IDENTIFICATION AND CHARACTERIZATION OF A NEURORETINA-SPECIFIC ENHANCER ELEMENT IN THE QUAIL PAX-6 (PAX-QNR) GENE

Using nuclear run-on assays, we showed that the tissue-specific expres sion of quail Pax-6 (Pax-QNR) P0-initiated mRNAs is due in part to reg ulation of the gene at the transcriptional level. Regulatory sequences governing neuroretina-specific expression of the P0-initiated mRNAs w ere investigated. By using reporter-based expression assays, we charac terized a region within the Pax-QNR gene, located 7.5 kbp downstream f rom the P0 promoter, that functions as an enhancer in neuroretina cell s but not in nonexpressing P0-initiated mRNA cells (quail embryo cells and quail retinal pigment epithelial cells). This enhancer element fu nctioned in a position- and orientation-independent manner both on the Pax-QNR P0 promoter and the heterologous thymidine kinase promoter. M oreover, this enhancer element exhibited a developmental stage-specifi c activity during embryonic neuroretina development: in contrast to ac tivity at day E7, the enhancer activity was very weak at day E5. This paralleled the level of expression of P0-initiated mRNAs observed at t he same stages. Using footprinting, gel retardation, and Southwestern (DNA-protein) analysis, we demonstrated the existence of four neuroret ina-specific nuclear protein-binding sites, involving multiple unknown factors. In addition we showed that the quail enhancer element is str ucturally and functionally conserved in mice. All of these results str ongly suggest that this enhancer element may contribute to the neurore tina-specific transcriptional regulation of the Pax-6 gene in vivo.