Endogenous Assays of DNA Methyltransferases: Evidence for Differential Activities of DNMT1, DNMT2, and DNMT3 in Mammalian Cells In Vivo

In eukaryotes, DNA methylation is an epigenetic encryption system that is essential for proper gene regulation (for reviews see references 2, 4, 7, 23, 29, and 30). Defects in methylation lead to diverse disorders from mental retardation to immune deficiencies, and there is particularly strong evidence that methylation defects create a favorable environment for malignant transformation (2, 3). Pharmacologic alterations in methylation of specific genes have also been correlated with tumor response to chemotherapy and patient survival; thus, methylation regulation and the enzymes that catalyze the process represent important areas for treating cancer (2, 30). The enzymatic machinery that mediates methylation involves a number of DNA (cytosine-5) methyltransferase (DNMT) isoforms, including DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b (and a host of DNMT3 splice variants) (4, 29, 30). Dnmt1, Dnmt3a, and Dnmt3b are independent genes and essential; embryos lacking both copies of Dnmt1 or Dnmt3b die before birth, whereas Dnmt3a-nulls survive about 4 weeks (18, 21). Heterozygous mutants appear normal and are fertile (18, 21). The murine DNMT3a and -3b enzymes appear to possess de novo methylation activity (based upon plasmid methylation), and there is evidence that they act on different DNA targets in vivo (12). No transmethylase activity has been found with DNMT2, and biallelic deletions appear to possess normal methylation patterns (8, 22). How different DNMTs are directed to specific sites in vivo is not well understood, although DNA sequence, chromatin structure, or ancillary interacting factors (or a combination of these) are obvious candidates. In order to sort out which specific DNMTs are responsible for methylation of selected DNA targets, one needs to examine individual methylases in vivo under physiological conditions. Achieving this objective is complicated when one considers that the most prevalent methylase is DNMT1, with DNMT3a and -3b being barely detectable (31), while there exist additional minor forms, including DNMT2 and DNMTb (5, 8, 13, 22). Recently, a cell line knockout of Dnmt1 was characterized (28) that displayed nearly normal global genomic methylation. Other methylases must be active in this case. For example, DNMT3b is thought to be the additional activity that cooperates with DNMT1 to maintain cellular DNA methylation patterns (28). Additional minor but active DNMTs that are physiologically relevant exist (8, 13, 22). Minor methylating activities may be less robust overall (relative to DNMT1) but have important consequences if key regulatory genes are their targets. We have developed a method to facilitate elucidation of the methylation machinery that acts in a chromosomal setting. The approach is based on studies showing that DNA methyltransferases have a fleeting covalent association with the DNA substrate; however, when 5-aza-2′-deoxycytidine (aza-dC) is present, the covalent DNA-protein intermediate is arrested, leading to adducts that have consequences in global methylation (9, 15, 33, 34). Consequently, active methylases become stoichiometrically removed from the active nuclear pool, leading to hypomethylation of the genome. We have used an antibody-based method to detect and quantify the physical interaction of several different DNMTs on the genome of the cell in vivo.