IRF8 regulates EBF expression. (A) Schematic arrangement of IRF8-binding sites (IECS, EICE, and ISRE) in the promoter of the Ebf1 gene. +1 corresponds to the first nucleotide of exon 1 of the gene. The nucleotides that were mutated at each IRF8-binding site are shown on the top. WT indicates wild type. (B) ChIP analysis of IRF8 binding with Ebf1. In vivo cross-linked IRF8-chromatin complexes from NFS 202 cells were analyzed using PCR primers that span the IRF8 sites in the Ebf1 gene. (C) IRF8 and PU.1 regulate EBF expression in a luciferase reporter assay. HeLa cells were cotransfected with a promoter reporter pGL4-Ebf1-WT and vectors expressing IRF8, PU.1, or both. An empty vector was used as a control. (D) Mutation of IRF8-binding sites in the Ebf1 gene impaired the expression of the Ebf1 reporter. The Ebf1 promoter reporter constructs containing mutated IRF8-binding sites were generated as illustrated in panel A and were cotransfected with plasmids expressing IRF8 and PU.1. Luciferase activities were measured after 22 hours. All data represent 3 to 4 independent experiments.