Figure 4

The Sp1/Myc-Max overlapping site is specific in BRD7 minimal promoter. (A) Top: The sense sequences of oligonucleotides used in electrophoretic mobility-shift assay. Bottom: Identification of specific E2F6 and Sp1/Myc-Max overlapping site in BDR7 minimal promoter. Oligonucleotides -260/-246, -243/-229 and -223/-198 containing a putative MYC-MAX, an E2F-6 binding site and a Sp1/Myc-Max overlapping site were respectively radiolabeled and incubated with 8 μg nuclear extracts of 5-8F cells in EMSA. For competition assay, 100-molar excess of unlabeled wild type or mutated oligonucleotide -260/-246, -243/-229 and -223/-198 were added to the reaction mixture, respectively, before the addition of radiolabeled probes. NP represents nuclear protein. In the supershift experiments, 2 μg antibody of Sp1 (Upstate Biotechnology), c-Myc (Sigma), E2F6 (Santa Cruz Biotechnology) and irrelevant anti-HA (Santa Cruz Biotechnology) was added to the reaction, respectively. (B) Transcription factor c-Myc and Sp1 are in vivo associated with BRD7 minimal promoter. Left panel: equivalent amounts of chromatin were immunoprecipitated in 5-8F cells by antibodies as indicated or an irrelevant antibody (Anti-HA) as a control. Right panel: equivalent amounts of chromatin were immunoprecipitated in 5-8F/Si-c-Myc cells by antibodies as indicated. PCR was performed using the indicated BRD7 primers or GAPDH primers.