Abstract
The gene encoding UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme that initiates the pathway for the biosynthesis of asparagine-linked glycoproteins, is ubiquitously expressed in eukaryotic cells. However, its expression in the mammary gland is developmentally and hormonally regulated; transcription of the mouse mammary GPT gene is stimulated by the lactogenic hormones, insulin, glucocorticoid, and prolactin. The involvement of cisacting elements in regulating the expression of the mouse GPT (mGPT) gene was investigated by transient transfections of various GPT promoter/luciferase (Luc) constructs into primary mouse mammary epithelial cells. A series of 5'-deletions of the GPT promoter identified a distal negative regulatory region (base pairs -1057 to -968) and deletion of this region results in enhanced hormonal induction (approximately 7-fold) with no effect on basal promoter activity. Electrophoretic mobility shift assays (EMSA) performed with nuclear extracts from different developmental stages of mouse mammary gland demonstrated that the binding activity of the nuclear proteins to the distal negative regulatory region was predominant in virgin stage as compared with pregnant and lactating stages. EMSA performed with nuclear extracts from virgin explants showed that the binding activity was markedly decreased after cultivation with the combination of the three lactogenic hormones. DNase I footprinting analysis identified two pentamer direct repeat motifs, AGGAA and GAAAC, within the negative regulatory region. EMSA competition experiments showed that mutations within the direct repeats failed to compete for binding of the nuclear proteins to labeled wild type oligonucleotide. Transcription from the promoter containing the mutated direct repeats was increased greatly, consistent with the conclusion that these motifs functions in vivo to repress GPT gene expression. These data suggest the importance of the negative regulatory region in hormonal control of mGPT gene expression in mammary gland.
Highlights
The gene encoding UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme that initiates the pathway for the biosynthesis of asparagine-linked glycoproteins, is ubiquitously expressed in eukaryotic cells
In this report we show that the pentamer direct repeat motifs, AGGAA and GAAAC, within the distal negative regulatory region of the mouse GPT (mGPT) promoter play an important role in developmental and hormonal regulation of GPT gene expression in mouse mammary gland
Using a promoterless Luc reporter vector, deletion mutations were targeted systematically to eliminate these potential binding sites to determine whether the sites and/or any other cis-acting elements were involved in developmental and hormonal regulation of GPT gene expression
Summary
11197–11203, 1996 Printed in U.S.A. Negative Regulatory Element Involved in the Hormonal Regulation of GlcNAc-1-P Transferase Gene in Mouse Mammary Gland*. Transcription from the promoter containing the mutated direct repeats was increased greatly, consistent with the conclusion that these motifs functions in vivo to repress GPT gene expression These data suggest the importance of the negative regulatory region in hormonal control of mGPT gene expression in mammary gland. The aim of our studies is to identify cis-acting elements involved in the developmental and hormonal regulation of GPT gene in the mouse mammary gland. In this report we show that the pentamer direct repeat motifs, AGGAA and GAAAC, within the distal negative regulatory region of the mGPT promoter play an important role in developmental and hormonal regulation of GPT gene expression in mouse mammary gland
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