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D. C. Otteson, L. Pillai, K. Zhu; The Mouse EphA3 Promoter Contains Multiple Conserved Binding Sites for Forkhead-Family Transcription Factors and Is Transcriptionally Active in QNR/D Embryonic Retinal Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2927.
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EPHA receptors are expressed in nasal/temporal gradients in the developing retina and play a key role in establishing positional information in the retina. Our goal is to clone and characterize the EphA3 promoter in mouse and identify the transcriptional mechanisms regulating graded and cell-specific patterns of EphA3 expression in the retina.
Genomic sequences spanning exon 1 and the 5’ flanking region of rodent and primate EphA3 were obtained from the NCBI and UCSC genome databases and aligned using AlignX (VectorNTI). Conserved transcription factor binding sites were predicted using DiAlign/Genomatix and TESS (Transcription Element Search System). Genomic fragments were amplified from mouse DNA using proofreading Taq polymerase with primers designed using Primer3 software, cloned into pSC-A (Stratagene) and sequence verified. For luciferase assays, EphA3 promoter fragments were subcloned into pGl2Basic and transfected into QNR/D cells (ATCC) using lipofectamine. Luciferase expression was quantified from total cell lysates using a SynergyHT plate reader (BioTek).
Regions of high sequence conservation between rodents and primates were present in the putative EphA3 promoter region. The proximal promoter region (-500 to +1) contained multiple conserved SP1/MAZ, E2F and forkhead binding sites. A more distal conserved region (-1500 to -1000) contained a BRN3b and multiple LEF1 consensus binding sites. When transfected into quail embryonic retinal cell line QNR/D, an EphA3 promoter-luciferase reporter construct, containing the region from -1700 to +120, showed a 20-fold increase in luciferase expression compared to un-transfected controls. Reporter constructs containing a smaller promoter fragment (-180 to +120) yielded an approximately 4-fold increase in luciferase activity, whereas a fragment containing only exon 1 sequences (+20 to +120) showed no activity.
We have cloned the promoter region for mouse EphA3 and identified an 1800 bp genomic fragment is transcriptionally active in quail embryonic retinal cells in vitro. The forkhead-containing transcription factors FOXD1/BF2 and FOXG1/BF are proposed regulators of nasal/temporal polarity in the developing retina, therefore they are candidates for directly regulating the EphA3 expression through the conserved forkhead binding sites. We have also identified multiple binding sites for LEF-1, a mediator of Wnt signaling, in the EphA3 promoter suggesting a potential role for Wnt signaling in regulating EphA expression during retinal development.
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