Fig 1: Targeted CRISPR screening identifies CUL4BDCAF8 ubiquitin ligase complexes essential for DNMT3A protein degradation. A, Schematic of targeted CRISPR screening to identify ubiquitin ligases essential for DNMT3A protein degradation. HEK293T cells were engineered to constitutively overexpress Cas9 and the indicated bicistronic DNMT3AW297Del reporter and then infected with sgRNA libraries targeting ubiquitin ligases. Nine days after infection, we sorted both the top and bottom 5% of cells for DNMT3A-GFP expression. B, The graph depicts the gene enrichment score and P value in targeted CRISPR screening for ubiquitin ligases. DCAF8, RBX1, and CUL4B genes (red) were enriched and statistically significant. C, The image depicts DCAF8 immunoprecipitation in DNMT3A-WT or W297del-expressing cells after incubation for 6 hours with an inhibitor of E1 ubiquitin-activating enzymes (MLN7243), followed by blotting using GFP, DCAF8, DDB1, and GAPDH antibodies. D, DNMT3AW297del-expressing HEK293T cells treated with sgRNA targeting DCAF8, proteasome inhibitor, and E1 ubiquitin–activating enzyme, followed by Western blot analysis for DNMT3A-GFP, DCAF8, and GAPDH. E, Cycloheximide (CHX) treatment with 0, 2, 4, and 6 hours of DNMT3AWT and DNMT3AW297Del HEK293T cells with or without DCAF8-KO followed by Western blot for DNMT3A-GFP, DCAF8, and GAPDH. Inhibitor treatments of proteasome and E1 ubiquitin–activating enzyme in DNMT3AWT and DNMT3AW297Del HEK293 cells serve as rescue control of experiment. F, Western blot analysis of DNMT3A, DCAF8, and GAPDH in DCAF8-KO DNMT3AWT LCLs. G, The image depicts the levels of ubiquitination in DNMT3AWT and DNMT3AW297del HEK293T cells with or without DCAF8-KO. DNMT3A-GFP was pulled down by a GFP antibody, followed by Western blot analysis using ubiquitin and GFP antibody. H, Western blotting analysis of heterozygous mESC unstable mutants (DNMT3AW293Del and DNMT3AG681R) and stable mutants (DNMT3AE752A DNMT3AW856R) with or without Dcaf8-KO. I, Stability of variants before and after DCAF8-KO measured as described in Fig. 2C. J, Schematic of the RBX1–CUL4B–DCAF8 complex serving as the ubiquitin ligase for DNMT3A protein. ***, P < 0.001 using the unpaired t test.