NF-Y CONTROLS TRANSCRIPTION OF THE MINUTE VIRUS OF MICE P4 PROMOTER THROUGH INTERACTION WITH AN UNUSUAL BINDING-SITE

Electrophoretic mobility shift assays performed with nuclear extracts from human fibroblasts revealed the formation of two major protein com plexes with an oligonucleotide (nucleotides 78 to 107) from the palind romic region located upstream from the minute virus of mice (MVM) P4 p romoter. It was shown that this oligonucleotide bound USF at the enhan cer E box CACATG. The second complex contained the transcription facto r NF-Y, whose association was surprising because its target sequence l acks the canonical CCAAT motif present in all mammalian NF-Y binding s ites identified so far. The MVM NF-Y recognition element instead conta ins the CCAAC sequence. USF and NF-Y had distinct but overlapping sequ ence requirements for binding, suggesting that their associations with MVM DNA were mutually exclusive. Because of the palindromic nature of MVM DNA terminal sequences, NF-Y associated with the three nucleotide configurations corresponding to the hairpin structure and to the exte rnal and internal arms of the extended duplex replication form, respec tively. However, owing to the imperfection of the palindrome, the bind ing of USF was restricted to the internal arm. Point mutations that su ppressed the in vitro binding of NF-Y to the internal palindromic arm reduced the activity of the resident P4 promoter, while those preventi ng complex formation with USF did not, as determined by transient expr ession assays using the luciferase reporter gene. The data led to the identification of a novel P4 upstream regulatory region capable of int eracting with two transcription factors, from which one (NF-Y) appeare d to upmodulate the activity of the promoter.