Figure 3
C/EBPβ binds to the chromatin embedded MAD1 promoter in cells. A. Schematic presentation of the MAD1 promoter relative to the major transcriptional start site with the homology region as defined in [17]. The positions of primer sets used for ChIP PCR are given (upper panel). U937 cells were used in ChIP assays to investigate the binding of C/EBPβ to the MAD1 promoter in response to TGFβ1. The cells were treated with 2.5 ng/ml TGFβ1 for 1.5 hrs prior to crosslinking and immunoprecipitation, and the purified DNA was analyzed by PCR with the indicated primers. The displayed experiment is representative of three independent experiments with similar outcome. B. C/EBPβ was expressed in HEK293 cells. Whole cell extracts were generated and incubated with [32P]-radiolabelled CCAAT box1 oligonucleotids derived from the MAD1 or the neutrophil elastase promoter (NE-CCAAT). Supershift experiments with C/EBPβ specific antibodies are indicated. The complexes were analyzed by EMSA and detected by autoradiography. Ø, indicates lanes without cell extracts; +, whole cell extracts of control transfected HEK293 cells; β, whole cell extracts of HEK293 cells transiently expressing C/EBPβ. C. The C/EBPα/β heterodimer DNA-binding activity to CCAAT box1 was determined by EMSA. HEK293 cell extracts overexpressing C/EBPα and C/EBPβ were generated as in panel B and incubated with radiolabelled CCAAT box1. Supershift experiments were performed with C/EBPα- or C/EBPβ-specific antibody. For control an irrelevant oligonucleotide was used. D. Re-ChIP experiment using C/EBPα- and C/EBPβ-specific antibodies in U937 cells treated with or without TGFβ1 for 1.5 hrs. The first immunoprecipitation was performed with antibodies specific for C/EBPβ. The protein-DNA complexes were then released and subjected to a second immunoprecipitation using C/EBPα or C/EBPβ specific antibodies. The isolated DNA was analyzed by quantitative PCR with the primer set P (panel A) amplifying a portion of the homology region of the MAD1 promoter. The signal intensities obtained with the specific immunoprecipitations were normalized to samples generated with control antibodies. For all panels one representative of typically at least three independent experiments are shown.