Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo.

Abstract

The dihydrofolate reductase (DHFR) gene encodes an enzyme important for metabolism and cell growth. We have found multiple DNA-protein interactions within the hamster DHFR gene promoter in vitro. These interactions occur over the consensus binding sites for two eucaryotic transcription factors, Spl and E2F. The DHFR E2F consensus site possesses a dyad symmetry and is unique in its location immediately 3' to the major transcription start site. The interaction of E2F with the DHFR promoter has been detected in HeLa nuclear extracts, confirmed by using partially purified E2F, and characterized by both enzymatic and chemical assays of the DNA-protein interaction. A mutation of the E2F recognition sequence which abolishes E2F binding to the DHFR promoter results in a two-to fivefold decrease of in vitro transcriptional activity and a fivefold reduction of DHFR promoter activity in transient-expression assays. Thus, the interaction of E2F with the DHFR promoter is required for efficient expression of the DHFR gene.Dihydrofolate reductase (DHFR) is a metabolic enzyme involved in the synthesis of purines, thymidylate, and glycine (23). It is considered a housekeeping enzyme, since it is found in small amounts in nearly all cell types and is required for cell growth (21). Although the gene encoding DHFR is widely expressed, this expression is influenced by several factors, including the growth state of the cells, the cell cycle, and the presence of viral infection (6,21,27). The DHFR promoter lacks TATAA and CCAAT DNA sequence motifs, which are regulatory sequence elements found in most eucaryotic promoters transcribed by RNA polymerase II. The DHFR promoter contains multiple copies of GC boxes, which are capable of interacting with the transcription factor Spl (5). An additional conserved sequence including the major transcription start site has been identified in the DHFR promoter (1).The eucaryotic transcription factor E2F was originally identified as a DNA-binding activity in HeLa nuclear extracts (12). This factor binds to two sites in a region of the adenovirus E2 promoter known to be required for E2 promoter activity. E2F binding activity increases upon infection of HeLa cells with wild-type adenovirus; this increase depends on the presence of a functional EIA gene (12). The adenovirus ElA gene product is known to trans-activate several viral and cellular genes by a variety of mechanisms. E2F is hypothesized to be one of the targets of the adenovirus ElA gene product early in adenovirus infection. E2F binding sites have also been identified in the ElA enhancer (13) and the c-myc promoter (10, 24); E2F binds to the sequence 5'-TTTCGCGC-3'. DHFR expression is increased by adenovirus infection, although the molecular level at which this increase occurs is unclear (7,27).One study of the mouse DHFR promoter reported that a fraction of HeLa nuclear extract enriched in Spl bound to the consensus Spl binding sites in the mouse DHFR promoter (5). It also showed that HeLa nuclear extracts depleted of this fraction were inc...

Interactions of the p107 and Rb proteins with E2F during the cell proliferation response.

“…Moreover, E2F appears to be a critical component of the activation of transcription of the cellular dihydrofolate reductase (DHFR) gene (Blake and Azizkhan, 1989) as cells progress from G1 into S phase (Means et al, 1992). Although previous experiments demonstrated the cell cycle regulation of complex formation involving E2F with cyclin A specifically during S-phase, several questions remained unanswered.…”

Identification of a Viral Kinase That Phosphorylates Specific E2Fs and Pocket Proteins

“…The wild-type DHFR promoter construct contains four Sp1 sites and the dyad E2F sites (7). DHFR⌬E2F contains a substitution of TA at positions Ϫ57 and Ϫ56 and does not bind E2F (6).…”

“…The gene encoding dihydrofolate reductase (DHFR) was the first cellular gene shown to contain a binding site for E2F (6), and transactivation of its promoter by adenovirus E1A was found to be E2F dependent (18). Subsequently, E2F was shown to be involved in the cell cycle regulation of several genes important in cellular growth control (for reviews, see references 23, 24, and 38).…”

Cell Cycle Regulation of the Human Polo-like Kinase (PLK) Promoter

“…The E2F/DP transcription factors are repressed in G 0 -G 1 due to their association with RB family members. In late G 1 , transcription of E2F-regulated genes such as DHFR, E2F-1, and DNA polymerase is activated following dissociation of E2F/DP complexes from RB (15)(16)(17). Similarly, transcription of cdc2 is repressed during G 1 by binding of E2F-4-p130 complexes at the CHR/CDE region (18).…”