Nucleic Acids Research, Vol 25, Issue 19 3930-3936, Copyright © 1997 by Oxford University Press
TJ Hadden, C Ryou and RE Miller
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.
ARTICLES
Elements in the distal 5'-flanking sequence and the first intron function cooperatively to regulate glutamine synthetase transcription during adipocyte differentiation
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
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