Protein-DNA Interactions at the Major and Minor Promoters of the Divergently Transcribed dhfr and rep3 Genes during the Chinese Hamster Ovary Cell Cycle

Abstract

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 during the G1-to-S phase transition of the cell cycle, whereas the levels of rep3 transcripts vary less than twofold during this time. To assess the role of DNA-binding proteins in transcriptional regulation of the dhfr and rep3 genes, the major and minor dhfr-rep3 promoter regions were analyzed by high-resolution genomic footprinting during the cell cycle. At the major dhfr promoter, prominent DNase I footprints over four upstream Sp1 binding sites did not vary throughout G1 and entry into the S phase. Genomic footprinting revealed that a protein is constitutively bound to the overlapping E2F sites throughout the G1-to-S phase transition, an interaction that is most evident on the transcribed template strand. On the nontranscribed strand, multiple changes in the DNase I cleavage pattern are observed during transit through G1 and entry into the S phase. By using gel mobility shift assays and a series of sequence-specific probes, two different species of E2F were shown to interact with the dhfr promoter during the cell cycle. The DNA binding activity of one E2F species, which preferentially recognizes the sequence TTTGGCGC, did not vary significantly during the cell cycle. The DNA binding activity of the second E2F species, which preferentially recognizes the sequence TTTCGCGC, increased during the G1-to-S phase transition. Together, these results indicate that Sp1 and the species of E2F that binds TTTGGCGC participate in the formation of a basal transcription complex, while the species of E2F that binds TTTCGCGC regulates dhfr gene expression during the G1-to-S phase transition. At the minor promoter, DNase I footprints at a consensus c-Myc binding site and three Sp1 binding sites showed little variation during the G1-to-S phase transition. In addition to protein binding at sequences 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.

Caspase-dependent apoptosis by ectopic expression of E2F-4

“…Band A composed of complexes containing E2F-4 and p107 was not aected by the ectopic expression of E2F-4. Bands B and C containing p130 and pRb (Wells et al, 1996(Wells et al, , 1997 were not visible in this experiment.…”

“…Induction of complex C was accompanied by decreases in other E2F DNA binding complexes. In E4-4 cells, E2F-4 expression increased the binding of complex D, which is composed of E2F-4 and DP-1 (Wells et al, 1996(Wells et al, , 1997. Band A composed of complexes containing E2F-4 and p107 was not aected by the ectopic expression of E2F-4.…”

The periodic down regulation of Cyclin E gene expression from exit of mitosis to end of G1 is controlled by a deacetylase- and E2F-associated bipartite repressor element

“…This ®nding is unusual in the present state of the art of the E2F ®eld. Indeed, while several studies have described variations in the in vivo occupation of E2F sites during the G 0 -S transition, none could reproduce these variations in vitro using EMSA (Campanero et al, 1999;Pfeifer, 1995, 1999;Wells et al, 1996;Zwicker et al, 1996). We previously hypothesized that CERM is a two-component element which displays a weak a nity for E2Fs unless they are associated with an unidenti®ed partner(s) within the CERC complex.…”

c-Myc initiates illegitimate replication of the ribonucleotide reductase R2 gene

“…Previous work has established that c-Myc deregulation initiates locusspeci®c gene ampli®cation (Denis et al, 1991;Mai, 1994;Mai et al, 1996aMai et al, , 1999, which is reversible in vitro (Mai et al, 1996a), and karyotypic instability (Mai et al, 1996a;Felsher and Bishop, 1999a), which is reversible in vivo (Felsher and Bishop, 1999b). c-Myc-dependent gene ampli®cation is associated with the binding of c-Myc/Max heterodimers to E-boxes located on c-Myc target genes (Mai and Jalava, 1994;Wells et al, 1996). c-Myc/Max heterodimers in cell-free extracts from proliferating cells and in vivo were shown to bind two adjacent 5'-anking E-box motifs of the dihydrofolate reductase (DHFR) gene (Mai and Jalava, 1994;Wells et al, 1996) and to four E-boxes located in the 5'-¯anking region of cyclin D2 (CCND2) .…”

“…c-Myc-dependent gene ampli®cation is associated with the binding of c-Myc/Max heterodimers to E-boxes located on c-Myc target genes (Mai and Jalava, 1994;Wells et al, 1996). c-Myc/Max heterodimers in cell-free extracts from proliferating cells and in vivo were shown to bind two adjacent 5'-anking E-box motifs of the dihydrofolate reductase (DHFR) gene (Mai and Jalava, 1994;Wells et al, 1996) and to four E-boxes located in the 5'-¯anking region of cyclin D2 (CCND2) . In these studies, c-Myc/Max heterodimer binding was correlated with cellular proliferation and DNA synthesis.…”