Figure 7

Chromatin immunoprecipitation (ChIP) assay for examining interactions in vivo of consensus binding sequences in the 5'promoter of MTG16. The forward and reverse primers used to amplify the proximal promoter region from -358 to -252 (primers A, solid arrows) and forward and reverse primers for a downstream region from 1700 to 1835 as control (primers B, dashed arrows) are shown. ChIP assays were carried out as described in Methods using chromatin isolated from erythroid HEL/TF-1, erythroid/megakaryocytic UT-7 and megakaryocytic MEG-01cells. PCR products were separated on a 2% gel and representative results are shown. Lane 1-2, no antibody and primers A or B; lanes 3-4, actin antibody and primers A or B; lanes 5-6, genomic DNA and primers A or B; lanes 7-11, GATA-1(top) or GATA-2 (bottom) antibody and primers A or B (lane 11). By use of the specific primers, a PCR product is generated both from the anti-GATA-1 and the anti- GATA-2 immunoprecipitated chromatin. No amplification is seen without antibody or in the presence of anti-actin. All experiments were repeated at least twice.