Abstract
Nuclear orphan receptors are known to be important mediators of neurogenesis, but the target genes of these transcription factors in the vertebrate nervous 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 expression. In our present studies, mutation of an 11-base pair element within this fragment resulted in a loss of nuclear protein binding and reverses negative regulation by the intron. Using yeast one-hybrid screening, we have identified intron-binding proteins from rat brain as COUP-TFI, EAR2, and NURR1. Gel shift studies with postnatal day 2 rat brain extract indicate the presence of COUP-TFs, EAR2, and NURR1 in the DNA-protein complex. Competition assays with GRIK5-binding site mutations show that the recombinant clones exhibit differential binding characteristics and suggest that the DNA-protein 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 tissue and developmentally regulated. Co-transfection assays showed that recombinant 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 role(s) in active and/or transrepression. Our findings are thus consistent with the notion that multiple nuclear orphan receptors can regulate the transcription of a widely expressed neurotransmitter receptor gene by binding a common element in an intron and directly modulating the activity of the transcription machinery.
Highlights
This indicates that the effect of recombinant TFIIB is dependent on the presence of endogenous DNA-bound nuclear orphan receptors and that this interaction is likely to contribute to the mechanism of negative regulation of GRIK5 promoter activity. These results suggest that the interaction of nuclear orphan receptors with TFIIB may form a general mechanism by which these receptors mediate transcriptional repression in vivo, and apparently additional factors may be involved in determining the specificity of action for members of the COUP-TF family in the context of the intact first intron (Fig. 6B) and the GRIK5 promoter (Fig. 6C)
Nuclear orphan receptors have been proposed to play a key role in regulating organogenesis, neurogenesis, and cellular differentiation during embryonic development
Genetic ablation of nuclear orphan receptors including COUP-TF and NURR1 is lethal, and perinatal mortality arises from severe deficiencies in nervous system maturation [14, 15]
Summary
Preparation of Nuclear Protein Extracts from Cultured Cells and Tissues—CG-4 cells were cultured as described previously by Louis et al [28] and Patneau et al [3] and were harvested 2 days after replating (80% confluency). One-hybrid Screen for Binding Protein(s)—The in vivo screening strategy was carried out using the Yeast Matchmaker One-hybrid system (CLONTECH) This required generating a yeast reporter strain containing three tandem copies of wild type or mutated probe 62 sequences (to eliminate false positives) cloned upstream of yeast promoters driving two different reporter genes, HIS3 and lacZ. 3– 6 l of crude translation mixture was mixed with 4 l of TFIIB antibody (Santa Cruz) and 20 l of protein A-agarose (Santa Cruz) in a total volume of 150 l, containing binding buffer of the following composition: 10% glycerol, 50 mM HEPES, pH 7.9, 100 mM KCl, 6 mM magnesium acetate, 5 mM EDTA, 0.5 mM dithiothreitol, 0.5% nonfat dry milk. The agarose pellets were resuspended in 20 l of SDS loading buffer and boiled prior to SDS-polyacrylamide gel electrophoresis analysis
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