Targeting of 5-aza-2'-deoxycytidine residues by chromatin-associated DNMT1 induces proteasomal degradation of the free enzyme

Abstract

5-Aza-2'-deoxycytidine (5-aza-dC) is a nucleoside analogue with cytotoxic and DNA demethylating effects. Here we show that 5-aza-dC induces the proteasomal degradation of free (non-chromatin bound) DNMT1 through a mechanism which is dependent on DNA synthesis and the targeting of incorporated 5-aza-dC residues by DNMT1 itself. Thus, 5-aza-dC induces Dnmt1 degradation in wild-type mouse ES cells, but not in Dnmt [3a(-/-), 3b(-/-)] mouse ES cells which express Dnmt1 but lack DNA methylation (<0.7% of CpG methylated) and contain few hemi-methylated CpG sites, these being the preferred substrates for Dnmt1. We suggest that adducts formed between DNMT1 and 5-aza-dC molecules in DNA induce a ubiquitin-E3 ligase activity which preferentially targets free DNMT1 molecules for degradation by the proteasome. The proteasome inhibitor MG132 prevents DNMT1 degradation and reduces hypomethylation induced by 5-aza-dC.

Figure 1.

5-Aza-dC induces the proteasomal degradation of DNMT1. (A) Western blot of DNMT1, DNMT3A, DNMT3B and KU70 (loading control) after treatment of HCT116 cells with 1 µM 5-aza-dC. Degradation is induced by 6 h of treatment. (B) Quantitative RT–PCR of DNMT1 relative to 18S RNA in HCT116 cells exposed to 0.1 and 1 µM 5-aza-dC for 24 h. (C) Western blot of DNMT1 levels and GAPDH (loading control) after treatment of HCT116 cells with cycloheximide (protein synthesis inhibitor) and 5-aza-dC. The DNMT1-lowering effect of 5-aza-dC in cycloheximide treated cells indicates that 5-aza-dC acts post-transcriptionally and post-translationally to lower DNMT1 levels. (D) Western blot of DNMT1 and ACTIN (loading control) to show the effect of a 12-h treatment with 5-aza-dC and MG132 (10 μM). The MG132 treatment was commenced 2 h before 5-aza-dC to ensure that proteasome blockade was effective prior to 5-aza-dC exposure. (E) CDH1/FZR1 was knocked down with siRNA in HCT116 cells before being exposed to 0.1 and 1 µM 5-aza-dC. Controls include a mock transfection control (no siRNA), to control for untoward effects of the transfection reagent, and a fluorescent nonsense siRNA, to ensure that transfection had taken place and to exclude non-specific effects. The graphs demonstrate that CDH1/FZR1 was knocked down to 40% of the level seen in the control cells.

Figure 2.

DNMT1 expression is highly sensitive to 5-aza-dC treatment. (A) Western blots of DNMT1 and GAPDH (loading control) after the indicated doses of 5-aza-dC in HCT116 cells (A) and SW620 cells (B). (C) and (D) Global DNA methylation analysis corresponding to the 5-aza-dC treatments in the HCT116 and SW620 cell lines. (E) DNMT1 protein levels, and (F) global DNA methylation levels, in HCT116 cells exposed to 5-aza-dC for 5 days.

Figure 3.

(A) Chromatogram of nucleoside standards (top panel) and nucleosides from digested DNA of cells treated with 5-aza-[6³H]-dC for 4-h post-medium replacement (middle panel). The bottom panel shows ³H-DPM of fractions collected in the panel above. Specific activity of DNA from HCT116 cells treated with aphidicolin (20 μg/ml) for 24 h followed by treatment with 5-aza-[6³H]-dC and [5-methyl-³H]-thymidine for 12 h. (B) Specific activity of 5-aza-[6³H]-dC and [5-Methyl-³H]-thymidine in HCT116 cells treated for 0.5, 2, 4 and 6 h. (C) Specific activities at 0.5, 2, 4 and 6 h after washout of labeling nucleotides. (D) The incorporation of 5-aza-[6³H]-dC and [5-methyl-³H]-thymidine into HCT116 cell DNA, with and without prior treatment with the DNA synthesis inhibitor aphidicolin at 20 μg/ml for 24 h.

Figure 4.

The DNA synthesis inhibitor aphidicolin prevents the 5-aza-dC induced degradation of DNMT1. Western blots of DNMT1 and GAPDH (loading control) in 5-aza-dC treated HCT116 cells (A) and SW620 cells (B) to show the effects of 5-aza-dC on its own (top panels) as compared with cells that had been treated with the DNA synthesis inhibitor aphidicolin 20 μg/ml for 24 h prior to treatment with 5-aza-dC (bottom panels). (C) Western blot and (D) cell-cycle analysis to show DNMT1 levels at various stages after synchronization and release from an aphidicolin block. DNMT1 protein levels are prominent in synthesis, and markedly reduced in G1 and G2 phases of the cell cycle. (E) Cell-cycle analysis of HCT116 cells exposed to the indicated concentrations of 5-aza-dC for 24 h.

Figure 5.

The DNMT1-degrading effects of 5-aza-dC are dependent on pre-existing DNA methylation (A) Global DNA methylation in wild-type (J1) ES cells and Dnmt[3a^–/–,3b^–/–] ES cells. (B) Dnmt1 protein levels of in wild-type and Dnmt[3a^–/–,3b^–/–] mouse ES cells treated with 1 μM 5-aza-dC for 12 h. The bar graph shows the averaged quantifications with standard deviations (by densitometry) of western blots from three independent experiments.

Figure 6.

Soluble DNMT1 is more sensitive to degradation that chromatin bound DNMT1. (A) upper panel, western blot of HCT116 cells treated with the indicated doses of 5-aza-dC for 12 h. Whole cell proteins were fractionated by differential solubility in PBS/0.5% Triton X-100. The soluble fractions (S) fractions were separated from the chromatin bound insoluble (INS) fractions by centrifugation. In the lower panel the effects of pre-treatment of the cells with MG132 are shown. GAPDH and Histone H4 serve as a loading controls for the soluble fraction and insoluble fractions respectively, and serve to verify that chromatin was separated as expected. (B) Pre-treatment with MG132 reduces the DNA hypomethylating effects of 5-aza-dC.

Figure 7.

Model to explain the effects of 5-aza-dC on DNA methylation and the proteasomal degradation of DNMT1. In normal cells undergoing replication DNMT1 binds to and remethylates the hemi-methylated DNA duplexes produced as a result of DNA synthesis. There is an exchange of DNMT1 between the chromatin and free compartments. This exchange is indicated by the arrows in the first panel. Maintenance methylation is linked to DNA replication and in normal untreated cells the amount of hemi-methylated DNA is low because of effective maintenance methylation. The addition of 5-aza-dC causes: 1, Incorporation of 5-aza-dC into DNA and trapping of DNMT1 enzymes in a dosage dependent manner. This leads to an increase in the amount of hemi-methylated DNA and fall in proportion of cytosines methylated. 2, 5-aza-dC also induces the proteasomal degradation of free DNMT1, even at very low dosage. The mechanism is unknown but involves the targeting of 5-aza-dC incorporated DNA by DNMT1. 3, Degradation of free DNMT1 prevents the re-methylation of the hemi-methylated DNA that accumulates as a result of trapping. Proteasomal degradation of DNMT1 contributes to the hypomethylation induced by 5-aza-dC.