Role of DNA methyltransferase 3b in neuronal cell differentiation

To elucidate the role of epigenetic reprogramming in cell or tissue-specific differentiation, we explored the role of DNA methyltransferases (Dnmts) in the NGF-induced differentiation of PC12 (pheochromocytoma) cells to neuronal cells. The mRNA and protein levels of de novo methyltransferase Dnmt3b increased whereas those of Dnmt3a and Dnmt1 decreased during NGF-induced neurite outgrowth. Dnmt3b localized in the nucleus as well as in the growing neurites. When the expression of Dnmt3b was inhibited by antisense or siRNA, PC12 cells continued to proliferate and failed to generate neurites. Cells depleted of Dnmt3b were unable to exit cell cycle even after 6 days of NGF treatment. Further, this failure in differentiation correlated with significant attenuation in tyrosine phosphorylation of TrkA (NGF receptor) and reduced expression of neuronal markers, Hu antigen and MAP2. The methyl CpG content of PC12 genome or the methylation status of repetitive elements was not significantly altered after differentiation and was not affected by Dnmt3b depletion. Further, the catalytic site mutant of Dnmt3b was able to induce differentiation in Dnmt3b depleted cells after NGF treatment. The Dnmt3b-mediated differentiation was attributed to its N-terminal domain that recruits histone deacetylase 2 (Hdac2), as demonstrated by (a) impediment of differentiation by the Hdac inhibitors, (b) facilitation of the differentiation process by overexpression of the N-terminal domain of Dnmt3b, (c) higher Hdac activity associated with Dnmt3b after NGF treatment and (d) co-immunoprecipitation and co-sedimentation of Dnmt3b specifically with Hdac2 in a glycerol density gradient. These data indicate a novel role of Dnmt3b in neuronal differentiation.; T-cadherin was one of the target genes of Dnmt3b, which was down-regulated during NGF-induced differentiation. Dnmt3b repressed T-cadherin promoter activity independent of its catalytic domain, which corroborated with the observation that no significant change in T-cadherin promoter DNA methylation upon Dnmt3b depletion as revealed by COBRA, CHIP-CHOP and bisulfite sequencing analysis. Overexpression of T-cadherin inhibited NGF-induced PC12 cell differentiation, which corroborated with its redistribution after NGF treatment. Extensive staining of T-cadherin at axon growth cone and restricted distribution on the plasma membrane was observed after NGF treatment in contrast to concentrated staining at cell-cell contact region in undifferentiated cells. In summary, the present study has demonstrated a key role for Dnmt3b and T-cadherin in the NGF-mediated differentiation of PC12 cells to neuronal cells.