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...

Numerous genes contain TATAA-less promoters, and the control of transcriptional initiation in this important promoter class is not understood. We have determined that protein-DNA interactions at three of the four proximal GC box sequence elements in one such promoter, that of the hamster dihydrofolate reductase gene, control initiation and relative use of the major and minor start sites. Our results indicate that although the GC boxes are apparently equivalent with respect to factor binding, they are not equivalent with respect to function. At least two properly positioned GC boxes were required for initiation of transcription. Abolishment of DNA-protein interaction by site-specific mutation of the most proximal GC box (box I) resulted in a fivefold decrease in transcription from the major initiation site and a threefold increase in heterogeneous transcripts initiating from the vicinity of the minor start site in vitro and in vivo. Mutations that separately abolished interactions at GC boxes II and III while leaving GC box I intact affected the relative utilization of both the major and minor initiation sites as well as transcriptional efficiency of the promoter template in in vitro transcription and transient expression assays. Interaction at GC box IV when the three proximal boxes were in a wild-type configuration had no effect on transcription of the dihydrofolate reductase gene promoter. Thus, GC box interactions not only are required for efficient transcription but also regulate start site utilization in this TATAA-less promoter.Transcription of eucaryotic promoters by RNA polymerase II involves multiple sequence elements and protein factors that associate with these sequences. Certain DNAprotein interactions regulate the efficiency of transcriptional initiation, while others have the additional role of specifying the transcriptional initiation site. In many class II gene promoters, a TATAA sequence element is located approximately 25 to 30 bp upstream of the transcription start site (5, 17); interaction of a factor(s) with this sequence specifies the site of initiation in many of these promoters (24, 41). However, in other eucaryotic promoters (7,25,47), interaction with TATAA appears not to specify the start site but rather to control the efficiency of transcription from a downstream initiation site. Another common important control element, CCAAT, is the target of factors that regulate the efficiency of transcription (13,24).A large subclass of polymerase II promoters lacks both TATAA and CCAAT sequence motifs but contains multiple GC boxes. This promoter class includes several housekeeping genes (e.g., the genes encoding dihydrofolate reductase [DHFR] (26), GCF-1 (27), and AP2 (35) have also been shown to interact with GC boxes. Determining the functional role of multiple GC boxes in the absence of TATAA and CCAAT motifs is crucial to the understanding of transcriptional regulation of this important class of promoters.It has been shown that GC boxes are required for efficient promoter activity in the...

Members of the transforming growth factor-␤ (TGF-␤)superfamily mediate a broad range of biological activities by regulating the expression of target genes. Smad proteins play a critical role in this process by binding directly to the promoter elements and/or associating with other transcription factors. TGF-␤1 up-regulates several genes transcriptionally through Sp1 binding sites; however, the mechanism of TGF-␤ induction of gene expression through Sp1 sites is largely unknown. Here we report the identification of a novel 38-base pair TGF-␤-responsive element in the human plasminogen activator inhibitor-1 (PAI-1) promoter, which contains two Sp1 binding sites, and is required for TGF-␤-induced Smad-dependent transcriptional activation. Three canonical Sp1 binding sites also support strong transcriptional activation by TGF-␤ and Smads from a minimal heterologous promoter. TGF-␤ induction of PAI-1 and p21 is blocked by the Sp1 inhibitor mithramycin, implicating Sp1 in the in vivo regulation of these genes by TGF-␤. We show that the association between endogenous Sp1 and Smad3 is induced by TGF-␤ in several cell lines; however, Smad4 shows constitutive interaction with Sp1. These data provide novel insights into the mechanism by which TGF-␤ up-regulates several gene expression by activating Sp1-dependent transcription through the induction of Smad/Sp1 complex formation.

Dihydrofolate reductase (DHFR) gene expression is required for cell growth. The DHFR gene promoter contains several GC elements capable of binding the transcription factor Sp1. In this report we have characterized the effect of protein(s) binding to these sequence elements in the Chinese hamster DHFR promoter on transcription. We have constructed a series of deletions containing from 896 to 103 bp 5' to the start of translation. The protein binding domains have been mapped by DNAse I footprint analysis using HeLa nuclear extract, and the function of the protein-binding elements has been assessed by in vitro transcription and transient CAT expression. Maximal transcription in vitro and CAT expression is obtained with a construct containing 3 GC elements extending to position -184. Removal of GC element binding factor(s), by competition with an oligonucleotide containing an Sp1 binding site, completely abolishes transcription in vitro and significantly diminishes CAT expression. Ten-fold higher molar excess of competitor is required to abolish SV40 early transcription, suggesting that the GC element interactions in the DHFR promoter are different from those in the SV40 early region. Co-transfection of a DHFR CAT construct with an expressor of Sp1 dramatically increased CAT expression in Drosophila cells.

We demonstrated that MIF-1, identified initially as a binding activity that associated with the intron I element of the c-myc gene, consists of two polypeptides, the myc intron-binding peptide (MIBP1) and the major histocompatibility class II promoter-binding protein, RFX1. Using a polyclonal antiserum directed against either oligonucleotide affinity-purified MIBP1 or a peptide derived from RFX1, we showed that MIBP1 and RFX1 are distinct molecules that associate in vivo and are both present in DNA-protein complexes at the c-myc (MIF-1) and major histocompatibility complex class II (RFX1) binding sites. We have also found that MIBP1 and RFX1 bind to a regulatory site (termed EP) required for enhancer activity of hepatitis B virus. In addition, we have identified MIF-1-like sequences within regulatory regions of several other viral genes and have shown that MIBP1 binds to these sites in cytomegalovirus, Epstein-Barr virus, and polyomavirus. We have also demonstrated that the MIF-1 and EP elements can function as silencers in the hepatocarcinoma HepG2 and the cervical carcinoma HeLa cell lines. These findings indicate that MIBP1 and EP/RFX1 can associate in vivo and may regulate the expression of several distinct cellular and viral genes.The product of the c-myc oncogene has been implicated in diverse cellular processes, including cell proliferation, differentiation, and tumorigenesis. The observation that c-Myc can heterodimerize with other cellular proteins to transactivate gene expression in vitro has suggested that c-Myc may function to modulate genes involved in cell growth and differentiation pathways (3,4,14). The regulation of the c-myc gene involves a complex interplay of cis-and trans-acting elements, and several nuclear proteins that interact with regulatory sequences found in the 5Ј upstream and exon I untranslated regions of the c-myc gene have been identified (16,31). In addition, a 20-bp region of intron I was defined as a binding site for a phosphoprotein, initially designated MIF-1, and it was demonstrated that binding to this site was abolished by a point mutation present in the corresponding region of a translocated Burkitt's lymphoma c-myc gene (34,35). Adjacent binding sites in c-myc intron I were also identified and designated MIF-2 and MIF-3 (33), and somatic mutations found in Burkitt's lymphoma samples were shown to be frequently clustered within discrete domains that define these recognition sequences (33). These findings suggested that the mutations clustering in this region of the c-myc gene in Burkitt's lymphoma may be targeting specific regulatory elements for c-myc regulation, although direct evidence for this is lacking.It was recently demonstrated that the RFX1 protein, which binds to the X box of the major histocompatibility complex (MHC) class II genes, the methylated DNA-binding protein (MDBP), and the EP protein, which binds to the enhancer of hepatitis B virus (HBV), all represent the same DNA binding activity (29,36). The X-box sequence has been shown to play a role in regula...