Fig. 4. Polycomb promoter hypermethylation following DNMT3A1 redistribution requires DNMT3A1 interaction with the nucleosome acidic patch and H2AK119Ub.

(A) Scatter plots showing the percentage of DNAme (by RRBS) at promoters [TSS ± 500 base pairs (bp)] in DNMT3A1^W330R, DNMT3A^W330R+181E, and DNMT3A1^{W330R+Ub mut} versus DNMT3A1^WT-expressing 10T cells. Each dot represents a single promoter. Significantly hypermethylated (>10% and q < 0.01) and hypomethylated (<−10% and q < 0.01) promoters are colored in red and blue, respectively (two biological replicates). (B) Metagene plot showing the percentage of DNAme over CGIs ± 5 kb (left), promoters (TSS ± 5 kb) (middle), and Polycomb-regulated promoters (H3K27me3⁺ and H2AK119Ub⁺) (right) in DNMT3A1^W330R, DNMT3A^W330R+R181E, and DNMT3A1^{W330R+Ub mut} compared to DNMT3A1^WT 10T cells. (C) Bar plot showing representation among all promoters, or 10T DNMT3A^W330R–hypermethylated promoters, for each group of promoters defined by histone PTM status in control cells as in fig. S6B. (D) Metagene plot showing enrichment of H2AK119Ub, H3K27me3, and H3K4me3 CUT&RUN reads from DNMT3A1^WT, DNMT3A1^W330R, DNMT3A^W330R+R181E, and DNMT3A1^{W330R+Ub mut} 10T cells, centered at TSS ± 5 kb, and grouped by all, hypermethylated, and hypomethylated promoters. (E) Genome browser view (chromosome 2, 74.5 to 75.0 Mb), showing differences in the percentage of DNAme (by RRBS) between DNMT3A1^WT and various mutant 10T cells. Bottom: H2AK119Ub, H3K27me3, and H3K4me3 CUT&RUN reads from DNMT3A1^WT and DNMT3A1^W330R 10T cells. Genes from RefSeq database are annotated at the bottom.