We have characterized the interactions between the TATA element and other sequence elements of a human heat shock protein 70 (hsp70) promoter by a mutational approach. Expression of a distal element of this promoter requires an intact TATA element in human cell lines. The hsp70 TATA element can be functionally replaced for this interaction by TATA elements from the simian virus 40 early and adenovirus EIIa promoters. The TATA element in this promoter therefore both determines the appropriate start site and determines strength by allowing function of the distal element. In contrast, three proximal upstream elements necessary for basal and heat-regulated transcription have no requirement either for a TATA element or for any other proximal element. The behavior of promoters multiply mutant in these proximal elements implies that these elements function independently. We examined the interaction between the heat shock element (HSE) and the TATA element as the distance between the two factor-binding sites was increased. It was necessary to create a mutant HSE with an extended consensus sequence in order for the HSE to function at a distance. Moving this extended HSE 500 bases upstream did not increase its dependence on the TATA element, suggesting that the TATA independence of this element is intrinsic to its function and is not determined by distance from the promoter.

One mechanism by which nuclear-localized oncogenes might transform cells is through an ability to regulate gene expression. We show that the c-myc protein stimulates the level of appropriately initiated expression from the human heat shock protein 70 (hsp70) promoter. Sequences required for full activation lie upstream of the transcription initiation site and are distinct from sequences necessary for basal expression. These sequences also appear distinct from promoter sequences necessary for heat induction, serum induction, and induction by the papovavirus T antigens. The c-myc protein inhibits appropriately initiated expression from the mouse metallothionein I (MT-I) promoter. A mutation that removes 138 amino acids of exon 2 produces a c-myc gene product that is capable of activating the hsp70 promoter but is no longer capable of inhibiting MT-I expression, suggesting that these two properties reside in different domains of the c-myc protein. Expression from the adenovirus EII promoter is slightly inhibited, while expression from the SV40 early promoter is minimally affected by the c-myc protein. Both the spectrum of promoters regulated by the c-myc protein and the sequence requirements for that regulation differ from those of previously characterized viral trans-activating proteins. The data suggest that the c-myc protein can both stimulate and inhibit transcription from mammalian promoters in a novel manner.

The human heat shock protein 70 (hsp70) gene is expressed constitutively in a wide variety of cells. Two separate promoter domains determine this basal level of hsp70 expression. The proximal domain is contained within 84 bases of the transcription initiation site and consists of three elements which appear to interact with the TATA factor(s) and CCAAT-box-binding transcription factor and SP1, respectively. The proximal domain is sufficient for near-maximal basal expression to rodent cell lines. The distal promoter domain consists of sequences upstream of -84 and is necessary in conjunction with the proximal domain for full basal expression in human cell lines. Although in BALB/c 3T3 cells the distal promoter domain plays little role in basal expression, it is functional as evidenced by the ability to compensate efficiently for mutations in the proximal CCAATC homology. The distal domain does not compensate as efficiently for proximal-domain mutations in HeLa cells. Basal expression of this human hsp70 promoter is, therefore, determined by multiple elements. Fewer elements are required for basal expression in rodent cell lines than in human cell lines, suggesting that there are significant differences between the rodent and human transcription apparatuses.