Fig 2: CRL4DDB2 components DDB2, DDB1 and CUL4A are required for restoring H3K56Ac post phleomycin treatment(A) LFS-041B and its derivative LFS-041B+DDB2 cells were treated with 60 μg/ml of phleomycin for 2 h. The treated cells were maintained in fresh medium for indicated time periods. Samples of whole cell Laemmli extracts were analyzed by Western blotting for DDB2 and H3K56Ac and other acetylated histone H3. (B) The blots from at experiments in A were quantitated using ImageJ software and the relative protein levels were plotted against time course. The immunoblots of H3K56Ac with GeneTex anti-H3K56Ac antibody were used for ImageJ quantitation. (C) Western blotting analysis of DDB1 and H3K56Ac in DDB1 siRNA-transfected NHFs upon phleomycin post-treatment. (D) Western blotting analysis of CUL4A and H3K56Ac in DDB1 siRNA-transfected NHFs upon phleomycin post-treatment. Actin blots serve as the loading controls.

Fig 3: L3MBTL3 interacts with CRL4DCAF5 and recruits DCAF5 onto the methylated DNMT1 protein. a Downregulation of DCAF5 or L3MBTL3 increases the K142-methylated DNMT1. HCT116 cells were transfected with 50 nM of control, DCAF5 and L3MBTL3 siRNAs. DNMT1 was immunoprecipitated by anti-DNMT1 antibody and western blotted by anti-methylated K142 or anti-DNMT1 antibodies. Student’s t-test was caculated for significant differences. **P < 0.01 and ***P < 0.001. b L3MBTL3 and DCAF5 interact. Endogenous DCAF5 and L3MBTL3 proteins were immunoprecipitated from HCT116 cells and western blotted with indicated antibodies. c The interaction between DCAF5 and DNMT1 is abolished by K142 mutation. Flag-WT-DNMT1 and its K142A mutant constructs were transfected into HCT116 cells and the interactions between DCAF5 and Flag-DNMT1 or the K142A mutant were analyzed by anti-DCAF5 immunoprecipitation and western blotting with anti-Flag and DCAF5 antibodies. d Downregulation of L3MBTL3 abolished the interaction between DNMT1 and DCAF5. Endogenous DNMT1 and DCAF5 proteins were immunoprecipitated by anti-DNMT1 antibodies from control and L3MBTL3 siRNA-mediated knockdown cells (42 h of siRNA and MG132 for additional 6 h). The immunoprecipitated proteins were blotted with anti-DCAF5 and DNMT1 antibodies. e L3MBTL3 is required for preferential binding of DCAF5 to the monomethylated K142 peptide. HCT116 extracts expressing a stable Flag-DCAF5 or Flag-DCAF5 and L3MBTL3 were prepared. 250 μg extract proteins were incubated with the monomethylated K142 or the cognate unmethylated peptide resins as in Fig. 5c. The binding of Flag-DCAF5 to the peptide resins with or without L3MBTL3 were examined by anti-Flag and L3MBTL3 antibodies. Student’s t-test was used for significant differences. *P < 0.05. f L3MBTL3 and CRL4DCAF5 target DNMT1 for polyubiquitination in vivo. Flag-WT DNMT1 and Flag-K142 A mutant expressing constructs were co-transfected into 293 cells together with vectors expressing HA-tagged ubiquitin (HA-Ub), SET7, CUL4A, CUL4B, and DDB1, in the presence or absence of L3MBTL3 and DCAF5 expressing constructs as indicated. Proteins were immunoprecipitated with anti-HA antibodies and western blotted with the anti-Flag-DNMT1 antibodies as indicated. Quantifications for all experiments were represented by bar graph with mean and S.D. for error bars from three replicates and Student’s t-test was used for significant differences, as described in Fig. 2a

Fig 4: UBR5-containing EDVP E3 ligase complex interacts and regulates MOAP-1 ubiquitylation and stability. (a) Flag-MOAP-1 was transfected into 293T cells, and lysates were prepared 48 h posttransfection. Co-IP with Flag M2 agarose beads were performed and immunoblotted with antibodies as indicated. n=3 independent experiments. (b) In vitro ubiquitylation assay was performed as Figure 2d in the presence or absence of recombinant DDB1, VprBP and Dyrk2. n=3 independent experiments. Original uncropped image is shown in Supplementary Figure S6. (c) siCtrl or siDyrk2 was transfected into H1299 or 293T cells, and Flag-MOAP-1 was transfected into each siRNA transfectant 24 h post-siRNA transfection. Cell lysates were prepared under denaturing condition; Flag-MOAP-1 was immunoprecipitated and immunoblotted with ubiquitin antibody. Same membrane was re-blotted with Flag antibody for Flag-MOAP-1. n=3 independent experiments. Asterisk in Dyrk2 blot indicates non-specific band. Quantification of MOAP-1 ubiquitylation is shown in Supplementary Figure S3B. (d) 293T cells transfected with siCtrl, siDyrk2 or siUBR5 were treated with CHX 48 h posttransfection. Cells were collected at the indicated times after CHX treatment, and lysates were prepared and immunoblotted as indicated (left). MOAP-1 protein level was quantified and plotted (right). The MOAP-1 abundance at 0 time point was set at 100%. n=4 independent experiments (means±s.e.m.). Molecular weight markers are in kDa.

Fig 5: Depletion of CRL4DDB2 components DDB2, DDB1 and CUL4A decrease H3K56Ac levels(A) DDB2, H3K56Ac, H3K9Ac, H3K27Ac, H3K27Ac and H3K14 levels were examined in LFS-041B cell line and its derivative cell line with re-established DDB2 expression. (B) Cytoplasmic and chromatin fractions of LFS-041B and its derivative were obtained through a cellular protein fractionation protocol. Samples containing identical protein amounts from each fraction were examined by Western blotting for DDB2, H3K56Ac and other acetylated histone H3. Actin and Lamin B blots serve as the loading controls. (C) NHFs were transiently transfected with control (Ctrl) siRNA or siRNA targeting DDB2, DDB1 or CUL4A. Levels of DDB2, DDB1 and CUL4A as well as H3K56Ac were detected in whole cell Laemmli extracts by Western blotting.