A DNA replication-related element (DRE)-binding factor (DREF) has been revealed to be an important transcription factor for activating promoters of cell proliferation and differentiation related genes. The amino acid sequences of DREF are conserved in evolutionary separate Drosophila species, Drosophila melanogaster (Dm) and Drosophila virilis (Dv) in three regions. In the present study, evidence was obtained that there are several highly conserved regions in the 5′ flanking region between the DmDREF and DvDREF genes. Band mobility shift assays using oligonucleotides corresponding to these conserved regions revealed that specific trans-acting factors can bind to at least three regions -554 to -543 (5′-TTTGTTCTTGCG), -81 to -70 (5′-GCCCACGTGGCT) and +225 to +234 (5′-GCAATCAGTG). Using a transient luciferase expression assay, we demonstrated that the region -554 to -543 functions as a negative regulatory element for DmDREF promoter activity, while the regions -77 to -70 (5′-ACGTGGCT) and +225 to +236 (5′-GCAATCAGTGTT) function as positive regulatory elements. In previous studies, we observed that expression of the homeodomain protein Zerknüllt (Zen) represses PCNA gene transcription, by reducing the DNA binding activity of DREF. Here we show Zen downregulates DREF gene promoter activity through action on the region between +241 and +254 (5′-AGAATACTCAACA). In addition, the DmDREF promoter contains five DREs. Using a double stranded RNA-mediated interference method, we generated evidence that expression of DmDREF could be auto-regulated by DREF through the third DRE located at +211 to +218. In living flies we obtained results consistent with those obtained in vitro and in cultured cells. The study thus indicates that DmDREF is effectively regulated via highly conserved regions between the DmDREF and DvDREF promoters, suggesting the existence of common regulatory factors, and that DmDREF can be positively regulated by itself via the third DRE located in its most highly conserved region.
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