Elements in the distal 5'-flanking sequence and the first intron function cooperatively to regulate glutamine synthetase transcription during adipocyte differentiation.
Glutamine synthetase (GS) expression increases dramatically during adipocyte differentiation of confluent 3T3-L1 cells. To identify differentiation-responsive cis-acting elements in the GS gene, several GSfusion genes were prepared and analyzed in stably transfected 3T3-L1 cells under conditions that trigger adipocyte differentiation. We find that the GS proximal 5'-flanking sequence lacks the regulatory elements required for differentiation-responsive expression. In contrast, a 2 kb intron 1 restriction fragment fused upstream of a heterologous promoter does drive reporter gene expression during hormone-triggered differentiation. The enhancer activity was localized to a 310 bp sequence near the middle of intron 1. Expression of fusion genes that include this 310 bp sequence does not temporally coincide with native gene expression. However, a composite gene that includes a far upstream GS sequence and the 2 kb intron 1 sequence yields a qualitatively different pattern of expression that closely resembles that of the native GS gene. The far upstream sequence alone exhibits no enhancer activity. Electrophoretic mobility shift analyses indicate that a 32 bp sequence within the 310 bp functional enhancer specifically binds differentiation-associated nuclear proteins. Although a C/EBP consensus sequence occurs in the 32 bp fragment, supershift analyses exclude C/EBP isoforms as the binding factor. In contrast, mutational analysis of the putative enhancer suggests that an HNF-3 isoform is involved. Thus our data indicate that elements in the distal 5'-flanking sequence and the first intron function cooperatively to regulate GS transcription and that HNF-3 may participate in that regulation.
Hadden
T J
TJ
Research Service of the Detroit VA Medical Center and Department of Internal Medicine and Center for Molecular Medicine and Genetics, School of Medicine, Wayne State University, Detroit, MI 48201, USA. thadden@cmb.biosci.wayne.edu
Ryou
C
C
Miller
R E
RE
D016475:3T3 Cells; D000273:Adipose Tissue / Q000166:cytology* / Q000201:enzymology*; D000818:Animals; D019677:Artificial Gene Fusion; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002454:Cell Differentiation; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015971:Gene Expression Regulation, Enzymologic; D005809:Genes, Regulator; D017930:Genes, Reporter; D005974:Glutamate-Ammonia Ligase / Q000235:genetics*; D007438:Introns; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D004247:DNA; D005974:Glutamate-Ammonia Ligase