Context-dependent gene expression: cis-acting negative effects of specific procaryotic plasmid sequences on eucaryotic genes.

A sequence element within pBR322 DNA mediates a cis-acting negative effect on expression from eucaryotic genes in transient expression assays. The negative element overlaps with sequences that inhibit DNA replication, but its effect is observed in the absence of detectable replication of transfected DNA.

Multiple hormone-inducible enhancers as mediators of differential transcription.

Sets of genes under a common regulatory control in a given cell type are often differentially transcribed. The possibility that this differential transcription can be modulated by the number or strength of cis-acting regulatory sequences associated with a given gene was tested by using the glucocorticoid-responsive enhancer element associated with the mouse mammary tumor virus promoter. Results indicate that differential levels of hormone-inducible gene expression can be modulated in an additive way by the number of glucocorticoid-responsive enhancers associated with this promoter. Realization of these effects shows little preference for position of the additional elements with respect to the promoter. When sequences that bind the glucocorticoid receptor in vitro with somewhat lower affinity than the enhancer were tested, these additive effects were not detected. The results support that differential transcription of genes subject to a common regulatory control can be mediated, at least in part, by the number or strength of their associated cis-acting regulatory sequences.

The mouse c-rel protein has an N-terminal regulatory domain and a C-terminal transcriptional transactivation domain.

We have shown that the murine c-rel protein can act as a transcriptional transactivator in both yeast and mammalian cells. Fusion proteins generated by linking rel sequences to the DNA-binding domain of the yeast transcriptional activator GAL4 activate transcription from a reporter gene linked in cis to a GAL4 binding site. The full-length mouse c-rel protein (588 amino acids long) is a poor transactivator; however, the C-terminal portion of the protein between amino acid residues 403 to 568 is a potent transcriptional transactivator. Deletion of the N-terminal half of the c-rel protein augments its transactivation function. We propose that c-rel protein has an N-terminal regulatory domain and a C-terminal transactivation domain which together modulate its function as a transcriptional transactivator.

Domain swapping reveals the modular nature of Fos, Jun, and CREB proteins.

The products of the Jun and Fos proto-oncogenes form a heterodimer that binds to and activates transcription from 12-O-tetradecanoylphorbol-13-acetate-responsive promoter elements (TGACTCA) and AP-1-binding sites (TGACATCA). These two proteins belong to a family of related transcription factors which contain similar domains required for protein dimerization and DNA binding but display different protein and DNA binding specificities. The basic region, required for DNA binding, is followed by a leucine zipper structure, a domain that mediates protein-protein interactions. To assess the role of these two domains in three related proteins, Fos, Jun, and CREB, we carried out extensive domain-swapping analysis. We found that (i) dimers formed by two Jun leucine zipper-containing proteins were unable to bind DNA as efficiently as a Fos-Jun combination, regardless of the source of the basic region; (ii) the Fos leucine zipper was unable to form either homo- or heterodimers with a chimeric protein containing a Fos leucine zipper; (iii) the Fos basic region was capable of binding to an AP-1 site; (iv) replacement of the Jun amino terminus with that of CREB had little effect on dimerization, whereas replacement with the amino terminus of Fos disrupted both protein-protein and protein-DNA interactions; (v) changes in relative affinities of the Fos and Jun basic regions for the AP-1 element were dependent on the secondary contributions of amino-terminal residues; and (vi) the Fos-Jun chimeric constructs cooperated in transcriptional transactivation of the Jun promoter in NIH 3T3 cells.

Transcriptional repression of a hormone-responsive promoter.

The activity of the mouse mammary tumor virus promoter was assessed in various sequence contexts with a transient transfection assay in which promoter activity was determined by way of expression of a linked gene encoding chloramphenicol acetyltransferase, as well as by direct analysis of RNA transcripts. The results indicate that the proviral long terminal repeat contains a negative transcriptional control element in addition to the glucocorticoid-responsive transcriptional enhancer that has been described previously. The negative element is able to function in both orientations and, at least to some extent, at multiple positions with respect to the regulated transcription unit. The effects on gene expression cannot be explained by alterations in transfection efficiency. The element has been localized to a 91 base pair fragment located immediately 5' of binding sites for the glucocorticoid receptor protein that have been defined in vitro. The role of the negative element may be to repress the inherent activity of the proviral promoter in the absence of glucocorticoids, resulting in an increased ratio of gene expression in the presence and absence of hormone.

Multipartite structure of a negative regulatory element associated with a steroid hormone-inducible promoter.

The steroid hormone-inducible promoter from mouse mammary tumor virus is associated with a distal negative regulatory element that represses its inherent basal activity. Deletion analysis localized the sequences required for repression to 64 base pairs of DNA between -427 and -364 with respect to the transcription initiation site. Transient transfection experiments with a series of linker scanning and small internal deletion mutations revealed two mutation-sensitive domains separated by a region of relative resistance to sequence alterations. DNase I footprinting and gel electrophoresis mobility shift experiments with crude nuclear extracts identified at least one protein-binding site within each of the two mutation-sensitive regions. An oligonucleotide corresponding to one of these sites is able to repress transcription, but only when linked to the promoter in multiple copies. This negative regulatory element functions synergistically with a promoter proximal negative element to mediate efficient promoter repression, selectively affecting basal relative to steroid hormone-induced transcription and thus increasing the ratio of promoter activity observed in the presence and absence of hormone.