Analysis of the promoter region of the gene encoding mouse cholesterol side-chain cleavage enzyme

The cholesterol side-chain cleavage enzyme (SCC) catalyzes the initial and rate-limiting step in the synthesis of steroid hormones. The mouse gene encoding SCC was cloned and the nucleotide sequence of its 5'-flanking region determined. This sequence includes an AP-1 motif at -319 and two motifs, AGGTCA at -70 and AGCCTTG at -40, that match elements proposed to be important in the expression of steroid 21-hydroxylase. When transfected into mouse Y1 adrenocortical tumor cells, 1.5 kilobase pairs of 5'-flanking region of the SCC gene directed high levels of expression of a growth hormone reporter gene; treatment of the transfected Y1 cells with 8-bromo-cAMP increased this expression by 5-fold. In contrast, transfected mouse MA-10 Leydig cells showed appreciably lower expression, suggesting that SCC expression in Leydig cells requires additional elements not contained in the 5'-flanking region of the SCC gene used in these experiments. Deletion experiments showed that 424 base pairs of 5'-flanking sequences were sufficient for regulated expression in Y1 cells and mapped two regulatory regions: one from -424 to -327 and a second from -219 to -77. DNase I footprinting and gel mobility shift analyses of these 424 base pairs defined several interactions between nuclear proteins and the SCC promoter, including footprints centered over the AP-1 motif, over a sequence at -120, and over the sequences (-70 and -40) that resemble 21-hydroxylase promoter elements. Finally, site-selected mutagenesis of the potential elements at -40, -70, or -120 decreased SCC promoter activity in transfected Y1 adrenocortical cells, thus establishing their importance in SCC expression.     

A cAMP-responsive element regulates expression of the mouse steroid 11 beta-hydroxylase gene

In Y1 mouse adrenocortical tumor cells, expression of steroid 11 beta-hydroxylase (11 beta-OHase) is stimulated by cAMP following a delay of 4-6 h. Our results demonstrate that a cAMP-responsive element (CRE) within the 11 beta-OHase promoter region is a major determinant of this induction. The 5'-flanking sequences from the mouse 11 beta-OHase gene were placed in front of a human growth hormone reporter gene and transfected into Y1 cells. Treatment of transfected cells with 8-bromo-cAMP increased expression directed by the 11 beta-OHase 5'-flanking region by 3.8-fold. In 5'-deletion analyses, 123 base pairs of 5'-flanking sequences were sufficient for cAMP induction, whereas cAMP treatment did not affect expression of a plasmid with only 40 base pairs of 5'-flanking sequence. Within these 123 base pairs, a region from -56 to -49 matched 7 of 8 bases comprising the consensus sequence for the CRE. 11 beta-OHase 5'-flanking sequences from -65 to -42, including the CRE-like sequence, conferred cAMP inducibility to promoters from the thymidine kinase and chorionic gonadotropin alpha-subunit genes. DNase I footprinting and Southwestern blotting analyses demonstrated that the protein which interacted with the CRE in the 11 beta-OHase promoter region was similar to the CRE-binding protein associated with other cAMP-regulated genes. Together, these results suggest that an interaction between the 11 beta-OHase CRE and CRE-binding protein mediates cAMP induction of the 11 beta-OHase gene.     

Tissue-specific and insulin-dependent expression of a pancreatic amylase gene in transgenic mice.

The regulatory properties of mouse pancreatic amylase genes include exclusive expression in the acinar cells of the pancreas and dependence on insulin and glucocorticoids for maximal expression. We have characterized a murine pancreatic amylase gene, Amy-2.2y, whose promoter sequence is 30% divergent from those of previously sequenced amylase genes. To localize sequences required for tissue-specific and hormone-dependent activation, we established two lines of transgenic mice. The first line contained a single copy of the complete Amy-2.2y gene as well as 9 kilobases of 5'-flanking sequence and 5 kilobases of 3'-flanking sequence. The second line carried a minigene which included 208 base pairs of 5'-flanking sequence and 300 base pairs of 3'-flanking sequence. In both lines the transgene was expressed at high levels exclusively in the pancreas. Both constructs were dependent on insulin and induced by dexamethasone. Thus, the transferred genes contained the sequences required for tissue-specific and hormonally regulated expression.     

Nuclear factor 1 regulates adipose tissue-specific expression in the mouse GLUT4 gene

Previous studies demonstrated that an adipose tissue-specific element(s) (ASE) of the murine GLUT4 gene is located between −551 and −506 in the 5′-flanking sequence and that a high-fat responsive element(s) for down-regulation of the GLUT4 gene is located between bases −701 and −552. A binding site for nuclear factor 1 (NF1), that mediates insulin and cAMP-induced repression of GLUT4 in 3T3-L1 adipocytes is located between bases −700 and −688. To examine the role of NF1 in the regulation of GLUT4 gene expression in white adipose tissues (WAT) in vivo, we created two types of transgenic mice harboring mutated either 5′ or 3′ half-site of NF1-binding sites in GLUT4 minigene constructs. In both cases, the GLUT4 minigene was not expressed in WAT, while expression was maintained in brown adipose tissue, skeletal muscle, and heart. This was an unexpected finding, since a −551 GLUT4 minigene that did not have the NF1-binding site was expressed in WAT. We propose a model that explains the requirement for both the ASE and the NF1-binding site for expression of GLUT4 in WAT.     

Expression and regulation of the human GLUT4/muscle-fat facilitative glucose transporter gene in transgenic mice.

To study the molecular basis of tissue-specific expression of the GLUT4/muscle-fat facilitative glucose transporter gene, we generated lines of transgenic mice carrying 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene (hGLUT4[2.4]-CAT). This reporter gene construct was specifically expressed in tissues that normally express GLUT4 mRNA, which include both brown and white adipose tissues as well as cardiac, skeletal, and smooth muscle. In contrast, CAT reporter activity was not detected in brain or liver, two tissues that do not express the GLUT4 gene. In addition, the relative levels of CAT mRNA driven by the human GLUT4 promoter in various tissues of these transgenic animals mirrored those of the endogenous mouse GLUT4 mRNA. Since previous studies have observed alterations in GLUT4 mRNA levels induced by fasting and refeeding (Sivitz, W. I., DeSautel, S. L., Kayano, T., Bell, G. I., and Pessin, J. E. (1989) Nature 340, 72-74), the regulated expression the hGLUT4[2.4]-CAT transgene was also assessed in these animals. Fasting was observed to decrease CAT activity in white adipose tissue which was super-induced upon refeeding. These alterations in CAT expression occurred in parallel to the changes in endogenous mouse GLUT4 mRNA levels. Although CAT expression in skeletal muscle and brown adipose tissue was unaffected, the endogenous mouse GLUT4 mRNA was also refractory to the effects of fasting/refeeding in these tissues. These data demonstrate that 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene contain all the necessary sequence elements to confer tissue-specific expression and at least some of the sequence elements controlling the hormonal/metabolic regulation of this gene.     

Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

Mutations were introduced in 7 kilobases of 5'-flanking rat alpha 1-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.