Control of insulin-like growth factor-II/mannose 6-phosphate receptor gene transcription by proximal promoter elements.

Abstract

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.

E-box-binding Repressor Is Down-regulated in Hepatic Stellate Cells during Up-regulation of Mannose 6-Phosphate/Insulin-like Growth Factor-II Receptor Expression in Early Hepatic Fibrogenesis

“…In C3H 10T1/2 cells, the two M6P/IGFIIR E-boxes promote transcription apparently through Myc binding (20). Hepatic stellate cells, on the other hand, use the same E-boxes to repress (and de-repress) transcription of M6P/IGFIIR through regulation of an E-box binding repressor.…”

“…This region was selected for study because it contains two E-boxes, which are often involved in transcriptional regulation (19,20). E-boxes are transcription factor binding elements that conform to a CANNTG motif and bind proteins that carry a basic helix-loop-helix structural domain, of which more than 200 have been found, categorized into five classes according to the basic helix-loop-helix domain amino acid sequence (19).…”

The IGF2 Receptor Is a USF2-specific Target in Nontumorigenic Mammary Epithelial Cells but Not in Breast Cancer Cells

“…USF-type E boxes A-C are in boldface and a fourth E box (BЈ) is in italics. Vertical arrows mark the transcription start sites for the mouse promoter (33), and the translation start is shaded. The 183-bp promoter region used for footprinting and transfection experiments is underlined.…”

“…essential for strong promoter activity (33). We inserted a 183-bp segment of the human IGF2R promoter containing the three USF-type E boxes into a plasmid upstream of luciferaseencoding sequences.…”

“…Deletion analysis of the mouse Igf2r gene identified a 5Ј region with two E boxes essential for promoter activity in liver cells (33). In hepatic stellate cells, Igf2r expression was down-regulated by a repressor binding at an E box of the mouse promoter (34).…”