A transcriptional regulatory element screening system reveals a novel E2F1/pRb transcription regulation pathway.

We developed a transcriptional regulatory element library which contains 160 independent known transcriptional regulatory elements linked to luciferase reporter vectors. That library proved valuable in the identification of p53 response elements and of E-box sequence preferences of several E-box binding proteins, and we used it to explore E2F1 target regulatory elements. Among those 160 elements, we found 3 E2F1 response elements, an E2F1 consensus sequence, an insulin response element which contained the E2F consensus sequence, and a basal level enhancer (BLE1) which had a nonconsensus E2F binding sequence. BLE1 functioned as multiple copy, with E2F1 in a dose-dependent manner, and had a sequence specificity for E2F1. Electrophoretic mobility shift assay revealed that BLE1 specifically interacts with E2F1 comparable to the E2F element. Interestingly, transactivation via five copies of BLE1 was not repressed but rather was stimulated by E2F1 in combination with the retinoblastoma tumor suppressor protein (pRb). The retinoblastoma control element (RCE) contains a direct repeated BLE1 in the c-fos gene promoter which also functioned like the multiple BLE1. Our data show that E2F1 has potential binding activity to the RCE and a different transcriptional regulation pathway which cooperates with pRb. Our transcriptional regulatory element screening system is useful for identifying novel transcriptional pathways.