The TEAD4216 Isoform Inhibits VEGF Expression Figure 3. The TEAD4216 isoform does not require the HRE sequence to function. The TEAD4216 isoform can repress expression from the human VEGF promoter (F2–R3) that lacks the HRE (p,0.01, n = 6). The TEAD4148 and TEAD4311 enchancer isoforms do not require the HRE to promote reporter gene expression (p,0.001). However the full length TEAD4434 did not significantly enhance expression (p.0.02). doi:10.1371/journal.pone.0031260.g003 The TEAD4216 Transcription Factor is Localized to the Cytoplasmic Region The TEAD4216 isoform lacks the putative nuclear localization sequence (NLS) situated at the end of the TEA DNA-binding domain (TEAD) (Figure 1). Enhancer isoforms contain the complete TEAD and are assumed to function within the nucleus by binding to cis-elements within chromosomal DNA. In the case of the VEGF promoter, the full-length TEAD4434 transcription factor binds to Sp1 elements situated within 100 bp upstream of the transcriptional start site. [12] To determine the cellular localization of the TEAD4148, TEAD4216 and TEAD4434 proteins, fusion constructs of each isoform linked to fluorescent tags were prepared and expressed within mammalian cells. Fluorescent imaging demonstrated that the enhancer isoforms were located within the nucleus whereas the TEAD4216 isoform was present in the cytoplasm (Figure 8). It would appear that the NLS is essential for nuclear positioning of TEAD4 isoforms and may determine whether the protein acts as an enhancer or repressor. The TEAD4216 Gains Enhancer Activity with a Nuclear Localization Sequence In order to dissect which domains within the TEAD4216 isoform affords repressor activity, mutant and chimeric constructs were generated (Methods S1) and their effect on the human VEGF promoter was analyzed (Figure S6). The repressor TEAD4216 containing the complete DNA binding domain including the NLS sequence (TEAD4216+NLS) enhanced promoter activity whereas the TEAD4148 without a NLS motif (TEAD4148-NLS) lost the ability to enhance. A chimeric containing the DNA binding Figure 4. The TEAD4216 isoform can competitively inhibit promoter activity. The TEAD4216 isoform can competitively repress expression from the human VEGF promoter in the presence of either the TEAD4434 or TEAD4148 enhancer isoforms (p,0.005, n = 6). doi:10.1371/journal.pone.0031260.g004 Figure 5. The TEAD4216 isoform can inhibit promoter activity under hypoxic conditions. The TEAD4216 isoform can still repress expression mediated from the human VEGF promoter (F1–R3) that includes the HRE sequence under conditions of hypoxia (p,0.001, n = 6). doi:10.1371/journal.pone.0031260.g005 PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e31260