Regulation of rat insulin 1 gene expression: evidence for negative regulation in nonpancreatic cells.

Abstract

Two cis-acting elements, the enhancer and the promoter,, independently contribute to the cell-specific expression of the rat insulin 1 gene. The activities of these elements are presumably mediated by trans-acting factors. We have performed intracellular competition experiments that suggest the presence of a negative factor(s) that represses the enhancer activity in cells that do not express the insulin gene.In these experiments fibroblast cells (COS-7) were transfected with two plasmids: a test plasmid containing the gene for chlorampheiiicol acetyltransferase under the control of the thymidine kinase promoter and the insulin enhancer; and a competitor plasmid containing insulin enhancer sequences and the simian virus 40 origin of replication to permit its replication in the recipient cells. The presence of the competitor plasmid led to a 5-to 6-fold -increase in chloramphenicol acetyltransferase activity as compared with the activity detected when insulin enhancer was absent from either the competitor or the test plasmid. A 5-fold increase in chloramphenicol acetyltransferase activity was also seen when the rat amylase enhancer was present on the competitor plasmid; in contrast the simian virus 40 enhancer exerted no effect. Efficient derepression required additional sequences downstream from those essential for enhancer activity. We propose that the activity ofthe rat insulin 1 enhancer is modulated by a negative tbans-acting factor(s) that is active in cells not expressing insulin but is overridden by the dominant positive trans-acting factor(s) present in insulinproducing cells.During cellular differentiation, cell types acquire the ability to stably express a characteristic set of genes. The. molecular mechanisms by which this occurs are poorly understood. Several genes whose expression is restricted to a specific subset of cells contain cis-acting sequences required for efficient transcription in the appropriate differentiated cells (1-6). We have demonstrated (7) that 5' flanking DNA sequences of the rat insulin 1 gene contain two such cellspecific elements. The properties of these elements are consistent with the hypothesis that differentiated cells contain positive trans-acting proteins (differentiators) that interact with the specific cis-acting sequences to stimulate the expression of the associated genes. This idea is also consistent with the observations and concepts of other workers (8-12). On the other hand, "extinction" of the differentiated phenotype after fusion of differentiated cells with fibroblasts is frequently observed (13). We have also noted that specific sequence deletions in the insulin gene 5' flanking sequences can lead to increases in expression in nondifferentiated cells (unpublished observations). These results are consistent with a role for repressor-like molecules in nonexpressing cells.

trans-acting factors interact with a cyclic AMP response element to modulate expression of the human gonadotropin alpha gene.

“…Competition assays for limiting cellular transcription factors allow insight into the complex interactions between the cis-acting regulatory elements of a gene and the trans-acting factors that modulate formation of an active transcription complex (4,8,31,35,(41)(42)(43)50). Importantly, these assays are performed in intact cells, and competition may therefore occur for a single or several different transcription factors.…”

Multiple 5'-flanking regions of the human alpha-skeletal actin gene synergistically modulate muscle-specific expression.

“…USA, in press). Furthermore, a number of recent studies on other tissue-specific mammalian promoters have emphasized their general complexity by identifying cis-acting elements which mediate both positive (59) and negative (33,46) modulation of transcription and that span many kilobases of 5'-flanking sequences.…”

Negative regulation of transcription in vitro by a glucocorticoid response element is mediated by a trans-acting factor.

“…Many examples of mammalian transcription units that contain cis-acting negative regulatory elements are now well documented. In particular, most of these genes are susceptible to induction by hormonal or environmental influences (12,31) or show very restricted patterns of tissue expression (17,25 genes. This mechanism would ensure that genes remain transcriptionally silent in the absence of the proper cellular signals or in inappropriate cell types.…”

“…Additionally, some cellular genes whose expression is regulated in a tissue-specific manner-for example, the rat insulin gene (25) and the immunoglobulin heavy-chain gene (17)-also contain cisacting negative elements.…”