The HIP1 binding site is required for growth regulation of the dihydrofolate reductase gene promoter.

Abstract

The transcription rate of the dihydrofolate reductase (DHFR) gene increases at the G1/S boundary of the proliferative cell cycle. Through analysis of transiently and stably transfected NIH 3T3 cells, we have now demonstrated that DHFR promoter sequences extending from -270 to +20 are sufficient to confer similar regulation on a reporter gene. Mutation of a protein binding site that spans sequences from -16 to + 11 in the DHFR promoter resulted in loss of the transcriptional increase at the G1/S boundary. Purification of an activity from HeLa nuclear extract that binds to this region enriched for a 180-kDa polypeptide (HIP1). Using this HIP1 preparation, we have identified specific positions within the binding site that are critical for efficient protein-DNA interactions. An analysis of association and dissociation rates suggests that bound HIP1 protein can exchange rapidly with free protein. This rapid exchange may facilitate the burst of transcriptional activity from the DHFR promoter at the GI/S boundary. Cellular proliferation is controlled by a programmed series of events termed the cell cycle. Neoplasia can result when quiescent or differentiated cells escape cell cycle control and begin inappropriate proliferation. Therefore, understanding the checks and balances used by the cell to control entrance into S phase of the cell cycle is important in understanding the loss of proliferation control that leads to neoplasia. One widely used model system to study cell cycle progression is the reentry of quiescent cells into the proliferative cell cycle elicited by an increase in the level of serum growth factors. Murine 3T3 fibroblasts withdraw from the proliferative cell cycle into a quiescent Go state when the concentration of serum in the culture medium is low. The addition of high concentrations of serum to these quiescent cells initiates a series of events that results in the transduction of extracellular signals to the cell nucleus, with a concomitant change in the expression of growth-responsive genes. The first genes that are activated by the serum growth factors are termed immediate-early-response genes, many of which are transcription factors. Several hours later, at the transition from G1 to S phase, late-response genes are activated, followed by the onset of DNA synthesis and the activation of histone gene expression. Among the late-response genes are the genes for DNA polymerase alpha, thymidine kinase, thymidylate synthase, carbamoyl phosphate synthase-aspartate carbamoyltransferase-dihydroorotase, and dihydrofolate reductase (DHFR). Many of these genes are involved in nucleotide biosynthesis; for example, the DHFR gene encodes an enzyme involved in the production of glycine, purines, and thymidylate.Although much is known about the cis elements and trans-acting factors that are responsible for the activation of the early-response genes (see reference 32 for a review), few studies have focused on the activation of the late-response genes. Recently, several groups have analyzed the ability of the promo...

Retinoblastoma protein associates with SP1 and activates the hamster dihydrofolate reductase promoter

“…The role of E2F in cell growth regulated transcription of the dhfr promoter has been emphasized by the groups of Azizkhan Wade et al, 1992) and Farnham (Means et al, 1992;Slansky et al, 1993;Li et al, 1994). On the other hand, Sp1 powerfully activates DHFR transcription (Farnham and Shimke, 1986;Swick et al, 1989;Blake et al, 1990;Ciudad et al, 1992), and given this essential role of Sp1 in activating transcription, we have explored the possibility of the interaction of Sp1 with the cell cycle regulator Rb.…”

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.…”

pRb and p107 regulate E2F activity during lens fiber cell differentiation

“…Phosphorylation of pRb by cyclin D/cdk4 prevents formation of the pRb/E2F complex, permitting transcription of genes with E2F binding sites in their promoters (Nevins, 1992;Ikeda et al, 1996). Genes thought to be E2F targets include protooncogenes (Heibert et al, 1989;Lam and Watson, 1993), G1 cyclins (Muller et al, 1994;Henglein et al, 1994;Ohtani et al, 1995), enzymes required for DNA synthesis (Pearson et al, 1991;Means et al, 1992;Fridovich-Keil et al, 1993), and the G2-speci®c cdk, cdc2 (Dalton, 1992;Tommasi and Pfeifer, 1995). Several recent studies suggest that E2F may be primarily a negative regulator of transcription, directing the binding of repressors, such as pRb, to speci®c target genes (Tommasi and Pfeifer, 1995;Field et al, 1996;Yamasaki et al, 1996;Zwicker et al, 1996).…”