Use of chromatin immunoprecipitation to clone novel E2F target promoters

We have taken a new approach to the identification of E2F-regulated promote rs. After modification of a chromatin immunoprecipitation assay, we cloned nine chromatin fragments which represent both strong and weak in vivo E2F b inding sites. Further characterization of three of the cloned fragments rev ealed that they are bound in vivo not only by E2Fs but also by members of t he retinoblastoma tumor suppressor protein family and by RNA polymerase II, suggesting that these fragments represent promoters regulated by E2F trans cription complexes. In fact, database analysis indicates that all three fra gments correspond to genomic DNA located just upstream of start sites for p reviously identified mRNAs. One clone, ChET 4, corresponds to the promoter region for beclin 1, a candidate tumor suppressor protein. We demonstrate t hat another of the clones, ChET 8, is strongly bound by E2F family members in vivo but does not contain a consensus E2F binding site. However, this fr agment functions as a promoter whose activity can be repressed by E2F1. Fin ally, we demonstrate that the ChET 9 promoter contains a consensus E2F bind ing site, can be activated by E2F1, and drives expression of an mRNA that i s upregulated in colon and liver tumors. Interestingly, the characterized C hET promoters do not display regulation patterns typical of known E2F targe t genes in a U937 cell differentiation system. In summary, we have provided evidence that chromatin immunoprecipitation can be used to identify E2F-re gulated promoters which contain both consensus and nonconsensus binding sit es and have shown that not all E2F-regulated promoters show identical expre ssion profiles.