Identification of nuclear orphan receptors as regulators of expression of a neurotransmitter receptor gene

Nuclear orphan receptors are known to be important mediators of neurogenesi s, but the target genes of these transcription factors in the vertebrate ne rvous system remain largely undefined, We have previously shown that a 500- base pair fragment in the first intron of the GRIK5 gene, which encodes the kainate-preferring glutamate receptor subunit KA2, down-regulates gene exp ression. In our present studies, mutation of an Ii-base pair element within this fragment resulted in a loss of nuclear protein binding and reverses n egative regulation by the intron. Using yeast one-hybrid screening, we have identified intron-binding proteins from rat brain as COUP-TFI, EAR2, and N URR1. Gel shift studies with postnatal day 2 rat brain extract indicate the presence of COUP-TFs, EAR2, and NURR1 in the DNA-protein complex. Competit ion assays with GRIK5-binding site mutations show that the recombinant clon es exhibit differential binding characteristics and suggest that the DNA-pr otein complex from postnatal day 2 rat brain may consist primarily of EAR2. The DNA binding activity was also observed to be enriched in rat neural ti ssue and developmentally regulated. Co-transfection assays showed that reco mbinant nuclear orphan receptors function as transcriptional repressors in both CV1 cells and rat CG4 oligodendrocyte cells. Direct interaction of the orphan receptors with and relief of repression by TFIIB indicate likely ro le(s) in active and/or transrepression. Our findings are thus consistent wi th the notion that multiple nuclear orphan receptors can regulate the trans cription of a widely expressed neurotransmitter receptor gene by binding a common element in an intron and directly modulating the activity of the tra nscription machinery.