Identification of T-cadherin as a novel target of DNA methyltransferase 3B and its role in the suppression of nerve growth factor-mediated neurite outgrowth in PC12 cells.
Previously we showed that DNA methyltransferase 3b (Dnmt3b) is required for nerve growth factor (NGF)-induced differentiation of PC12 cells to neuronal phenotype. The present study identified T-cadherin (T-Cad) as one of the targets of Dnmt3b by chromatin immunoprecipitation (ChIP) assay. Combined bisulfite restriction analysis and bisulfite sequencing showed that T-Cad promoter was sparsely methylated in PC12 cells. ChIP-CHOP analysis demonstrated that Dnmt3b is associated with T-Cad promoter irrespective of its methylation status. The mRNA and protein levels of T-Cad were markedly elevated in cells depleted of Dnmt3b by antisense or small interfering RNA. Suppression of T-Cad promoter activity by Dnmt3b was independent of its catalytic activity, which was consistent with the insignificant change in T-Cad promoter methylation status in Dnmt3b-depleted cells. In contrast, deletion of its N-terminal ATRX and PWWP domain abolished its repressor function. Association of histone deacetylase 2 (Hdac2) with T-Cad promoter and restoration of the promoter activity from Dnmt3b-mediated suppression upon treatment with Hdac inhibitor indicated involvement of histone deacetylation in this process. NGF-induced neurite outgrowth was inhibited in a dose dependent manner upon ectopic expression of T-Cad in PC12 cells. Immunofluorescence studies showed that T-Cad was redistributed upon NGF treatment, as evident from its concentration in axon growth cones as opposed to its localization at cell-cell contact region in undifferentiated cells. These results demonstrate a novel role of T-Cad in the NGF-mediated differentiation of PC12 cells to neuronal phenotype.
Bai
Shoumei
S
Department of Molecular and Cellular Biochemistry, College of Medicine, Ohio State University, Columbus, Ohio 43210.
Ghoshal
Kalpana
K
Department of Molecular and Cellular Biochemistry, College of Medicine, Ohio State University, Columbus, Ohio 43210.
Jacob
Samson T
ST
Department of Molecular and Cellular Biochemistry, College of Medicine, Ohio State University, Columbus, Ohio 43210. Electronic address: jacob.42@osu.edu.
D000818:Animals; D015820:Cadherins / Q000378:metabolism*; D002454:Cell Differentiation; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D015536:Down-Regulation; D015971:Gene Expression Regulation, Enzymologic; D020932:Nerve Growth Factor / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D013379:Substrate Specificity; D000097584:DNA Methyltransferase 3B
D015820:Cadherins; D004248:DNA (Cytosine-5-)-Methyltransferases; D020932:Nerve Growth Factor; D000097584:DNA Methyltransferase 3B; C099972:H-cadherin