Investigating the Role of Methylation in Silencing of Gene Expression in Normal Cells During Hematopoiesis and in Their Leukemic Counterparts

(1) : Vitamin D receptor (VDR) is present in multiple types of blood cells, and its ligand, 1,25-dihydroxyvitamin D (1,25D), is important for the proper functioning of the immune system. Activity of VDR is higher in hematopoietic stem and progenitor cells than in fully differentiated blood cells of mice and humans. In some human acute myeloid leukemia (AML) blasts, the expression of the gene is also high. The mechanism of silencing the gene expression during differentiation of blood cells has been addressed in this work. (2) : The cells have been obtained using fluorescence activated sorting from murine tissues and from human umbilical cord blood (UCB). Then, the expression of the gene and transcriptional activity of the VDR protein has been tested in real-time polymerase chain reaction (PCR). Eventually, the methylation of promoter regions was tested using bisulfite sequencing. (3) : The CpG islands in promoters were not methylated in the cells studied both in mice and in humans. The use of hypomethylating agents had no effect toward expression of human transcripts, but it increased expression of the VDR-target gene, . (4) : The expression of the gene and transcriptional activity of the VDR protein varies at successive stages of hematopoietic differentiation in humans and mice, and in blasts from AML patients. The experiments presented in this case indicate that methylation of the promoter region of the gene is not the major mechanism responsible for these differences.