cPRC1 and PRC2 cooperate to maintain the repression of IGF2BP1/3 and LIN28B in adult erythroid cells. (A) Volcano plots of DEGs identified from EZH2 depletion RNA-seq in HUDEP2 cells by DESeq2. Cutoff threshold: fold change > 2; FDR < 0.1; n = 2. (B-C) RT-qPCR analysis of the ratio of HBG:(HBB+HBG) mRNA levels (B), representative immunoblots (C) of EZH2, IGF2BP1, LIN28B, BMI1, H2AK119ub1, H3K27me3, and γ-globin protein in AsCas12a-expressing HUDEP2 cells that were transduced with lentivirus expressing indicated sgRNAs. Values are presented as mean ± SEM. RT-qPCR data were normalized to AHSP (n = 2). GAPDH was used as loading control in immunoblot experiment. (D) Schematic of EZH2 inhibition experiment in primary erythroid cells. (E) Representative immunoblots of IGF2BP1, IGF2BP3, H3K27me3, histone H3, and γ-globin in drug-treated primary adult erythroblasts. GAPDH were used as the loading control. (F) Relative IGF2BP1, IGF2BP3, and HBG mRNA level in primary adult erythroblasts (at day 10 or 12 of differentiation [equal to 3 or 5 days after drug treatment]) as detected by RT-qPCR. Values are presented as mean ± SEM. Data were normalized to AHSP; n = 4. (G) Summary of PRC1/2 perturbation experiments. In HUDEP2 cells, genetic perturbation of BMI1, CBX, or EZH2 led to upregulation of LIN28B and IGF2BP1, and thus, HbF activation. In primary adult erythroblasts, BMI1 depletion and EZH2 inhibition both resulted in derepression of IGF2BP1/3, and elevation of HbF. However, the molecular link between the 3 RNA binding proteins and HbF activation remains to be defined. Our observations support a major role of BCL11A in mediating the function of these RNA-binding proteins, especially in HUDEP2 cells, however we cannot rule out additional, yet to be identified targets of 3 RNA binding proteins involved in HbF activation.