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Gillian Shaw, Cynthia Berlinicke, Donald Zack; Transcriptional regulation of synaptotagmin 11 in retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6205.
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© ARVO (1962-2015); The Authors (2016-present)
Gene regulation is essential for development and maintenance of different cell types. Compared to other retinal cell types, relatively little is known about transcriptional regulation in retinal ganglion cells (RGCs). As a model for studying RGC transcriptional regulation, we have been analyzing the cis-elements and trans-factors that regulate the expression of the RGC-enriched gene synaptotagmin 11 (Syt11).
5’-upstream fragments from seven RGC enriched genes (Syt11, Slc17a6, Sema6b, Sncg, Prph1 Nefm and Nrn1) were inserted into reporter plasmids and transfected into primary rat RGCs and dissociated whole retina. Deletion analysis of the Syt11 promoter reporter followed by the bioinformatic analysis of the active regions identified putative transcription factor (TF) binding sites (BSs). This set of TFs was cross-referenced to microarray data of isolated rodent RGCs. The predicted BSs of two RGC-expressed TFs, Deaf1 and Sp1, were mutated in the promoter reporter construct and their activity was compared to the unmutated construct. The effect of Deaf1 and Sp1 knockdown on endogenous Syt11 expression was also analyzed. Optimization of chromatin immunoprecipitation for Deaf1 and Sp1 is underway.
Of the seven RGC enriched genes tested, the Syt11 construct showed the highest activity in RGCs compared to whole retina. Promoter analysis showed that the region between -205 and -54bp contains important sequences for promoter activity in primary RGCs. This region contains two overlapping predicted BSs for each of the RGC-expressed TFs, Deaf1 and Sp1. Mutation of the Sp1 and Deaf1 BSs caused the greatest decrease in promoter reporter activity. Decreased expression of Deaf1, and to a lesser extent Sp1, was achieved with siRNA knockdown, the effect of which on Syt11 expression is being analyzed. Confirmation of Deaf1 and Sp1 binding to the promoter region of Syt11 in vivo is also being analyzed.
As a step in understanding transcriptional regulation in RGCs, a system to analyze promoter reporters in cultured primary rat RGCs was established. Using this system, we determined regions of Syt11 that are important for its RGC expression. Bioinformatic analysis of these regions identified BSs for Deaf1 and Sp1 and mutating these sites revealed that they are important for promoter activity. Analysis of the effect of Deaf1 and Sp1 knockdown and confirmation of their binding to Syt11’s promoter is underway.
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