Transcriptional regulation of GABAA receptor gamma2 subunit gene.
We have cloned the promoter regions of the genes for the mouse and human gamma2 subunits of the type A receptors for gamma-aminobutyric acid (GABA). For the mouse, the two major transcription start sites were at +1 (by definition) and +43, as established by rapid amplification of cDNA ends (RACE) and primer extension. This numbering places the start methionine at +297. There was no TATA or CCAAT box. Both mouse and human sequences have a candidate neuron-restrictive silencer element (NRSE) site in the first intron (+956 in mouse). We made assorted mouse-based promoter/reporter (luciferase) constructs starting from a core extending from -331 to +136, varying sizes at both ends, and including and excluding the putative NRSE and more proximal sequences. These were tested by transient transfection in several neuron-like and non-neuronal cell lines. Both proximal and distal downstream elements appeared to help direct expression to neuron-like cells, the NRSE in the intron, by repression in non-neurons, and a 24-bp portion of the 5' untranslated region starting at +113 (named GPE1) by preferentially promoting expression in neuron-like cells. Cotransfected human NRSF (transcription factor for NRSE) reduced reporter expression in neuron-like cells for constructs containing the NRSE in two locations. In gel mobility shift assays, the mouse gamma2 NRSE and a consensus NRSE both bound in vitro translated NRSF very similarly, and the NRSF gave the same major shifted band with the mouse gamma2 NRSE as was observed with nuclear extracts.
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Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201-1559, USA.
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D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001569:Benzodiazepines / Q000494:pharmacology; D001923:Brain Chemistry / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary; D005786:Gene Expression Regulation / Q000502:physiology; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000502:physiology; D017434:Protein Structure, Tertiary; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000502:physiology
D017931:DNA Primers; D018076:DNA, Complementary; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D005680:gamma-Aminobutyric Acid; D008156:Luciferases