Control of insulin-like growth factor-II/mannose 6-phosphate receptor gene transcription by proximal promoter elements.
The insulin-like growth factor-II/cation-independent mannose 6-phosphate receptor (IGF-II/MPR) is a multifunctional protein that binds IGF-II and ligands containing a mannose 6-phosphate recognition marker. Recent studies have shown that this receptor plays a critical role in mammalian development, and that its expression is controlled by both epigenetic and tissue-specific factors. Our laboratory has cloned the 93-kilobase mouse gene and characterized its 48 exons. In this report we describe the structure and function of the IGF-II/MPR gene promoter. To study promoter function, a series of chimeric plasmids linking different segments of IGF-II/MPR 5' flanking DNA to the reporter gene, firefly luciferase, were transiently transfected into HepG2 and C3H 10T1/2 cells. Promoter activity was orientation-specific and was maximal (550- to 4250-fold above promoterless control) with a plasmid containing 266 base pairs (bp) of IGF-II/MPR DNA. The fusion gene accurately directed transcription as measured by ribonuclease protection assay using RNA extracted from transfected cells. DNA-protein binding studies by in vitro DNase I footprinting revealed an extended 54-bp footprint within the proximal promoter that contained two E-boxes and potential binding sites for transcription factors Sp1, NGF-IA, and related proteins. Gel mobility shift experiments with double-stranded oligonucleotides containing this region gave rise to several specific DNA-protein complexes, and the addition of specific antibodies indicated that proteins antigenically related to Sp1 and c-Myc were components of one or more of these bands. Deletion of this 54-bp segment led to an 8-fold decline in promoter activity, and its transfer to a heterologous promoter stimulated gene expression by nearly 7-fold. Mutational analyses indicated that each E box contributed to more than half of the enhancer's activity. These results define a strong minimal IGF-II/MPR promoter of no more than 266 bp and identify a 54-bp enhancer within this promoter fragment. Our observations thus represent a first step toward characterizing the developmental, epigenetic, and tissue-specific factors that control IGF-II/MPR gene expression.
Liu
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Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, Saint Louis, Missouri 63110, USA.
Mittanck
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DW
Kim
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Rotwein
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P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D018983:DNA Footprinting; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation; D005813:Genes, Synthetic; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D008099:Liver / Q000166:cytology; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017527:Receptor, IGF Type 2 / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D017527:Receptor, IGF Type 2; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D007335:Insulin-Like Growth Factor II