Analysis of the GAD1 promoter: Trans-acting factors and DNA methylation converge on the 5′ untranslated region

GAD67 corresponds to one of two enzymes that decarboxylates glutamate to produce γ-aminobutyric acid, the main inhibitory neurotransmitter in the mammalian central nervous system, hence defining the cellular phenotype of a diverse set of inhibitory interneurons of the brain. Reduced cortical GAD67 mRNA levels have consistently been reported in schizophrenia and bipolar disorder with psychosis. The human gene encoding GAD67, GAD1, is located on chromosome 2q31.1 and the transcriptional start site resides within a large CpG island that spans a region extending from upstream through the first exon. We have analyzed the GAD1 promoter using transient transfection analysis of upstream and downstream sequences in NT2 cells, a human neuroprogenitor cell line. Interestingly, results from these studies show that cis-acting regulatory elements are located downstream of the RNA start site and are in the region corresponding to the first exon. Trans-acting factors such as Pitx2 and the Dlx family of transcription factors are active in promoting downstream reporter expression even when all of the 5′ flanking sequences are removed. However, those constructs that contain an internal deletion from +66 to +173 bp fail to support expression even when these factors are provided in trans. We have previously shown that the Class I histone deacetylase inhibitor MS-275 potently activates GAD1 mRNA expression in NT2 cells suggesting the possibility that the promoter is sensitive to drugs that induce chromatin remodeling. Using methyl DNA immuneprecipitation of MS-275-treated NT2 cells, we provide data showing that Class I HDAC inhibition mediated an increase in GAD1 expression and that this was accompanied by decreased GAD1 promoter methylation. Moreover, the reduced levels of GAD1 DNA methylation are highest in those regions proximal to the location of the in vitro defined cis-acting regulatory elements. Our data suggest that changes in promoter methylation associated with gene regulation are not random but overlap the locations of proximal cis-acting elements. This article is part of a Special Issue entitled 'Trends in Neuropharmacology: In Memory of Erminio Costa'.