and Ser-311, eliminating the first eleven codons of exon 10. The splicing event results in removal of the third alpha helix and a nuclear localization signal (NLS), within the TEA DNA-binding domain, the proline rich domain (PRD) and a hydroxyl containing amino acid rich STY domain (Figure 1). The combination of specific protein motifs present in the TEAD4216 isoform is novel when compared with the previously identified TEAD4311 and TEAD4148 isoforms. [11]. The TEAD4216 Isoform Represses VEGF Promoter Activity The full-length TEAD4 isoform (TEAD4434) acts as a transcriptional enhancer of VEGF in bovine aortic endothelial cells. [12] Reporter assays indicate that the TEAD4311 and TEAD4148 isoforms increase human VEGF promoter activity. [11] To test whether the TEAD4216 isoform was able to effect VEGF promoter-driven transcription, co-transfection experiments in 293T cells with a reporter plasmid that placed the secretable alkaline phosphatase (SEAP) gene under the transcriptional control of the VEGF promoter (1,136 bp of the 59 proximal VEGF promoter and 54 bp of the VEGF gene 59UTR), and an expression plasmid containing the TEAD4216 cDNA, were performed. The TEAD4216 isoform repressed VEGF promoter activity approximately 50% below background (P,0.05, Figure 2). The TEAD4216 Isoform Does not Require the HRE Sequence to Repress VEGF Promoter Activity The hypoxia-inducible factor (HIF)-1 subunits require the presence of a specific sequence the HRE, situated between –985 and –939 bp within the human VEGF 59 promoter to function as an enhancer of transcription. The HIF-1 induced VEGF expression is initiated under conditions of cellular oxygen starvation as observed in tumor tissue and ischemic disease.[13– 16] Full-length human TEAD4 (TEAD4434) stimulates VEGF production independent of the HIF-1/HRE pathway in bovine endothelial cells. [12] To assess whether the TEAD4216 isoform requires the HRE sequence to repress VEGF activity, a reporter plasmid containing a truncated promoter fragment (lacking the HRE sequence) was generated, as described previously. [11] Using this plasmid, we demonstrated that the enhancer isoforms increased expression in 293T cells, whereas the TEAD4216 isoform retained its ability to inhibit VEGF promoter activity (Figure 3). The HRE-independent, TEAD4216-mediated repres- Figure 2. The TEAD4216 isoform represses VEGF promoter activity. The TEAD4216 isoform can repress expression from the full length human VEGF promoter F1–R3 (p,0.01, n = 9) in contrast to the TEAD4434 and TEAD4148 enhancers. doi:10.1371/journal.pone.0031260.g002 The TEAD4216 Isoform Inhibits VEGF Expression sion of VEGF promoter activity was approximately 50% below background control levels. The full length TEAD4 requires four consecutive Sp1 sequences (Sp1–1 to Sp1–4) situated between 2100 and 240 bp upstream of the human VEGF transcription initiation site to function as an enhancer. [11,12] We show that TEAD4216 and TEAD4148 can also interact with the Sp1 sequences situated within the VEGF promoter (Figure S3, Methods S1). The TEAD4216 Isoform Competitively Inhibits VEGF Promoter Activity Cotransfection experiments were performed to test whether the TEAD4216 isoform competitively inhibited VEGF promoter- directed transcription in the presence of stimulating isoforms. Equal copy numbers of TEAD4216 expression plasmid and one enhancer isoform were co-transfected into 293T cells with a pSEAP reporter vector under the control of the human VEGF promoter. The TEAD4216 isoform was able to significantly inhibit VEGF promoter activity in the presence of stimulatory isoforms (Figure 4). A dose response trend was observed in experiments that included the most potent stimulatory isoform (TEAD4148) (Figure S4). To ensure that expression plasmids containing each of the TEAD4 isoforms were capable of expressing protein and not degraded within the time frame of the transfection assays, a western blot confirmed presence of TEAD4434, TEAD4216 and TEAD4447 (Figure S5). The TEAD4216 Isoform Represses VEGF Promoter Activity Under Hypoxic Conditions Under hypoxic conditions, HIF-1 subunits bind to the HRE within the VEGF promoter to enhance gene expression. To test whether the TEAD4216 isoform represses promoter activity in a low oxygen environment, 293T cells were co-transfected with the pSEAP vector containing the human VEGF promoter and an expression plasmid containing one of each of the TEAD4 isoforms. Transfected cells were then cultured under hypoxic conditions. The TEAD4216 isoform repressed expression in the hypoxic environment suggesting inhibitory effect in pathophysio- logically relevant conditions (Figure 5). The TEAD4216 Isoform Decreases Native VEGF Production and Inhibits Cell Proliferation The 165 amino acid isoform of VEGF (VEGF165) is considered a key protein in the development of neovascular disease and neoplasia. [17] To test whether expression of the TEAD4216 isoform inhibits native VEGF production, cells were transfected with expression plasmids containing the TEAD4216 gene, and secreted VEGF165 was determined 48 hours following transfection by ELISA. Relative to the control, the TEAD4216 treated cells produced less VEGF protein (Figure 6). This reduction in native VEGF expression was observed in both 293T (Figure 6A) and retinal pigment epithelial (RPE) cells (Figure 6B). Lentiviral- mediated expression of the TEAD4216 isoform within RPE cells similarly reduced VEGF production (Figure 6C). A proliferation assay was performed to determine whether lentiviral mediated expression of the TEAD4216 isoform inhibits cell proliferation. Cells transduced with either a lentiviral vector containing the TEAD4216 isoform and GFP or GFP alone were FAC sorted and seeded at equal density into 96 well plates. Cells were cultured for four days in the presence or absence of serum and subsequently assayed for cell number. Cells expressing the TEAD4216 isoform proliferated at reduced rates regardless of the presence of serum (Figure 7). PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e31260