13:149-162. method to facilitate IDO/TDO-IN-1 elucidation of the methylation machinery that acts in a chromosomal setting. The approach is based on studies showing that IDO/TDO-IN-1 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 usually 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 conversation of several different DNMTs around the genome of the cell in vivo. MATERIALS AND METHODS Reagents. The topoisomerase I (topo I) antibody was isolated from serum of scleroderma patients and was donated by TopoGEN, Inc. (www.topogen.com) (Columbus, Ohio). Anti-DNMT1 rabbit antibody was prepared by using a commercial antibody production support (Research Genetics) and was raised against a synthetic peptide derived from the N-terminal region. Anti-DNMT1b rabbit antibody was prepared against a peptide unique to the additional exon not present in DNMT1 (5, 13). All peptide antibodies were immunoaffinity purified and tested by enzyme-linked immunosorbent assay and Western blotting for appropriate reactivity. Anti-DNMT2 antibody prepared against DNMT2 was provided by X. Cheng and Anti-DNMT3a and -3b antibodies (specific for mouse DNMT3) were provided by K. Robertson. All DNMT antisera were verified for specificity by Western blotting by Rabbit polyclonal to Piwi like1 using crude extracts. Camptothecin (CPT) and etoposide (VP16) were provided by TopoGEN, (in 100% dimethyl sulfoxide), and aza-dC was from Sigma Chemical Co. (St. Louis, Mo.). aza-dC was prepared fresh just prior to use. Cell culture. HeLa, WI-38, HCT-116 (wild type), and HCT-116 and (28), the DNMT1b splice variant would also be absent; however, based upon the ICM, this variant may contribute to the global methylation patterns in vivo in wild type cells. Understanding isoform-specific methylation in vivo is usually further complicated by studies showing the presence of multiple isoforms of DNMT3b, some of which are inactive in vitro (1). In our present study, we could not examine different DNMT3b isoforms due to the lack of specific antisera; however, the ICM has the potential to resolve these issues in a biologically relevant context. The availability of monospecific mouse antibodies allowed us to compare several DNMT isoforms in the murine system. Our data show that DNMT1, -3a, -3b, and -2 stably and specifically bind aza-dC-substituted genomes, suggesting that these are all active transmethylases in mouse P19 embryonic carcinoma cells (Fig. ?(Fig.6).6). Based upon the ICM assay, it appears that, after 8 h of aza-dC treatment, DNMT3b displayed considerably higher (four- to fivefold) global activity than did DNMT2 and -3a; however, DNMT3b expression was about half the levels of DNMT3a and DNMT2. A possible explanation is that the endogenous catalytic activity of each DNMT is usually regulated by other proteins or by accessibility of a DNA target in chromatin (recently reviewed in reference 29). For example, DNMT3b colocalizes to pericentromeric heterochromatin, which, as a repetitive element, may represent a localized sink for DNMT3b activity. Alternatively, this may simply be a unique feature of embryonic carcinoma cells; however, recent results showing that DNMT3b works coordinately with DNMT1 in maintaining genomic methylation says (28) support the notion that DNMT3b and -1 are operating at higher levels in vivo. In the two species tested (human and mouse), our data suggest that DNMT2 is usually a catalytically active transmethylase in IDO/TDO-IN-1 a chromosomal setting. DNMT2 is usually.