Fig. 6. A proposed regulation of DNA methylation. In normal cells, hDnmt1 is regulated by Rb, thus maintenance methylation is carried out only at the hemimethylated CGs (filled lollipop, methyl cytosine) during replication, and excess Dnmt1 is sequestered by Rb. DNA in cancer cells lacking functional Rb may facilitate aberrant methylation patterns.

Fig. 2. Interacting regions between Rb and hDnmt1. (A) Upper panel: the GST–Rb fusion constructs along with the amino acid numbers. Lower panel: the GST pull-down results using purified full-length hDnmt1 protein (+) and anti-N-terminal hDnmt1 antibody. (B) Upper panel: the catalytically active deletion mutants of hDnmt1. Deleted amino acids are indicated on the right. Lower panel: pull-down assay with either GST or GST–Rb ABC is indicated (+), along with the hDnmt1 and mutants at the top.

Fig. 5. Luciferase gene expression and methylation analysis of methylated pGL3 promoter (reporter gene construct) in COS-7 cells. (A) Luciferase gene expression in the transfected COS-7 cell extracts. Transfected DNAs are indicated: Um, unmethylated pGL3 promoter; Me, methylated pGL3 promoter; Me+Ad, methylated pGL3 promoter and adeno construct; and Me+Ad-Rb, methylated pGL3 promoter and adeno construct containing Rb. Luciferase expression was normalized with β-galactosidase internal control and was taken as 100% for unmethylated pGL3 promoter. (B) Changes in methylation pattern of the luciferase gene in the presence of exogenous Rb: Southern blot of total DNA probed with random-primed luciferase gene. Each lane represents either MspI (M) or HpaII (H) restriction digest as indicated. The combination of transfected DNA is as in (A). HpaII specific bands with significant change in intensity (lane 8) are indicated on the right by arrows.

Fig. 3. Rb inhibits hDnmt1 via disruption of a DNA–hDnmt1 binary complex. (A) MBP–Rb fusion proteins used for methyltransferase assay. (B) Inhibition of methyltransferase activity in the presence of various fragments of Rb. The enzyme used for the assay was either full-length hDnmt1, indicated by gray bars, or a 336 amino acid deletion mutant of hDnmt1, indicated by black bars. (C) Rb ABC also inhibits a hybrid Dnmt1–HhaI methyltransferase, but not M.HhaI alone. (D) Gel-shift assay showing binary complex formation between hDnmt1 (40 nM), radiolabeled DNA (1.4 nM) and various competitor proteins (200 nM) as indicated. Arrows indicate the relative positions of various complexes. (E) Gel-shift assay with a fixed concentration of hDnmt1 (40 nM) and an increasing concentration of Rb C (0, 1, 2, 3, 4, 5, 6× molar concentration, lanes 2–9). Arrow indicates the relative position of DNA–hDnmt1 complexes. Lane 1 shows input radiolabeled oligonucleotide duplexes. ‘+’ indicates presence of protein/DNA in the reaction mix.

Fig. 1. Human Dnmt1 associates with Rb in vivo. (A) Diagram representing full-length hDnmt1 and Rb pockets. Amino acid numbers for the Rb A, B and C pockets are indicated. Regions associated with specific function are indicated as: PCNA, proliferating cell nuclear antigen binding; RF, replication foci directing, and Zn, zinc binding. N- and C-termini are from amino acid residues 1–1108 and 1122–1616, respectively, for hDnmt1. Conserved motifs are in roman numerals. (B) Methyltransferase activity co-immunoprecipitates with Rb in a recombinant insect cell expression system using poly(dI–dC)·poly(dI–dC) as substrate DNA. Antibodies used for immunoprecipitation are indicated at the bottom. Pre-immune serum is indicated as PI. (C) hDnmt1 co-immunoprecipitates with Rb in recombinant Sf9 cell extracts. Expression of protein is indicated by a ‘+’. Immunoprecipitation with rabbit IgG is abbreviated as IgG. Positive control retinoblastoma (C Rb) and human maintenance methyltransferase (C hDnmt1) are indicated. (D) Left panel: co-immunoprecipitation of hDnmt1 in HEK 293 cell nuclei extracts using antibody against either the N- or C-terminus of Rb. Control rabbit IgG does not bind to hDnmt1. Right panel: association of Rb and Dnmt1 in the presence (+) or absence (–) of serum using C-terminus antibody for Rb. For both panels, HEK 293 cell nuclear extract was used as a control to show expression of Rb as well as hDnmt1 in cells. Cells used for the study (NIH 3T3 and HEK 293) are indicated at the top.

Fig. 4. Transfections of adenovirus mock and adenovirus-Rb DNA into Saos-2 cells. (A) Western blots of the transfected Saos-2 cell extracts using anti-hDnmt1 and anti-Rb antibodies. Endogenous hDnmt1 and Rb along with adenovirus-Rb (Adeno-Rb)-transfected cell extracts are indicated. Lanes 1 and 5 represent 10 μg of HEK 293 cell extracts. HEK 293 cells do express both Dnmt1 and Rb. Note that Saos-2 cell extracts transfected with adenovirus mock (Adeno) do not express endogenous Rb, but HEK 293 and adeno-Rb do (lanes 1, 3, 4 and 5). Lanes 3 and 4 represent extracts from two independent transfections. Equal amounts of cell extracts were loaded in each lane. (B) McrBC digestion of the genomic DNA from adenovirus mock and adenovirus-Rb-transfected Saos-2 cells. The total DNAs were digested by McrBC with or without GTP as indicated above. Molecular weight markers are in kbp. The source of DNA is also indicated at the the top of the gel. (C) Global methylation assay of adeno- and adeno-Rb-transfected cellular DNA. Available CpG target sites were methylated with M.SssI in the presence of tritiated AdoMet. The amount of ³H incorporated into adeno-transfected DNA (c.p.m.) was normalized as 100% methylation. Each DNA was assayed three times in duplicate. (D) Global hemimethylation assay of adeno- and adeno-Rb-transfected cellular DNA. Available hemimethylated CpG target sites in the genome were methylated with hDnmt1 in the presence of tritiated AdoMet. The amount of ³H incorporated into adeno-transfected DNA (c.p.m.) was normalized as 100% methylation. Each DNA was assayed three times in duplicate. Note the value represented here is an overestimate, since hDnmt1 methylation values also represent the addition of a few de novo methylation events by the enzyme.