Analysis of genomic DNA methylation and gene transcription modulation induced by DNMT3A deficiency in HEK293 cells

Abstract

DNA methylation is an important epigenetic modification associated with transcriptional repression, and plays key roles in normal cell growth as well as oncogenesis. Among the three main DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), DNMT3A mediates de novo DNA methylation with partial functional redundancy with DNMT3B. However, the general effects of DNMT3A and its downstream gene regulation profile are yet to be unveiled. In the present study, we used CRISPR/Cas9 technology to successfully create DNMT3A deficient human embryonic kidney cell line HEK293, with frameshift mutations in its catalytic domain. Our results showed that the cell growth slowed down in DNMT3A knockout cells. UPLC-MS analysis of DNMT3A deficient cells showed that the genome-level DNA methylation was reduced by 21.5% and led to an impairment of cell proliferation as well as a blockage of MAPK and PI3K-Akt pathways. Whole genome RNA-seq revealed that DNMT3A knockout up-regulated expression of genes and pathways related to cell metabolism but down-regulated those involved in ribosome function, which explained the inhibition of cell growth and related signal pathways. Further, bisulfite DNA treatment showed that DNMT3A ablation reduced the methylation level of DNMT3B gene as well, indicating the higher DNMT3B activity and thereby explaining those down-regulated profiles of genes.