Figure 6

Chromatin immunoprecipitation (ChIP) assay for examining interactions in vivo of consensus GATA binding sequences in the 5'promoter of ETO. The forward and reverse primers used to amplify the proximal promoter region from -684 to -595 (primers A, solid arrows) and forward and reverse primers for a downstream region from 1929 to 2032 as control (primers B, dashed arrows) are shown. ChIP assays were carried out as described in Methods using chromatin isolated from HEL and MEG-01 cells. PCR products were separated on a 2% gel and representative results are shown. Top and bottom gel figures represent MEG-01 and HEL cells, respectively, except that lane 10 in top gel represents HEL cells. Lane 1, 100-bp ladder; lane 2, actin antibody and primers A; lane 3, actin antibody and primers B; lane 4, no antibody and primers A; lane 5, no antibody and primers B; lane 6, GATA-2 antibody and primers A; lane 7, GATA-2 antibody and primers B; lane 8, genomic DNA and primers A; lane 9, genomic DNA and primers B. GATA-1 precipitated chromatin amplified with primers A in HEL and MEG-01 cells is shown in lane 10 and lane 11, respectively (top gel). By using primers specific for the evolutionary conserved region of the ETO promoter, a PCR product is generated both from the anti-GATA-1 and the anti-GATA-2 immunoprecipitated chromatin. No amplification is seen in the absence of antibody or in the presence of anti-actin. The experiment was repeated twice.