CORRECT USAGE OF MULTIPLE TRANSCRIPTION INITIATION SITES AND C EBP-DEPENDENT TRANSCRIPTION ACTIVATION OF THE RAT XDH/XO TATA-LESS PROMOTER REQUIRES DOWNSTREAM ELEMENTS LOCATED IN THE CODING REGION OF THE GENE/

In the present study, we have shown that a downstream element located in the coding region of the TATA-less rat xanthine dehydrogenase/oxida se (XDH/XO) gene (-7 to +42) plays an important role in transcription initiation and C/EBP transcriptional activation. Previous work from ou r laboratory has shown that the promoter is organized with multiple in itiator elements (Inr 1, 2, 3 and 4) which are important for transcrip tion initiation. Additionally, we had identified two C/EBP binding sit es upstream of this promoter. Deletional and mutational studies reveal ed that C/EBP binding was not essential for the basal level of transcr iptional initation. However when XO-luciferase constructs include down stream sequence extending to +42 there is development of C/EBP sensiti vity as well as a shift in the initiator usage. In the absence of the downstream element, primer extension analyses reveals Inr 3 and 4 to b e the major start sites but in the presence of this additional sequenc e the usage is shifted to Inr 1 and 2, This shift in Inr usage more cl osely resembles that seen in intact macrophages or liver cells. Gel mo bility shift assays indicate the presence of several binding factors l ocated in this downstream region, one of which has been identified as YY-1, We postulate that YY-1 allows DNA bending which permits the upst ream C/EBP elements to exhibit a transcriptional activation which is n ot seen when the downstream element is absent. This study presents a p otential model for regulation of the XDH/XO promoter.