Bisulfite analysis and mRNA expression from Runx1 P1 promoter. (A) Methylation patterns of the Runx1 P1 promoter in cells from in vivo–derived E14.5 MEFs, E8.5 YS CD41⁺ (E8.5 YS), E14.5 FL Lin-Sca-1⁺CD48⁻CD150⁺ (E14.5 FL), and adult marrow Lin-c-Kit⁺Sca-1⁺CD150⁺CD48⁻ (KLS + SLAM). Sequencing reactions of individual amplicons are represented by each row of circles. Open circles denote unmethylated CpGs, and solid circles represent methylated CpGs. (B) Quantification of percent CpG methylation at P1 in hematopoietic populations derived in vivo. ***P < .001. (C) Methylation patterns in mESCs, day 6 EB c-Kit⁺CD41⁺ (D6 EB) and from OP9 cocultures: GFP⁺c-Kit⁺CD45⁺ cells isolated from IRES-GFP control group at day 6 (D6 + 6 control) and HOXB4-IRES-GFP group at day 6 (D6 + 6 HoxB4) and day 11 (D6 + 11 HoxB4). (D) Quantification of percent CpG methylation at P1 in cell populations isolated during hematopoietic differentiation of mESCs. ***P < .001; *P < .05. (E) P1/P2 mRNA levels after normalization to Gapdh over the course of hematopoietic differentiation in vitro.