ELEMENTS IN THE DISTAL 5'-FLANKING SEQUENCE AND THE FIRST INTRON FUNCTION COOPERATIVELY TO REGULATE GLUTAMINE-SYNTHETASE TRANSCRIPTION DURING ADIPOCYTE DIFFERENTIATION

Glutamine synthetase (QS) expression increases dramatically during adi pocyte differentiation of confluent 3T3-L1 cells. To identify differen tiation-responsive cis-acting elements in the GS gene, several GS fusi on 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 re quired for differentiation-responsive expression. In contrast, a 2 kb intron 1 restriction fragment fused upstream of a heterologous promote r does drive reporter gene expression during hormone-triggered differe ntiation. The enhancer activity was localized to a 310 bp sequence nea r the middle of intron 1. Expression of fusion genes that include this 310 bp sequence does not temporally coincide with native gene express ion. However, a composite gene that includes a far upstream GS sequenc e and the 2 kb intron 1 sequence yields a qualitatively different patt ern of expression that closely resembles that of the native GS gene. T he far upstream sequence alone exhibits no enhancer activity, Electrop horetic mobility shift analyses indicate that a 32 bp sequence within the 310 bp functional enhancer specifically binds differentiation-asso ciated 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 enha ncer suggests that an HNF-3 isoform is involved. Thus our data indicat e that elements in the distal 5'-flanking sequence and the first intro n function cooperatively to regulate GS transcription and that HNF-3 m ay participate in that regulation.