Abstract
Direct reprogramming of fibroblasts to neurons induces widespread cellular and transcriptional reconfiguration. Here, we characterized global epigenomic changes during the direct reprogramming of mouse fibroblasts to neurons using whole-genome base-resolution DNA methylation (mC) sequencing. We found that the pioneer transcription factor Ascl1 alone is sufficient for inducing the uniquely neuronal feature of non-CG methylation (mCH), but co-expression of Brn2 and Mytl1 was required to establish a global mCH pattern reminiscent of mature cortical neurons. Ascl1 alone induced promoter CG methylation (mCG) of fibroblast specific genes, while BAM overexpression additionally targets a competing myogenic program and directs a more faithful conversion to neuronal cells. Ascl1 induces local demethylation at its binding sites. Surprisingly, co-expression with Brn2 and Mytl1 inhibited the ability of Ascl1 to induce demethylation, suggesting a contextual regulation of transcription factor - epigenome interaction. Finally, we found that de novo methylation by DNMT3A is required for efficient neuronal reprogramming.
Highlights
Mesoderm originated fibroblast cells can be reprogrammed to ectodermal induced neuronal cells by the overexpression of proneural transcription factor (TF) Ascl1 (Chanda et al, 2014; Vierbuchen et al, 2010)
We analyzed the methylomes of control MEFs, cells at initial (Ascl1 2d) and intermediate stages of reprogramming (Ascl1 5d and Brn2 and Mytl1 with Ascl1 (BAM) 5d) and fully reprogrammed induced neuronal (iN) cells (Ascl1 22d and BAM 22d). 5d and 22d cells were isolated by fluorescence-activated cell sorting (FACS) using a neuronal TauEGFP reporter
Robust mCH accumulation suggests that iN cells recapitulates a major epigenomic signature that starts to emerge during the second postnatal week of mouse brain development and supports the notion that fully reprogrammed iN cells resemble differentiated neurons (Lister et al, 2013; Vierbuchen et al, 2010)
Summary
Mesoderm originated fibroblast cells can be reprogrammed to ectodermal induced neuronal (iN) cells by the overexpression of proneural transcription factor (TF) Ascl (Chanda et al, 2014; Vierbuchen et al, 2010). Co-expression of Brn and Mytl with Ascl (BAM), enhances reprogramming efficiency by suppressing a competing myogenic program (Chanda et al, 2014; Treutlein et al, 2016). Ascl acts as a pioneer factor by binding to and opening closed chromatin regions, as well as modulating the binding of Brn (Wapinski et al, 2013), while Myt1l serves as multi-lineage repressor that maintains neuronal identity (Mall et al, 2017).
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