Figure 6.

BAHD1 interacts functionally and physically with the PRC2 complex. (A) Cas9-expressing F11KO HUDEP-2 cells were transduced with a lentiviral vector library encoding sgRNAs against 496 epigenetic modifier genes, selected in puromycin, then induced to undergo maturation for 5 days. Band3^high and Band3^low erythroblasts were FACS purified and analyzed by deep sequencing to compare the sgRNA representation. (B) Differential gene rank by RRA score from MAGeCK analysis^32,33 of Band3^high and Band3^low erythroblasts. The PRC2 complex genes EED, EZH2, and RBBP4 are indicated. (C) WT and F11KO (clone F11KO #2) HUDEP-2 cells expressing Cas9 plus individual EZH2, SUZ12, EED, or control (Ctrl) sgRNAs were analyzed for Band3 expression after 5 days of induced erythroid maturation. The graph shows the mean ± SEM from 4 biological replicate experiments. (D) Pellets of cells described in panel C after 5 days of induced maturation. (E) 293T cells were transfected with plasmids encoding BAHD1-MYC and EZH2 (left panel) or BAHD1-MYC and EED (right panel). After 48 hours, cell lysates were immunoprecipitated with anti-MYC or IgG antibody, and western blot analysis was performed with the antibodies indicated at the right of each panel. The input represents 10% of the immunoprecipitated samples. (F) GSEA of differentially expressed genes in F11KO vs WT HUDEP-2 cells (left panel) or BAHD1-OE vs control HUDEP-2 cells (right panel), showing enrichment for a PRC2 target gene set. (G) Model showing the regulation of erythroid gene expression by FBXO11-mediated degradation of BAHD1. In early-stage erythroblasts, many erythroid genes are maintained in a bivalent state associated with concomitant H3K4me3 and H3K27me3 promoter marks and low-level expression. The heterochromatin-associated protein BAHD1 binds H3K3me3, recruits repressor proteins, and physically interacts with the PRC2 complex (an H3K27me3 writer). Recruitment of the erythroid transcription factor GATA1 is inhibited by BAHD1-induced heterochromatin. FBXO11 relieves transcriptional repression, facilitates GATA1 binding and promotes transition to a monovalent activating state by stimulating ubiquitin-mediated proteolysis of BAHD1, either before or after its binding to DNA and/or its interaction with corepressor proteins. NGS, next-generation sequencing. **P < .01; ***P < .001; ****P < .0001 (unpaired Student t test).