Differentiation of umbilical cord blood derived mononuclear cells to endothelial cells is accompanied by massive changes in gene expression. Although methylation and demethylation of DNA likely play crucial roles in regulating gene expression, their interplay during differentiation remains elusive. To address this question, we performed deep sequencing of DNA methylation and mRNA expression to profile global changes in promoter methylation and gene expression during differentiation from mononuclear cells to outgrowing cells. We identified 61 downregulated genes with hypermethylation, including CD74, VAV1, TLR8, and NCF4, as well as 21 upregulated genes with hypomethylation, including ECSCR, MCAM, PGF, and ARHGEF15. Interestingly, gene ontology analysis showed that downregulated genes with hypermethylation were enriched in immune-related functions, and upregulated genes with hypomethylation were enriched in the developmental process and angiogenesis, indicating the important roles of DNA methylation in regulating differentiation. We performed polymerase chain reaction analyses and bisulfite sequencing of representative genes (CD74, VAV1, ECSCR, and MCAM) to verify the negative correlation between DNA methylation and gene expression. Further, inhibition of DNA methyltransferase and demethylase activities using 5′-aza-dc and shRNAs, specific for TET1 and TET2 mRNAs, respectively, revealed that DNA methylation was the main regulator of the reversible expression of functionally important genes. Collectively, our findings implicate DNA methylation as a critical regulator of gene expression during umbilical cord blood derived mononuclear cells to endothelial cell differentiation.
Bibliographical noteFunding Information:
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology [ NRF-2015R1A2A1A05001859 ; NRF2013M3A9B6046563 ; NRF-2015K1A4A3047851 ; NRF-2015M3A9B6066967 ], by the Bio and Medical Technology Development Program of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (MEST) [ NRF-2015M3A9B6066967 ], by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare of the Republic of Korea [ H16C1501 ], and by the Technology Innovation Program of the Ministry of Trade, Industry and Energy , Republic of Korea [Grant 10050154 ].
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