NG2 is a direct target of MLL-AF4 fusion protein in iB-ALL. (A) scRNA-seq heat map depicting the expression of various B-cell differentiation markers and NG2 in the indicated populations across human fetal B-cell differentiation (data from O'Byrne et al⁴⁰). (B) Box plots showing NG2 expression in the indicated normal or leukemic B cells (n = 44 patients, data from Agraz-Doblas et al²⁹). (C) Quantitative PCR (qPCR) analysis comparing NG2 expression in leukemic cells from patients with B-ALL with MLL germ line or MLL-AF4 (n = 6). (D) qPCR analysis of NG2 expression in MLL-AF4⁺ genome-edited fetal live (FL) and cord blood (CB) CD34⁺ cells. (E) Methylation levels (β value) across NG2 in normal B-cell progenitors (BCPs) and MLL germ line and MLL-AF4 leukemic cells, inferred from whole-genome bisulfite sequencing (WGB-seq) data (data from Tejedor et al²⁸). Bar plots depict the average DNA methylation level of the differentially methylated regions (DMRs) or differentially methylated promoters (DMPS) observed in the indicated conditions. (F) Scatter plot showing the correlation between DNA methylation and gene expression of NG2. (G) Experimental design of the dCas9-DNMT3A methylation editing approach. (H) DNA methylation levels at the indicated CpG residues (C066 and C1) in dCas9-DNMT3A^WT-targeted SEM cells (WT + sg) and nontargeted SEM cells (WT NT) (left panel). Right panel shows NG2 expression by qPCR in dCas9-DNMT3A^WT-targeted and nontargeted SEM cells. (I) DNA methylation levels (left panel) and NG2 expression (right panel) of dCas9-DNMT3A^MUT-targeted SEM cells (MUT + sg) and nontargeted SEM cells (MUT NT). (J) DNA methylation levels in MLL-AF4⁺ genome-edited CD34⁺ HSPCs. (K) Representative MLL chromatin immunoprecipitation–sequencing (ChIP-seq) tracks at NG2 gene in MLLr primografts, MLL-AF4⁺ B-ALL blasts, and healthy CB and fetal bone marrow (FBM) (data from Godfrey et al^42-46). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, unpaired Student t test. Ns, not significant.