splicing of TEAD4 in human cells and regulation of non-muscle specific genes is less well characterized. Recently, alternatively spliced transcripts for TEAD4 have been identified in human retinal vascular endothelial cells. [11] Shie and colleagues showed that full length TEAD4 protein was able to bind to Sp1 sequences within the VEGF promoter and enhance VEGF gene expression in bovine aortic endothelial cells. [12] TEAD4 expression was up-regulated under hypoxic conditions, and TEAD4 mediated stimulation of VEGF expression was independent of the classic hypoxia responsive element (HRE) and hypoxia-inducible factor (HIF-1) mechanism. [12] We identified two new human isoforms, 936 (TEAD4311) and 447 (TEAD4148), which enhanced VEGF promoter activity. The TEAD4148 isoform was the most potent enhancer relative to other isoforms. Interestingly, the TEAD4148 isoform was identified in human ocular endothelial cells cultured under hypoxic conditions, suggesting that environment-specific alternate splicing may occur within human tissue to generate specific transcription factors with altered functions. In this study, we report an alternatively spliced TEAD4 transcript that generates a novel isoform (TEAD4216) able to significantly repress promoter activity. This novel protein reduces native VEGF production and cell proliferation. We show that the TEAD4216 isoform can function in the absence of the HRE sequence and overcomes the enhancement mediated by the other TEAD4 isoforms. In addition we demonstrate the presence The TEAD4216 Isoform Inhibits VEGF Expression of TEAD4 protein in choroidal neovascular membrane in human age-related macular degeneration, suggesting a role in human disease. Results Sequence Comparison of TEAD4216 to Other TEAD4 Isoforms Amplification from cDNA prepared from pooled total RNA isolated from primary cultures of human choroidal (PCVEC), retinal (PRVEC) and iris (PIVEC) vascular endothelial cells, with primers to full length TEAD4, detected the previously described TEAD4434 and TEAD4311 isoforms as well as a novel 651 bp product (Figure S1A). Amplification of specific isoforms not only varied between REC, CEC and IEC, but also differed under hypoxic conditions (Figure S1B). The 651 bp product was consistently amplified in all donors and ECs tested in this study. In addition, TEAD434 and other isoforms were detected by western blot analysis of primate retinal-choroidal endothelial cells (Figure S2). The 651 bp (TEAD4216) isoform is generated by an inframe splice event within exons 3 and 10 that results in loss of the 39 end of exon 3, complete deletion of exons 4 to 9, and absence of the 59 end of exon 10 (Figure 1). The donor splice site occurs at the junction of Thr-92 and Arg-93, removing the last five codons of exon 3, and the acceptor site occurs between Gly-310 Figure 1. Schematic diagram of the full length TEAD4 (TEAD4434) and the novel TEAD4216 isoform. The previously identified TEAD4148 isoform is also shown for comparison. Schematic indicates the TEA DNA-binding domain (TEAD), a putative nuclear localization signal (NLS), a proline rich domain (PRD) and serine-threonine-tyrosine (STY) domains. doi:10.1371/journal.pone.0031260.g001 PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e31260