E2F4 directly binds to promoter regions of Ccna2 and Mcm2 but notCcnd2. (A) Fold difference in RNA expression between E2f4^+/+ and E2f4^–/– E15.5 FLs determined by microarray analysis (##; n = 7) and real-time PCR (▪; n = 3), the latter normalized to GAPDH expression. Data represent 2 to 3 individual FLs pooled per experiment. (B) Relative abundance of Mcm2, Ccna2, and Ccnd2 mRNA transcript in E2f4^+/+ and E2f4^–/– erythroid cells at 0 and 24 hours after induction of differentiation, normalized to β 2M expression. Data shown are representative of 3 independent experiments. (C) For ChIP experiments, real-time PCR primers were designed to detect genomic DNA spanning putative E2F binding sites present in the Mcm2, Ccna2, and Ccnd2 promoters (Mcm2.E, Ccna2.E, and Ccnd2.E), in addition to nonspecific upstream sites (Mcm2.U, Ccna2.U, and Ccnd2.U), as marked. (D) ChIP experiments were performed on wild-type erythroid cells 24 hours after induction of differentiation in vitro. Cross-linked DNA/protein was immunoprecipitated using nonimmune rabbit serum (NRS), or rabbit polyclonal antibodies recognizing E2F4 or acetylated histone H3 (AcH-H3). Precipitated DNA samples were amplified with primers recognizing upstream promoter nonspecific sites (U) and the E2F consensus sites (E) of candidate genes. Quantification of precipitated DNA was measured by real-time PCR, where the amount of immunoprecipitated promoter DNA is depicted as a percentage of the amount present in the total input chromatin sample. Data shown are representative of 5 independent experiments. Error bars indicate standard deviation.