Isolation and characterization of the human A-myb promoter: regulation by NF-Y and Sp1

The A-myb transcription factor shows a restricted tissue distribution and i s cell cycle regulated. Furthermore its deregulation has profound effects o n the growth and/or differentiation of the cells in which it is normally ex pressed. We have therefore characterized its promoter. A 12 kb genomic clon e was isolated that comprises the first exon, part of the first intron as w ell as upstream regulatory sequences. Multiple transcription start sites ha ve been identified which operate in both B lymphocytes and epithelial cells and the upsteam region was shown to have promoter, activity. The boundarie s of the minimal promoter region (-183-14), of a positive upstream (-538-18 3) and a negative downstream regulatory region (NRE) (+83+374) have been de fined. The NRE is promoter- and orientation-independent but position specif ic. The A-myb minimal promoter is GC-rich, does not contain any TATA box bu t has a functional CCAAT box. The! CCAAT box and minimal promoter is highly conserved in the corresponding murine sequence, The CCAAT box efficiently binds the NF-Y complex and its mutation decreases basal promoter activity b y 50%, Two Sp1 binding sites are present upstream from the CCAAT box which can bind Sp1 and contribute to A-myb promoter activity by 70 and 30%, respe ctively. The two Sp1 sites and CCAAT box together contribute to over 80% of A-myb basal promoter activity and are therefore the major regulatory eleme nts. Finally, we show that the promoter is cell cycle regulated and that th e SP1 and CCAAT elements are required for S phase induction.