Abstract
Aminopeptidase N/CD13 is a metallopeptidase found in many tissues. Aminopeptidase N activity is high in the small intestinal mucosa, moderate in the liver, and low in the spleen. Using DNase I footprinting and electrophoretic mobility shift assays with nuclear extracts from these tissues, three cis elements (DF, LF-B1, UF) were identified in the aminopeptidase N promoter. The DF region (-53 to -30) interacts with the ubiquitously expressed transcription factor Sp1. The LF-B1 region (-85 to -58) interacts with the liver transcription factor LF-B1 (HNF-1) which was detected as well in nuclei from small intestinal mucosa. The UF region (-112 to -90) interacts with nuclear factors which seem to be expressed differentially in the liver and the small intestine. Transfection of promoter deletions into HepG2 cells showed that the LF-B1 region is necessary for high expression of the aminopeptidase N gene in liver cells. LF-B1 could not be detected in spleen nuclei. In accordance with this, RNA analysis demonstrated that the aminopeptidase N promoter operating in the small intestine and in the liver is inactive in the spleen. In this tissue initiation of transcription from the aminopeptidase N gene occurs from an upstream promoter.
Highlights
Aminopeptidase N/CD13 isa metallopeptidase found chored to the membranes lining the bile canaliculi
APN is alsoexpressed in the spleen the small intestinal mucosa, moderate in theliver, and where it is present incells expressing the cluster differentialow in the spleen
Using DNase I footprinting and tion marker CD13 which has been identified as APN (Look electrophoretic mobility shift assays withnuclear ex- et al, 1989)
Summary
Protection with small intestinal and liver RNA results in t.he generation of a number of closely spaced, intense hands migrating slightlybelow the 267-base pair DNA marker (Fig. 1, lanes 3-6). 440 nucleotides (Fig. 1, lanes 7 and 8). in both the small intestine and in the liver the major transcriptional initiation. - 234 site is the one previouslyidentified and whichwasused to mark position +1 in the gene (Olsen et ai., 1989). - 213 spleen, the initiation of transcription occursa t least 185 nucleotides upstream from this position. The Potential LF-B1 Binding Site from Positio-8n3 to -63 I s Necessaryfor High A P N Expression in Liver Cells-An APN promoter fragment(-1122 to +28)was inserted in front of thebacterialchloramphenicolacetyltransferase(CAT) gene, and the construct was used to transfect human hepa-
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