Increased H3K9me3 suppresses EBF1 expression. (A) A snapshot showing histone modification enrichment at gene locus EBF1. The gained H3K9me3 peak in CLL is highlighted. (B) Downregulated EBF1 but not PAX5 expression in CLL B cells. Gene expression is determined by RNA-seq. (C) Validation of the expression of EBF1 in the indicated samples by Reverse Transcritpion-Polymerase Chain Reaction (RT-PCR). (D) Validation of the expression of EBF1 in the indicated samples by western blot. (E) Scheme of CRISPR dCas9-Krab–mediated (Dead Cas9-Kruppel-associated box) epigenetic silencing (H3K9me3 deposition). (F) Histone modification profiles at the EBF1 locus at indicated samples. The cells with infection of indicated single-guide RNAs (sgRNAs) were incubated with 0.25 mg/mL doxycycline for 7 days to induce dCas9-Krab expression before the cells were harvested. (G) RT-qPCR analysis of EBF1 expression after the introduction of H3K9me3 at the indicated region by CRISPR. (H) Western blot analysis of EBF1 expression after the introduction of H3K9me3 at the indicated region by CRISPR. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; sgRNA, small guide RNA; ns, not significant; sgGFP, small guide RNA targeting Green fluoresent protein (GFP).