Figure 5. Acetylation of KG^DNMT1 impairs DNMT1–USP7 interaction and promotes the degradation of DNMT1.

(a,b) Acetylation of KG^DNMT1 impairs DNMT1–USP7 interaction. HEK293T cells were transfected with Tip60 and treated with HDACi (TSA, MS-275 and NAM). Endogenous (a) or exogenous (b) DNMT1 was immunoprecipitated with antibodies against DNMT1 and Flag, respectively. The protein levels were analysed by immunoblotting. Uncropped blots are shown in Supplementary Fig. 7. (c) Acetyl-DNMT1 is less stable compared with DNMT1. HEK293T cells were transfected with Flag–DNMT1 and Tip60, followed by treatment with HDACi (TSA and MS-275). The cells were treated with CHX (100 μg ml⁻¹) and harvested at the indicated time. The relative protein levels of acetyl-DNMT1 were determined and indicated below. Uncropped blots are shown in Supplementary Fig. 7. (d) Negative correlation between DNMT1 and acetyl-DNMT1 in PANC-1 (low E-cadherin and high Vimentin) and Capan-1 (low Vimentin and high E-cad) pancreatic cancer cells. Scale bar, 20 μm. (e) HDACi (TSA, MS-275 and NAM) treatment increases the protein levels of acetylation of KG^DNMT1 and E-cad, and decreases the protein level of DNMT1 and Vimentin in PANC-1 cells. Scale bar, 20 μm. (f,g) Negative correlation between DNMT1 (f) and acetyl-DNMT1 (g) in differentiated neuronal cells. Neuronal cells were stained with an anti-Tuj1 antibody, and DNA was visualized with DAPI staining. The representative staining is shown and the percentage of the cells (among 100 cells) with representative staining are indicated. Differentiated neuronal cells and non-neuronal cells are indicated in yellow circles and squares, respectively. Scale bar, 20 μm.