PROTEIN-DNA INTERACTIONS AT THE MAJOR AND MINOR PROMOTERS OF THE DIVERGENTLY TRANSCRIBED DHFR AND REP3 GENES DURING THE CHINESE-HAMSTER OVAR, CELL-CYCLE

In mammals, two TATA-less bidirectional promoters regulate expression of the divergently transcribed dihydrofolate reductase (dhfr) and rep3 genes. In CHOC 400 cells (dhfr) mRNA levels increase about fourfold d uring the G(1)-to-S phase transition of the cell cycle, whereas the le vels of rep3 transcripts vary less than twofold during this time. To a ssess the role of DNA-binding proteins in transcriptional regulation o f the dhfr and rep3 genes, the major and minor dhfr-rep3 promoter regi ons were analyzed by high-resolution genomic footprinting during the c ell cycle. At the major dhfr promoter, prominent DNase I footprints ov er four upstream Sp1 binding sites did not vary throughout G(1) and en try into the S phase, Genomic footprinting revealed that a protein is constitutively bound to the overlapping E2F sites throughout the G(1)- to-S phase transition an interaction that is most evident on the trans cribed template strand On the nontranscribed strand, multiple changes in the DNase I cleavage pattern are observed during transit through G( 1) and entry into the S phase, By using gel mobility shift assays and a series of sequence-specific probes, two different species of E2F wer e shown to interact with the dhfr promoter during the cell cycle, The DNA binding activity of one E2F species, which preferentially recogniz es the sequence TTTGGCGC, did not vary significantly during the cell c ycle, The DNA binding activity of the second E2F species, which prefer entially recognizes the sequence TTTCGCGC, increased during the G(1)-t o-S phase transition. Together, these results indicate that Sp1 and th e species of E2F that binds TTTGGCGC participate in the formation of a basal transcription complex, while the species of E2F that binds TTTC GCGC regulates dhfr gene expression during the G(1)-to-S phase transit ion, At the minor promoter, DNase I footprints at a consensus c-Myc bi nding site and three Sp1 binding sites showed little variation during the G(1)-to-S phase transition. In addition to protein binding at sequ ences known to be involved in the regulation of transcription, genomic footprinting of the entire promoter region also showed that a protein factor is constitutively bound to the first intron of the rep3 gene.