Hormone response of rodent phenylalanine hydroxylase requires HNF1 and the glucocorticoid receptor.

Expression of the rodent phenylalanine hydroxylase (PAH) gene is dependent upon hormones. Induction by glucocorticoids and cAMP occurs slowly and maximal stimulation is obtained by a synergistic effect of the two compounds. Hormone responsiveness is conferred by the tissue-specific HSIII enhancer and involves (i) protein kinase A mediating the cAMP response, even though a consensus sequence for binding of the cAMP response element binding protein is not present; (ii) other serine/threonine kinases as deduced from inhibitor studies; (iii) glucocorticoid receptor protein bound to glucocorticoid response element half sites; and (iv) binding of the liver-enriched transcription factor hepatocyte nuclear factor 1 (HNF1) to sites in the enhancer. Glucocorticoid receptor and HNF1, bound to their cognate sites, cooperatively increase the glucocorticoid response of the PAH gene, this response being synergistically enhanced by cAMP after long-term treatment.

The activity of the highly inducible mouse phenylalanine hydroxylase gene promoter is dependent upon a tissue-specific, hormone-inducible enhancer.

Expression of the phenylalanine hydroxylase gene in livers and kidneys of rodents is activated at birth and is induced by glucocorticoids and cyclic AMP in the liver. Regulatory elements in a 10-kb fragment upstream of the mouse gene have been characterized. The promoter lacks TAATA and CCAAT consensus sequences and shows only extremely weak activity in transitory expression assays with phenylalanine hydroxylase-producing hepatoma cells. No key elements for regulation of promoter activity are localized within 2 kb of upstream sequences. However, a liver-specific DNase I-hypersensitive site at kb -3.5 comprises a tissue-specific and hormone-inducible enhancer. This enhancer contains multiple protein binding sites, including sites for ubiquitous factors (NF1 and AP1), the glucocorticoid receptor, and the hepatocyte-enriched transcription factors hepatocyte nuclear factor 1 (HNF1) and C/EBP. Mutation revealed that the last two sites are critical not only for basal activity but also for obtaining a maximal hormone response. Efficient transcription from the highly inducible promoter shows absolute dependence upon the enhancer at kb - 3.5, which in turn requires HNF1 and C/EBP as well as hormones. The regulatory region of the mouse phenylalanine hydroxylase gene differs totally from that of humans, even though the genes of both species are expressed essentially in the liver. Furthermore, the phenylalanine hydroxylase gene of mice shows an expression pattern very similar to those of the rodent tyrosine aminotransferase and phosphoenolpyruvate carboxykinase genes, yet each shows a different organization of its regulatory region.

Activation of phenylalanine hydroxylase expression following genomic DNA transfection of hepatoma cells.

Genomic DNA from cells producing the liver-specific enzyme phenylalanine hydroxylase (PAH) should contain, in active form, genes encoding regulators of PAH expression. We have transfected genomic DNA from PAH-producing rat hepatoma cells to PAH-deficient mouse hepatoma cells, and selected in tyrosine-deficient medium for cells producing the enzyme. The frequency of colonies obtained was similar to that for transfer of a single-copy gene. Genomic DNA from the primary transfectants permitted the isolation in tyrosine-free medium of secondary transfectants. Control experiments, using donor DNA from PAH-negative rat or mouse hepatoma cells also permitted the isolation of PAH-expressing cells, but at a frequency 10-30 times lower. The transfectants isolated in tyrosine-deficient selective medium all produced PAH mRNA. This transcript was from the previously silent mouse gene, which had not undergone amplification or gross rearrangement. Most of the transfectants contained less than 0.1% rat DNA. A search for other functions that might have been simultaneously activated was negative. It is concluded that the mouse transfectants acquired from the PAH+ rat donor some sequences whose presence permits activity of the previously silent PAH gene.