Elements in the distal 5'-flanking sequence and the first intron function cooperatively to regulate glutamine synthetase transcription during adipocyte differentiation
- Authors
- Hadden, Timothy J.; Ryou, Chongsuk; Miller, Richard E.
- Issue Date
- Oct-1997
- Publisher
- Oxford University Press
- Citation
- Nucleic Acids Research, v.25, no.19, pp.3930 - 3936
- Indexed
- SCIE
SCOPUS
- Journal Title
- Nucleic Acids Research
- Volume
- 25
- Number
- 19
- Start Page
- 3930
- End Page
- 3936
- URI
- https://scholarworks.bwise.kr/erica/handle/2021.sw.erica/47028
- DOI
- 10.1093/nar/25.19.3930
- ISSN
- 0305-1048
- Abstract
- Glutamine synthetase (QS) expression increases dramatically during adipocyte differentiation of confluent 3T3-L1 cells. To identify differentiation-responsive cis-acting elements in the GS gene, several GS fusion 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.
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