Tissue-specific DNaseI hypersensitive sites in the 5''-flanking sequences of the tryptophan oxygenase and the tyrosine aminotransferase genes.

The genes for tryptophan oxygenase (TO) and tyrosine aminotransferase (TAT) are expressed in a tissue- and development-specific manner and are regulated by glucocorticoids (TO and TAT) and glucagon or its intracellular mediator cAMP (TAT) in rat liver. We have analyzed the chromatin structure of these genes in the vicinity of the 5' ends with regard to DNaseI hypersensitivity and have found DNaseI hypersensitive sites upstream of each of the promoters. Mapping of this region reveals three closely spaced cleavage sites near the TO promoter and a doublet of sites near the TAT promoter. In both genes additional cleavage sites are found further upstream. All hypersensitive sites of both genes are absent in kidney nuclei and therefore appear to be specific for the tissue expressing the genes. A correlation of expression and modified chromatin structure was also observed in a hepatoma cell line expressing TAT but not TO: hypersensitive sites are present in TAT but not in TO chromatin. Upon glucocorticoid induction an additional hypersensitive site is detected approximately 2 kb upstream of the TAT promoter in liver and hepatoma cells.     

Insulin-mediated inhibition of the induction of tyrosine aminotransferase by dexamethasone.

Insulin-mediated regulation of glucocorticoid-induced expression of the liver-specific gene tyrosine aminotransferase was studied in a clone of the Reuber rat hepatoma cells. Insulin inhibited dexamethasone-induced chloramphenicol acetyltransferase expression from approximately 4 kb of TAT 5' flanking sequence. The degree of this inhibition was comparable to the response of the endogenous gene. A construct of approximately 3 kbp of 5' flanking sequence exhibited no significant basal expression but retained sensitivity to glucocorticoids and to insulin inhibition of the glucocorticoid response. Results of further analysis of the insulin response in deletion constructs and constructs containing glucocorticoid responsive elements ligated to a heterologous promoter suggest that in addition to the glucocorticoid response elements a region close to the start site in the TAT promoter is necessary for insulin to inhibit glucocorticoid-mediated induction of expression.     

Unlinked regulation of the sensitivity of primary glucocorticoid-inducible responses in mouse mammary tumor virus infected Fu5-5 rat hepatoma cells

The enzyme tyrosine aminotransferase (TAT) is induced by unusually low concentrations of glucocorticoids in Fu5-5 cells. We have isolated clones of Fu5-5 cells infected with mouse mammary tumor virus (MMTV) in order to simultaneously compare the glucocorticoid regulation of the host cell gene, TAT, with that of another primary inducible gene, MMTV. In the two clones that were examined in detail, MMTV RNA induction occurred at 4- to 11-fold higher concentrations of dexamethasone than those needed for induction of TAT mRNA. Furthermore, the amount of agonist activity displayed by the irreversible antiglucocorticoid dexamethasone 21-mesylate was greater for the induction of TAT mRNA than for MMTV RNA. These results extend our previous observations of unequal sensitivity of induction of TAT enzyme activity in two hepatoma cell lines and show that differential glucocorticoid regulation of gene induction within the same cell can occur at a pretranslational step. The present data also indicate that the unusual properties of TAT gene induction are not shared by all primary, glucocorticoid-inducible responses of the same cell and imply that additional factors mediating differential regulation of glucocorticoid-responsive genes are involved.     

Pollen- and anther-specific chi promoters from petunia: tandem promoter regulation of the chiA gene.

We have analyzed the spatial and temporal activities of chalcone flavanone isomerase (chi) A and B gene promoters from petunia. To study the tandem promoter regulation of chiA, various chiA promoter fragments were fused with the beta-glucuronidase (GUS) reporter gene. Analysis of transgenic plants containing these chimeric genes provided definitive proof that the chiA coding region is regulated by two distinct promoters (designated PA1 and PA2). We also showed that both promoters can function independently and that the chiA PA1 promoter is expressed in limb (epidermal and parenchyma cells), tube (inner epidermal and parenchyma cells), seed (seed coat, endosperm, and embryo), sepal, leaf, and stem. The use of chiA and chiB promoters in the regulation of anther- and pollen-specific gene expression has been studied. By analyzing transgenic plants containing chimeric genes consisting of chiA and B promoter fragments and the GUS reporter gene, we were able to identify a 0.44-kilobase chiA PA2 promoter fragment that drives pollen-specific gene expression and a 1.75-kilobase chiB PB promoter fragment that confers anther-specific (pollen and tapetum cells) expression to the GUS gene.     

Regulation of chimeric phosphoenolpyruvate carboxykinase genes by the trans-dominant locus TSE1.

Extinction of phosphoenolpyruvate carboxykinase (PCK) gene expression in hepatoma x fibroblast hybrids is mediated by a trans-acting genetic locus designated tissue-specific extinguisher 1 (TSE1). To identify PCK gene sequences required for extinction, hepatoma transfectants expressing PCK-thymidine kinase (TK) chimeric genes were fused with TK- fibroblasts and PCK-TK expression in the resulting hybrids was monitored. Expression of a PCK-TK chimera containing PCK sequences between base pairs -548 and +73 was extinguished in four of five hepatoma transfectants tested, although hybrids derived from one transfectant clone failed to extinguish PCK-TK expression. In contrast, crosses between hepatoma transfectants expressing the herpesvirus TK gene from its own promoter and TK- fibroblasts produced TK+ hybrids; extinction of the transfected TK gene was not observed. Thus, rat PCK gene sequences between base pairs -548 and +73 are sufficient for tissue-specific extinction in hybrid cells. Extinction of PCK-TK gene expression in transfectant microcell hybrids mapped specifically to human chromosome 17, the site of human TSE1.     

Cryptic human growth hormone gene sequences direct gonadotroph-specific expression in transgenic mice

Our laboratory reported previously that chimeric genes encoding either rat somatostatin (SS) or human GH (hGH), but containing the identical mouse metallothionein-I (MT) promoter/enhancer sequences and hGH 3'-flanking sequences, were selectively expressed in the gonadotrophs of transgenic mice. The experiments reported here were designed to identify the DNA sequences responsible for this unexpected cell-specific expression within the anterior pituitary. We produced new transgenic mice expressing fusion genes that tested separately the requirement of the MT or 3'-hGH sequences for gonadotroph expression. A fusion gene that retained the original MT and SS sequences, with a simian virus 40 polyadenylation signal exchanged for the 3'-hGH sequences, no longer directed strong pituitary expression, but was active in the liver. In contrast, a cytomegalovirus promoter/enhancer-SS-hGH fusion gene was expressed at the same high level in the anterior pituitaries of transgenic mice as the originally studied MT-SS-hGH gene. Immunohistochemical analysis indicated that pituitary expression of the cytomegalovirus promoter/enhancer-SS-hGH fusion gene was also restricted to gonadotroph cells in adult mice. These studies indicate that sequences within the 3'-flanking region of the hGH gene can direct expression of chimeric genes to pituitary cells that do not normally produce growth hormone.     

Modulation of the agonist activity of antisteroids by a novel cis-acting element.

The amount of agonist activity displayed by the antiglucocorticoid dexamethasone mesylate (Dex-Mes) for the induction of tyrosine aminotransferase (TAT) in rat hepatoma cells is greater than for glutamine synthetase and varies over a period of weeks. This variation, which has been reproduced over a period of 40 h by changing the density of the cells, suggests the involvement of a trans-acting factor. The target of this proposed trans-acting factor has now been localized to the region between -3.9 to -2.9 of the rat TAT gene from experiments with cells that were stably transfected with hybrid TAT/CAT constructs. Deletion experiments with transiently transfected TAT/tk promoter/CAT constructs revealed that this entire activity could be conveyed by a 21 bp sequence of the TAT gene. Gel shift experiments support the binding of a factor(s) to this 21 bp sequence. Thus the activity of the antagonist Dex-Mes is relatively independent of steroid structure and is largely determined by the further interactions of a trans-acting factor with the cis-acting sequence. We call this novel sequence a glucocorticoid modulatory element. A model is advanced which accounts for almost all of the results concerning TAT induction by glucocorticoids. This same model may also be useful in explaining why the amount of agonist activity of most antisteroids varies, even for different genes within the same cell.