Transcription Factor Sp4 Is Abundantly Expressed in Photoreceptors and Other Retinal Neurons and Together With Crx Co–Occupies the Promoters of Rod–Specific Opsin and ß–PDE Genes.

Abstract

Abstract: : Purpose: Transcription factors Sp4 and Crx have been implicated in the expression of a number of photoreceptor–specific genes including the ß–subunit of PDE (ß–PDE) and rod opsin genes. We reported previously that Sp4, Sp1 and Sp3 each show functional synergy with Crx on the rod opsin promoter in transient transfections, although Sp4–mediated activation was the most significant. Sp4, Sp1 and Sp3 bind Crx in co–immunoprecipitation experiments in vitro, and their zinc finger domains as well as the Crx homedomain are necessary and sufficient for these interactions. The purpose of the current study was to further test whether these transcription factors are able to interact with each other in vivo suggesting a novel mechanism for combinatorial regulation of certain retina–specific genes. Methods: For in situ hybridization, various–sized fragments of the mouse Sp4 cDNA were used for the generation of three different probes (1588 bp, 776 bp and 222 bp). The eyes were obtained from 2 month–old C57BL/6 mice, fixed in 4% paraformadehyde in PBS, and embedded for sagittal sectioning. For immunohistochemistry, the eyes from adult wild–type C57BL/6 mice were fixed overnight in 10% formalin/PBS, and processed for paraffin embedding. Following heat–induced epitope retrieval, retinal sections were probed with anti–Sp4 antibodies. Chromatin immunoprecipitation (ChIP) assays were performed using adult mouse retinas (n=6–8) or liver (30 mg) collected from adult wild–type C57BL/6 mice. Chromatin binding proteins were cross–linked by treating the tissue with 1% formaldehyde. Results: In situ hybridization and immunohistochemistry demonstrated that Sp4 is abundantly expressed in various retinal neurons of all retinal layers, and thus co–localizes or overlaps with multiple retina–restricted and –enriched genes, its putative targets. Chromatin immunoprecipitation showed that the rod opsin and ß–PDE promoters are targets of both Sp4 and Crx, suggesting the possibility of Sp4–Crx interactions in vivo in the context of retinal chromatin environment. Conclusions: Our results indicate that photoreceptor–specific gene transcription is controlled by the combinatorial action of Sp4 and Crx. These data suggest the potential importance of Sp4 in retinal neurobiology and pathology.

Keywords: gene/expression • transcription factors • retina