Negative Regulatory Element Involved in the Hormonal Regulation of GlcNAc-1-P Transferase Gene in Mouse Mammary Gland (∗)

• Published in: The Journal of biological chemistry 1996-05, Vol.271 (19), p.11197-11203
• Main Authors: Ma, Jie, Saito, Hiroshi, Oka, Takami, Vijay, Inder K.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Cell Nucleus - metabolism; Cloning, Molecular; Female; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic - drug effects; Hydrocortisone - pharmacology; Insulin - pharmacology; Lactation; Luciferases - biosynthesis; Mammary Glands, Animal - enzymology; Mice; Molecular Sequence Data; Polymerase Chain Reaction; Pregnancy; Prolactin - pharmacology; Prolactin - physiology; Promoter Regions, Genetic; Recombinant Proteins - biosynthesis; Reference Values; Regulatory Sequences, Nucleic Acid; Repetitive Sequences, Nucleic Acid; Restriction Mapping; Sequence Deletion; Transcription, Genetic; Transfection; Transferases (Other Substituted Phosphate Groups) - biosynthesis; Transferases (Other Substituted Phosphate Groups) - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.19.11197

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 cis-acting 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 (~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.