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...

Because of the large number of growth-regulated genes containing binding sites for the transcription factors Sp1 and E2F and the reported ability of E2F to mediate cell cycle (growth) regulation, we studied interactions between E2F1 and Sp1. In transient transfection assays using Drosophila melanogaster SL2 cells, transfection with both Sp1 and E2F1 expression vectors resulted in greater than 85-fold activation of transcription from a hamster dihydrofolate reductase reporter construct, whereas cotransfection with either the Sp1 or E2F1 expression vector resulted in 30-or <2-fold activation, respectively. Therefore, these transcription factors act synergistically in activation of dihydrofolate reductase transcription. Transient transfection studies demonstrated that E2F1 could superactivate Sp1-dependent transcription in a promoter containing only Sp1 sites and that Sp1 could superactivate transcription of promoters through E2F sites, further demonstrating that these factors functionally interact with one another. Coimmunoprecipitation studies revealed that Sp1 and E2F1 are physically associated in Drosophila cells transfected with Sp1 and E2F1 expression vectors and in human cells, with maximal interaction detected in mid-to late G 1 . Additionally, E2F1 and Sp1 interact in vitro through specific domains of each protein, and the physical interaction and functional synergism appear to require the same regions. Taken together, these data demonstrate that E2F1 and Sp1 both functionally and physically interact; therefore, through this interaction, Sp1 and E2F1 may regulate transcription of genes containing binding sites for either or both factors.

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...

. (1997) J. Cell. Biochem. 67, 24 -31). To investigate mechanisms underlying this induction, effects of serum stimulation on regulation of Sp1 were examined. In Balb/c 3T3 cells, serum stimulation did not affect Sp1 synthesis or the relative binding of Sp1 family members to DNA; however, it did result in a rapid, ϳ2-fold increase in Sp1 levels and an ϳ3-fold increase in specific Sp1 phosphorylation in mid-G 1 . In normal human diploid fibroblasts, serum stimulation also increased Sp1 phosphorylation in mid-G 1 but did not affect Sp1 levels. Therefore, Sp1 phosphorylation is regulated in a growth/cell cycle-dependent manner which correlates temporally with induction of dihydrofolate reductase transcription. Further studies revealed a kinase activity specifically associated with Sp1 in a growth-regulated manner. This activity is distinct from purified kinases previously shown to phosphorylate Sp1 in vitro and phosphorylates Sp1 between amino acids 612 and 678 in its C terminus, a region also phosphorylated in mid-G 1 in vivo. Therefore, this study indicates that phosphorylation of the C terminus of Sp1 may play a role in the cell cycle regulation of its transcriptional activity.Expression of a large number of genes associated with DNA synthesis, such as dihydrofolate reductase (DHFR), 1 is tightly regulated with cell growth and the cell cycle. Many of these genes have promoters which lack a TATAA element but contain binding sites for the transcription factors Sp1 and E2F (1). Although the role of E2F sites in growth/cell cycle regulation of transcription and the regulation of E2F by retinoblastoma protein (pRB) and related pocket proteins have been extensively characterized (see Refs. 2-4, for review), a role for Sp1 sites in growth/cell cycle regulation of transcription is only beginning to emerge (e.g. Refs. 5 and 6). We have determined that Sp1 and E2F sites have distinct roles in the growth/cell cycle regulation of the hamster DHFR promoter (6). Although complete repression of DHFR transcription in G 0 and early G 1 requires E2F sites, its induction in late G 1 is mediated by Sp1 sites. A direct role of Sp1-dependent transcription in growth regulation of transcription is supported by targeting of Sp1 by viral oncoproteins (e.g. Refs. 7-9), down-regulation of Sp1 expression, and/or DNA binding activity upon differentiation in some systems (10), and increased Sp1 expression during events associated with transformation (11).Sp1 is a ubiquitous, 778-amino acid transcription factor that recognizes GC-rich sequences present in many promoters (see Refs. 1 and 2, for review). Although Sp1 has been viewed as a constitutive transcriptional activator which acts as a basal factor for TATAA-less promoters, an increasing number of studies indicate that Sp1-dependent transcription is regulated in response to a variety of signals. For example, in addition to their role in growth/cell cycle regulation of transcription, Sp1 sites are involved in induction of DHFR transcription in response to methotrexate (12), induction of CYP1...

Migration of capillary endothelial cells is an important component of angiogenesis in vivo. Increased numbers of mast cells have been associated with several types of angiogenesis. We have used a quantitative assay in vitro to demonstrate that mast cells release a factor that significantly increases bovine capillary endothelial cell migration. The factor is present in medium conditioned by mast cells as well as lysates of mast cells. The stimulatory effect of mast cells on migration is specific for capillary endothelial cells. Furthermore, mast cells have no mitogenic activity for capillary endothelial cells. Of all the secretory products of mast cells tested, only heparin stimulated capillary endothelial cell migration in vitro. Heparin preparations from a variety of sources stimulated capillary endothelial cell migration to the same degree but did not stimulate migration of several other cell types. The migration activity of heparin and mast cell conditioned medium was blocked by specific antagonists of heparin (protamine and heparinase), but not by chondroitinase ABC. The migration activity of mast cell conditioned medium was resistant to heat (100 degrees C) and incubation with proteolytic enzymes. These results suggest that the role of mast cells in angiogenesis may be to enhance migration of the endothelial cells of growing capillaries.