O-GlcNAcylation of FOXK1 co-opts BAP1 to orchestrate the E2F pathway and promote oncogenesis

The E2F transcription factors constitute a core transcriptional network that governs cell division and oncogenesis in multi-cellular organisms, although their molecular mechanisms remain incompletely understood. Here, we show that elevated expression of the transcription factor FOXK1 promotes transcription of E2F target genes and cellular transformation. High expression of FOXK1 in patient tumors is also strongly correlated with E2F gene expression. Mechanistically, we demonstrate that FOXK1 is O-GlcNAcylated, and loss of this modification impairs FOXK1 ability to promote cell proliferation and tumor growth. We also show that expression of FOXK1 O-GlcNAcylation-defective mutants results in reduced recruitment of the H2AK119 deubiquitinase and tumor suppressor BAP1 to E2F target genes. This event is associated with a transcriptional repressive chromatin environment and reduced cell proliferation. Our results define an essential role of FOXK1 O-GlcNAcylation in co-opting the tumor suppressor BAP1 to promote cancer cell progression through orchestration of the E2F pathway. Chromatine immunoprecipitation and DNA-sequencing (ChIPseq) for endogenous FOXK1, FOXK2 in IMR90 cells as well as ChIPseq of exogenous 3Flag tagged FOXK1, FOXK2 and FOXK1 O-GlcNAc mutants FOXK1-7A, and FOXK1-11A. ChIPseq for endogenous FOXK1 and Flag tag FOXK1, FOXK1-7A in U2OS cells. ChIPseq for flag tag FOXK1 and FOXK2 in K562 cells. Cut&Run for BAP1, H3K4me1 and H2Aub performed in IMR90 cells expressing either FOXK1, FOXK1-7A or FOXK1-11A O-GlcNAc mutants. Cut&Run for FOXK1 and FOXK2 during cell cycle progression in synchronized IMR90 and U2OS cells. Cut&Run for BAP1 in U2OS knock-out for endogenous FOXK1 expression and expressing either FOXK1 or FOXK1-11A O-GlcNAc mutant. Chromatine immunoprecipitation and DNA-sequencing (ChIPseq) for endogenous FOXK1, FOXK2 in IMR90 cells as well as ChIPseq of exogenous 3Flag tagged FOXK1, FOXK2 and FOXK1 O-GlcNAc mutants FOXK1-7A, and FOXK1-11A. ChIPseq for endogenous FOXK1 and Flag tag FOXK1, FOXK1-7A in U2OS cells. ChIPseq for flag tag FOXK1 and FOXK2 in K562 cells. Cut&Run for BAP1, H3K4me1 and H2Aub performed in IMR90 cells expressing either FOXK1, FOXK1-7A or FOXK1-11A O-GlcNAc mutants. Cut&Run for FOXK1 and FOXK2 during cell cycle progression in synchronized IMR90 and U2OS cells. Cut&Run for BAP1 in U2OS knock-out for endogenous FOXK1 expression and expressing either FOXK1 or FOXK1-11A O-GlcNAc mutant. Chromatin accessibility (ATACseq) performed on U2OS cells expressing FOXK1 or FOXK1-7A and treated with siRNA targeting endogenous FOXK1 for knock-down. Transcriptomic analysis (RNA-seq) conducted on IMR90 cells expressing either a control vector, FOXK1, or FOXK2.