introduced either TpG (‘2T’ reporter plasmid, representing methylated DNA) or CpG (‘2C’ reporter plasmid, representing unmodified DNA) within the Klf4 binding sites upstream of the lux luciferase gene (Figure 3A).…”

“…pMXs-full length Klf4 (WT, E446P and E446D) (28), and pcDNA3.1-Flag-full length Klf4 (WT, E446P and E446D), pGL4.2-Basic-2XCR4 (2C) and pGL4.2-Basic-2XCR4 (2T) were generated. Two copies of CR4, which contains binding sites of mouse Klf4, Oct4, and Sox2/Nanag were subcloned into pGL4.2-[Luc/Puro] vector (generating 2C), and the Klf4 binding site mutated Cyt-to-Thy by PCR (generating 2T).…”

The novel application of cordycepin in maintaining stem cell pluripotency and increasing iPS cell generation efficiency

“…To date, there were several strategies to enhance the reprogramming efficiency, including knockdown of p53 gene 9 , hypoxic conditions 10,11 , epigenetic modification 12 , regulation of microRNAs 13 and addition of small molecular compounds 14,15 . In 2003, one group reported a near 100% reprogramming efficiency in mouse and human cells via OKSM transduction and Mbd3 depletion 16 .…”

5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts

“…Based on preliminary Whole Transcriptome Analysis, obtained with an Applied Biosystems SOLiD 5500xl Sequencer, we investigate in details changes in transcriptions of the TET family genes, that affect methylation2122 and play an essential role in pluripotency regulation of ESC2122 and in the very early stage of somatic cell reprogramming toward induced Pluripotent Stem Cells (iPSCs)23. We study whether upregulation of TET2 results in increased levels of 5-Formylcytosine (5fC) and 5-Carboxylcytosine (5caC), which are both products of TET2 enzyme-mediated oxidation of 5-methylcytosine.…”