MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6
Fig 5
L3MBTL2 and E2F6 recruit PRC1.6 differentially in a promoter-specific manner.
(A) Left panel, scatter plot comparing the extent of reduction (fold change of normalized tag counts) of MGA binding in L3MBTL2ko cells with the extent of reduction in E2F6ko cells. Right panel, scatter plot comparing the extent of reduction of E2F6 binding in L3MBTL2ko cells with the extent of reduction of L3MBTL2 in E2F6ko cells. The E2F6-dependent RNF130 and the L3MBTL2-dependent ZFR promoters are indicated for clarity. (B) Genome browser screenshots of ChIP-seq tracks showing binding of MGA, L3MBTL2, E2F6 and PCGF6 to the RNF130 and ZFR promoters in wild type cells (WT), and in MGAko, L3MBTL2ko, E2F6ko and PCGF6ko cells. (C) ChIP-qPCR analysis of MGA binding to selected promoters in two different L3MBTL2ko (L2ko cl10 and L2ko cl14, upper panel) and in two different E2F6ko (E2F6ko cl1 and E2F6ko cl11, lower panel) cell clones. The CDC7 -2kb region served as a negative control region. Percent of input values represent the mean of at least three independent experiments +/- SD. (D) Expression of L3MBTL2 in L3MBTL2ko cells rescues binding of PRC1.6. Left, Western blot for L3MBTL2. Right, ChIP-qPCR data showing binding of exogenous L3MBTL2 and of endogenous MGA, E2F6 and PCGF6 to representative PRC1.6 target promoters. Percent of input values represent the mean of at least three independent experiments +/- SD. (E) Expression of wild type E2F6 but not of a DNA binding-deficient E2F6 mutant (E2F6mut) in E2F6ko cells rescues binding of PRC1.6. Left, Western blot for E2F6. Right, ChIP-qPCR data showing binding of exogenous E2F6 (wild type or DNA-binding deficient mutant) and of endogenous MGA and L3MBTL2 to representative PRC1.6 target promoters. Percent of input values represent the mean of at least three independent experiments +/- SD. (F) Venn diagram showing the overlap of E2F6 peaks in L3MBTL2ko cells and L3MBTL2 peaks in E2F6ko cells. Logos of the enriched sequence motifs were obtained by running MEME-ChIP with 300 bp summits of the ChIP-seq peaks. (G) GO analyses of biological functions of E2F6-dependent and of L3MBTL2-dependent PRC1.6 target genes. Enriched GO terms were retrieved using Enrichr. p values are plotted in -log2 scale.