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B. Appukuttan, T.J. McFarland, Y. Zhang, L.–O. Atchaneeyasakul, T. Acott, J. Stout; Functional Characterization of Novel Human Ocular Specific Isoforms of RTEF–1 a Regulator of VEGF Gene Expression . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4893.
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Recently a TEA domain transcription factor RTEF–1 was identified that activates and enhances the production of VEGF in bovine aortic endothelial cells. Whether this 1200bp RTEF–1 transcript is present in the retina and the role of this activator/enhancer in the eye has not been investigated. We have identified the 1200bp RTEF–1 product as well as previously undiscovered alternate spliced isoforms of RTEF–1, a 900bp and 450bp transcripts, within human retinal vascular endothelial cells. We aim to characterize whether these isoforms are capable of regulating the VEGF gene.
The immediate 5’ flanking region of the human VEGF promoter was cloned into a pSEAP reporter construct. The 1200bp RTEF–1 cDNA and the 900bp and 450bp alternate spliced cDNA products were also cloned into an expression plasmid. Various deletion constructs of the VEGF promoter linked to the reporter gene were also cloned, to identify the cis–elements required for regulation by the TEA domain factors. The expression plasmids containing the various RTEF–1 isoforms and VEGF reporter constructs were coelectroporated into either human 293T cells or vascular endothelial cells. Expression levels of the reporter protein were assayed and compared.
Twelve exons are predicted to code for the full length 1200bp RTEF–1 mRNA product. Sequencing analysis revealed that the 900bp and 450bp transcript from human endothelial cells are alternate spliced products of the 1200bp factor. Four of the 11 translated exons that are predicted to code for the 1200bp transcript are spliced out of the 900bp transcript. The 1200bp and 900bp transcripts were identified in cultured human retinal endothelial cells under normoxic and hypoxic conditions whereas the 450bp product was only present in RNA isolated from cells maintained in a hypoxic environment. Reporter protein analysis indicate that the full length 1200bp product enhances expression from the VEGF promoter about 4–fold greater than background, whereas the 900bp and the 450bp isoforms enhance expression by 3x and 15x greater than background expression, respectively. Analysis with deletion promoter constructs determined that all isoforms required the presence of Sp1 elements for activation.
A gene from the TEA/ATTS DNA family is expressed within human retinal vascular endothelial cells. Alternative isoforms exist that may confer different transcriptional activity of the VEGF protein.
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