Epigenetic modifiers are necessary but not sufficient for reprogramming non-myelinating cells into myelin...

Modifications on specific histone residues and DNA methylation play an essential role in lineage choice and cellular reprogramming. We have previously shown that histone modifications or combinatorial codes of transcription factors (TFs) are critical for the differentiation of multipotential progenitors into myelinating oligodendrocytes. In this study we asked whether combining global manipulation of DNA methylation and histone acetylation together with the expression of oligodendrocyte-specific TFs, was sufficient to switch the identity of fibroblasts into myelin gene-expressing cells.

Transfection of six oligodendrocyte-specific TFs (Olig1, Olig2, Sox10, Mash1, E47 and Nkx2.2) into NIH3T3 fibroblasts was capable of inducing expression of myelin gene promoter-driven reporters, but did not activate endogenous myelin gene expression. These results suggested the existence of a transcriptionally incompetent chromatin conformation in NIH3T3 fibroblasts. Using chromatin immunoprecipitation (ChIP) analysis, we compared the histone code on the conserved regions of myelin genes (i.e. Mbp and Mag) in differentiating oligodendrocyte progenitors and NIH3T3 fibroblasts. Chromatin at myelin gene loci was characterized by the presence of repressive histone modifications (me3K9H3 and me3K27H3) in NIH3T3 fibroblasts and active histone marks (me3K4H3 and AcH3) in oligodendrocyte lineage cells. To induce a transcriptionally competent chromatin signature, NIH3T3 fibroblasts were treated with 5-azadeoxy-citidine (5-AzaC) to decrease DNA methylation, and trichostatin A (TSA) or sirtinol, to favor histone acetylation. Treatment with 5-AzaC/TSA but not sirtinol, resulted in the detection of endogenous myelin gene transcripts in fibroblasts, although not to the levels detected in myelinating cells. Transfection of oligodendrocyte-specific TFs after 5-AzaC/TSA treatment did not further increase myelin gene expression, nor did it reprogram the transcriptional network of NIH3T3 fibroblasts into that of oligodendrocytes.

These results suggest that reprogramming of fibroblasts into myelin gene-expressing cells not only requires transcriptional activation, but also chromatin manipulations that go beyond histone acetylation and DNA methylation.