Involvement of a cis-acting element in the suppression of carbamoyl phosphate synthetase I gene expression in the liver of carnitine-deficient mice

The expression of carbamoyl phosphate synthetase I (CPS) gene is suppressed in the liver of carnitine-deficient juvenile visceral steatosis (JVS) mice at weaning and under starvation at adult age. To clarify the suppression mechanism, we produced CPSL transgenic JVS mice carrying a transgene composed of the chloramphenicol acetyltransferase (CAT) gene with the upstream region (-12 kb to +138) of the rat CPS gene and CPSE transgenic JVS mice carrying a transgene composed of the luciferase gene with minimal promoter (299 bp from -161 to +138) and enhancer (469 bp around -6.3 kb) fragments of the rat gene. The expression of the CAT gene as well as the endogenous CPS was suppressed in CPSL transgenic JVS mice, but luciferase gene expression was not suppressed in CPSE transgenic JVS mice. We isolated the 5'-upstream region of the mouse CPS gene and identified an activator protein-1 (AP-1) site downstream of the minimum enhancer region of both rat and mouse CPS genes. In conjunction with the 313-bp mouse promoter region, the 714-bp mouse enhancer fragment conferred a cell-type-dependent hormone responsiveness. In rat primary cultured hepatocytes, the addition of oleic acid suppressed reporter gene expression induced by dexamethasone in the construct containing the enhancer fragment of 714 bp with the AP-1 site, but not in its AP-1 site mutants or in 519 bp without the AP-1 site. These results strongly suggest that direct protein-protein interaction between AP-1 and glucocorticoid receptor is not involved in the suppression of the CPS gene in JVS mice and that the AP-1 element is the cis-element which is responsible for the suppression.     

Regulation of amylase gene expression in diabetic mice is mediated by a cis-acting upstream element close to the pancreas-specific enhancer.

We localized the cis-acting sequences that mediate the regulation of a pancreatic amylase gene, Amy-2.2, in diabetic mice. We constructed three hybrid genes containing sequences from the 5'-flanking region of the amylase gene upstream of the heterologous elastase promoter linked to the CAT structural gene. These constructs were transferred to the germ line of transgenic mice by microinjection of fertilized eggs. The amylase sequences had two effects on expression of the elastase promoter: Basal expression was increased, and expression in diabetic animals was reduced to approximately 2% of basal levels. A 30-bp amylase fragment was sufficient to transfer both of these regulatory functions to the elastase promoter. Sequences within this 30-bp fragment are included in the binding site for the pancreatic nuclear protein PTF1. The close association of the PTF1-binding site and the regulatory functions is consistent with a mechanism based on interference with activation by PTF1 in diabetic pancreas. PTF1-binding activity is not reduced in diabetic pancreas. The data presented here demonstrate that the 5'-flanking region of the pancreatic amylase gene contains a novel insulin-dependent element (IDE) that mediates the loss of expression in diabetic animals.     

Transgenic analysis of the anterior eye-specific enhancers of the zebrafish gelsolin-like 1 (gsnl1) gene.

The anterior segment of the eye includes such structures as the cornea, lens, iris, and ciliary body and is essential for many visual and physiological functions of the eye. The zebrafish gelsolin-like 1 (gsnl1) gene encodes an actin regulatory protein and is expressed in the anterior segment of the eye. We report the transgenic analyses of the gsnl1 promoter and enhancer that are required for expression in the anterior segment of the eye. A 6.4-kb genomic fragment upstream from the translation initiation site (ATG) was capable of driving green fluorescent protein (GFP) expression in transient transgenic embryos and stable transgenic adult fish, which mimics the endogenous gsnl1 expression. The GFP expression was localized in the corneal epithelium (CE) and the annular ligament (AL) at the iridocorneal angle. A unique enhancer for each of these two tissues was identified at 3.7-kb upstream from the ATG. The 60-bp AL and 25-bp CE enhancers were separated by 100-bp and functioned independently from each other. Deletion analysis indicated that the proximal promoter was located 1.6-kb upstream from the ATG. Stable GFP transgenic lines were established for future studies of genetic regulation in the anterior segment of the fish eye.     

Identification of an enhancer-type sequence that is responsive to Z and R trans-activators of Epstein-Barr virus

We studied the regulatory region of the open reading frame BHRF1 of Epstein-Barr virus (EBV) DNA that was responsive to two trans-activators (Z and R) encoded by the BZLF1 and BRLF1, respectively. The 200-bp sequence, nucleotide numbers 53,617 to 53,817 on the EBV map, was sufficient for conferring responsiveness to Z and R. This 200-bp sequence also enhanced expression of the chloramphenicol acetyltransferase (CAT) gene from the simian virus 40 promoter in response to both Z and R, even when inserted downstream of the cat gene. The results indicate that the Z and R response sequence upstream of the BHRF1 has the properties of an enhancer.     

Mutations of the chloramphenicol acetyl transferase transgene driven by the immunoglobulin promoter and intron enhancer

Transgenic mice containing the chloramphenicol acetyl transferase(CAT) gene driven by the immunoglobulin (lg) V_H promoter andthe heavy chain intron enhancer were prepared to increase ourunderstanding of the mechanism responsible for somatic mutation.The transgene showed similar tissue specificity in terms ofexpression as endogenous lg genes. Hybridomas were preparedafter multiple Immunization of a transgenic mouse with 4-hydroxy-3-nitrophenyl)acetyl-chickengammaglobulin, a thymus-dependent antigen. We analyzed mutationsin the CAT coding region as well as in the region 5' upstreamof the promoter after amplification of DNA using the PCR followedby sequencing of cloned DNA. Mutation detection enhancementgel electrophoresls was also used to detect mutations. Onlya single band was observed in PCR products from the region 5'upstream of the promoter and from the enhancer, whereas in thosefrom the CAT coding region, three out of 11 hybridomas showedmultiple bands. In DNA sequences of the CAT coding region fromthese three hybrldomas, a total of six nucleotlde substitutionswere observed, but none In the region 5' to the promoter. Theresults of DNA sequencing and the electrophoresls were in goodagreement, suggesting that mutation occurred only in the CATgene but not in the region 5' to the promoter or the enhancer.The rate of mutation in the CAT gene was estimated to be 1.6x10^–5perbase pair per cell division, which was a lower limit of therate for somatic mutation reported for lg genes. Location anddistribution of mutations were similar to that of the lg gene,suggesting that (i) mutation in the CAT gene was induced bya mechanism similar to that occurring in the lg gene and (ii)the mutator mechanism can operate in a non-lg gene providedit is flanked by the V_H promoter and heavy chain intron enhancer.     

The human beta-globin gene 3' enhancer contains multiple binding sites for an erythroid-specific protein

We have shown that the minimal enhancer fragment present in the 3'-flanking region of the human beta-globin gene contains four regions that bind nuclear proteins in vitro. By using gel mobility shift and DNase I footprinting assays, we were able to show that each of these regions binds an erythroid-cell-specific nuclear factor which we name NF-E1. This factor is present in erythroid cells at different developmental stages of globin gene expression. The recognition sequence of this protein (A/C Py T/A ATC A/T Py) is also present in the intragenic enhancer and the promoter of the beta-globin gene as well as in the promoter of other erythroid-cell specific genes. In addition to NF-E1, each of the four binding regions interacts with at least one other protein factor.     

Identification of Regulatory Elements Necessary for the Expression of the COL1A1 Promoter in Murine Odontoblasts

Recent studies have indicated that odontoblasts and osteoblasts have unique regulatory mechanisms that control COL1A1 gene expression. We are currently examining the regulation of COL1A1 gene expression in odontoblasts and have produced transgenic mice containing various collagen promoter constructs fused to the indicator gene, chloramphenicol acetyl transferase (CAT). Mandibular first molars were removed from jaws of transgenic mice. Some teeth were assayed for CAT activity (CAT diffusion assays), others were fixed and prepared for immunohistochemistry (CAT antibodies).

Our results indicate the CAT activity was present in tooth germs containing promoter constructs longer than 1.719 kb. Immunoreactivity to CAT was confined to the odontoblast cell layer. No CAT activity was present in tooth germs containing a 1.670 kb construct.

These data suggest that there are important regulatory elements located between -1.719 kb and -1.670 kb on the collagen promoter in odontoblasts. Examination of sequences in this region of the promoter demonstrates consensus with those known to be involved with binding of translation products of homeobox genes.     

Evidence for a complex regulatory array in the first intron of the human adenosine deaminase gene

Adenosine deaminase (ADA) is expressed ubiquitously by diverse mammalian cells and tissues but at levels that vary according to tissue and species. In humans, the thymus exhibits levels of the enzyme up to 100-fold higher than most other tissues. Using transgenic mice, we identified human ADA gene regulatory domains. Up to 3.7 kb of 5'-flanking and first exon DNA from the human ADA gene failed to promote the expression of a chloramphenicol acetyl transferase (CAT) reporter gene in an efficient, reproducible, or tissue-appropriate manner in transgenic mice. However, when 12.8 kb of DNA from the first intron of the human ADA gene was placed 3' of CAT-coding and -processing sequences, transgenic mice reproducibly expressed CAT activity in most tissues, with profoundly high levels in the thymus. DNase I hypersensitivity studies demonstrated that among transgenic mouse tissues, human thymus, and a variety of human cell lines, a region of the intron 4-10 kb downstream of the first exon exhibited an array of hypersensitive sites that varied according to tissue and cell type. Deletion of this region from the gene construction eliminated high-level expression in transgenic mice. In transfection-transient expression assays, the 12.8-kb intron fragment exhibited enhancer activity in several cell types. A 1.3-kb fragment encompassing two of the hypersensitive sites exhibited some of these activities. The results of these studies suggest that the diverse pattern of human ADA gene expression is determined, in part, by a cluster of cis-regulatory elements contained within its large first intron.     

An albumin enhancer located 10 kb upstream functions along with its promoter to direct efficient, liver-specific expression in transgenic mice

Transgenic mice were used to locate the cis-acting DNA elements that are important for efficient, tissue-specific expression of the mouse albumin gene in the adult. Chimeric genes with up to 12 kb of mouse albumin 5'-flanking region fused to a human growth hormone (hGH) reporter gene were tested. Remarkably, a region located 8.5-10.4 kb upstream of the albumin promoter was essential for high-level expression in adult liver and the region in between -8.5 and -0.3 kb was dispensable. The far-upstream region behaved like an enhancer in that its position and orientation relative to the albumin promoter were not critical; however, it did not function well with a heterologous promoter. Two of four DNase hypersensitive sites found in the 5'-flanking region of the albumin gene map to the far-upstream and promoter regions; the others may reflect regions involved in developmental or environmental control of this gene.