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L. V. Wolf, Y. Yang, Y. Zavadil, Q. Xie, A. Cvekl; Genome-Wide Identification of Genes Regulated by Pax6 in Mouse Lens. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2792. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Pax6 is an essential regulatory gene that controls embryonic lens development. To date, there is a paucity of known direct targets of Pax6 in the lens. In this study we have taken two genome-wide approaches to identifiy novel downstream targets of Pax6 in mouse lens.
RNA microarray analyses was conducted with Pax6 normal and heterozygous postnatal day 1 (P1) mouse lenses. Due to the inability in distinquishing between direct and indirect targets using RNA microarray, ChIP-Chip promoter assays were additionally conducted with cultured mouse lens epithelial αTN4 cells. Potential target genes were validated wiht qtRT-PCR, along with quantitative chromatin immunoprecipitation (qChIP). A representative number of Pax6 binding sites were confirmed in vitro by gel shift assays.
We have identified 562 genes which are differentially expressed in Pax6 heterozygous P1 lens. Nine genes were chosen for validation based on potential Pax6 binding to the promoter regions of these genes revealed with ChIP-Chip. Of these nine genes, six genes: Mab21l2, Tgfb2, Sparc1, Olfm3, Cspg2, Igfbp5 are related to the eye, while Spon1 plays a role in cell adhesion. We also analyzed Nr2f2, a transcription factor and Spag5 which is involved in cell cycle regulation as these genes have been shown to be differentially expressed in the Pax6 null forebrain (1). Validation with qtRT-PCR confirmed a similar trend of differential expression of these genes as determined by the microarray. Analysis of these genes with qChIP revealed Mab21l2, Tgfb2, Sparc1 and Spag5 to be direct targets of Pax6. Computational studies have also determined common pathways are involved in both eye and forebrain development.
Using a combinatorial approach, we have identified Mab21l2, Tgfb2, Sparc1 and Spag5 to be direct Pax6 targets in P1 lens. In addition, bioinformatic comparison between microarray data in the P1 lens and Pax6 forebrain have revealed common pathways are involved in both lens and forebrain development.
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