Transcription Factor Pax6 Contributes to Induction of GLT-1 Expression in Astrocytes Through an Interaction with a Distal Enhancer Element

摘要

The Na⁺-dependent glutamate transporter, GLT-1 (EAAT2), shows selective expression in astrocytes, and neurons induce expression of GLT-1 in astrocytes. In unpublished analyses of GLT-1 promoter reporter mice, we identified an evolutionarily conserved domain of 467 nucleotides ~8 kb upstream of the GLT-1 translation start site that is required for astrocytic expression. Using in silico approaches, we identified Pax6 as a transcription factor that could contribute to the control of GLT-1 expression by binding within this region. We demonstrated expression of Pax6 protein in astrocytes in vivo. Lentiviral transduction of astrocytes with exogenous Pax6 increased expression of enhanced green fluorescent protein (eGFP) in astrocytes prepared from transgenic mice that use a bacterial artificial chromosome (BAC) containing a large genomic region surrounding the GLT-1 gene to control expression of eGFP. It also increased GLT-1 protein, and GLT-1-mediated activity, while there was no effect on the levels of astroglial glutamate transporter, GLAST. Transduction of astrocytes with an shRNA directed against Pax6 reduced neuron-dependent induction of GLT-1 or eGFP. Finally, we confirmed Pax6 interaction with the predicted DNA binding site in electrophoretic mobility assays (EMSA) and chromatin immunoprecipitation (ChIP). Together, these studies show that Pax6 contributes to regulation of GLT-1 through an interaction with these distal elements and identify a novel role of Pax6 in astrocyte biology.