Figure 2.

Direct activation of the Ebf1 proximal β promoter by Runx1. (A) Real-time PCR analysis showing messenger RNA (mRNA) expression levels of several B cell signature genes in subsets of pro-B cells (HSA⁺ BP-1⁻, HSA⁺ BP-1⁺, and HSA^hi BP-1⁺ in the order of development) from Runx1^F/F;mb-1-cre mice. Data were normalized to HPRT and are shown as fold changes to wild-type control. (B) A ChIP-on-chip assay was performed with anti-Cbfβ antibody to evaluate Runx–Cbfβ binding to the promoter regions of E2a, Pax5, and Ebf1 genes in B220⁺ bone marrow cells. Positions of putative RRSs within the Ebf1 proximal β promoter are indicated. One representative of two independent experiments is shown. (C) Analytical ChIP assay using B220⁺ bone marrow cells. The mb1 promoter was used as a positive control for Cbfβ binding. One representative result from three independent experiments is shown. (D) Schematic overview of the Ebf1 proximal β promoter is shown with three predicted RRSs. +1 indicates the transcription start site. The top panel shows the structure of each reporter construct, and the bottom panel indicates relative luciferase activity from each construct in a transfection assay in the Ba/F3 cell line. Values are shown in relative light units (RLU). (E) Relative H3K4Me3 (K4) and H3K27Me3 (K27) histone modification levels at the Ebf1 proximal β promoter, the mb1 promoter, and ThPOK silencer regions in Runx1-deficient pro-B cells relative to wild-type pro-B cells. Data are represented as relative fold changes as in A. (A, D, and E) Error bars represent mean ± SD of three independent experiments.