Histone

Gene

Cell biology

SOX2

KLF4

Molecular biology

Induced pluripotent stem cell

Biology

Pioneer factor

Enhancer

Demethylase

Demethylation

DNA demethylation

DNA methylation

Genetics

Chromatin

Embryonic stem cell

Methylation

Transcription factor

5-Hydroxymethylcytosine

Cell

Gene expression

Reprogramming

Cell stem cell

Here, we report DNA methylation and hydroxymethylation dynamics at nucleotide resolution using C/EBPα-enhanced reprogramming of B cells into induced pluripotent cells (iPSCs). We observed successive waves of hydroxymethylation at enhancers, concomitant with a decrease in DNA methylation, suggesting active demethylation. Consistent with this finding, ablation of the DNA demethylase Tet2 almost completely abolishes reprogramming. C/EBPα, Klf4, and Tfcp2l1 each interact with Tet2 and recruit the enzyme to specific DNA sites. During reprogramming, some of these sites maintain high levels of 5hmC, and enhancers and promoters of key pluripotency factors become demethylated as early as 1 day after Yamanaka factor induction. Surprisingly, methylation changes precede chromatin opening in distinct chromatin regions, including Klf4 bound sites, revealing a pioneer factor activity associated with alternation in DNA methylation. Rapid changes in hydroxymethylation similar to those in B cells were also observed during compound-accelerated reprogramming of fibroblasts into iPSCs, highlighting the generality of our observations.

Transcription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate