ZNF423 expression is regulated by DNA methylation. (A) Transactivation of ZNF423 isoform-specific promoters in dependence of central CGI. Reporter gene assay using ZNF423 promoters (α and β) with or without CGI after transfection in 293T cells. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent SD from three technical replicates. Significance is calculated by Student’s t test, comparing indicated samples (***, P ≤ 0.001). Data were reproduced in three independent experiments. (B) Methylation map of CpGs in the central CGI at the ZNF423 locus. Genomic DNA from primary ALL (n = 58), matched BM MNCs in complete continuous remission (MNC [CCR]; n = 58), H1, HES2 ESC lines, and normal hematopoietic cells at various stages of differentiation (n = 42) from healthy donors were sequenced after bisulfite conversion. Each row represents one cytosine in a CG dinucleotide of the analyzed sequence. For cell lineage abbreviations, refer to Figure 1. Significance is calculated by Student’s t test, comparing primary ALL to immunologically characterized control samples (in brackets; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). Color code represents degree of methylation in percent. White boxes indicate no sequencing data available. (C) ZNF423 promoter activity in dependence of CPG mutational status. Disruption of CpGs was performed by site-directed mutagenesis as indicated by red marks. Combined deletion mutant (pCpGL-CGI-del_comb_α-prom) represents deletions at CGI positions 102, 183, 220, 250. Wild-type and mutated plasmids were treated with DNA-methylase M.SssI and transfected into 293T. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent SD from three technical replicates. Significance is calculated by Student’s t test, comparing M.SssI-treated wild-type and mutated samples (***, P ≤ 0.001). Data were reproduced in three independent experiments. (D) DNA methylation pattern of central CGI. Diagram was created with BiQ Analyzer software from Max-Planck-Institute of Informatics. Drawing is to scale. White lollipop, unmethylated; black lollipop, methylated. Marked CpGs refer to heatmap in Fig. 4 B. (E and F) Expression of ZNF423 transcripts upon DNA demethylation and BMP2 stimulation. SEM cells (E) and CD34⁺ cord blood cells (F) were treated with 3 µM 5A2D for 24, 48, and 72 h. In SEM cells, additional BMP2 treatment was performed 6 h before lysis. Relative fold induction was measured by qPCR (2^−ΔΔCt) using a ZNF423 isoform-specific primer design in SEM cells. mRNA levels were normalized to B2M and solvent control. Error bars represent SD out of three technical replicates. Significance is calculated using 2^−ΔCt values by Student’s t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; n.s., not significant). Data were reproduced in three independent experiments.