Purchase this article with an account.
Trevor J. Mcfarland, Matthew Hartzell, Andrew Stempel, Yuzhen Pan, Justine Smith, John T. Stout, Binoy Appukuttan; Ocular Derived Tead4 Isoforms Differentially Bind Yap1. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1615.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The TEAD family (TEAD 1-4) are cell specific transcription factors that require essential cofactors such as Yes-associated protein (YAP1) to mediate gene expression. YAP1 is involved in various cellular pathways, including proliferation, apoptosis and organ growth. The activity of full-length TEAD4 (1305) is dependent on binding to YAP1. We have previously identified human ocular derived isoforms of TEAD4 (651 and 447) that can either enhance or repress VEGF-A promoter activity. Whether TEAD4 isoform mediated gene regulation within ocular cells requires YAP1 and/or other cell-specific cofactors is unknown. In order to test whether these new isoforms of TEAD4 bind YAP1, computer modeling and ocular cell derived protein pull-down assays were employed.
To test for the presence of TEAD4 and YAP1, RT-PCR was performed on various ocular derived cells, including Muller (MIO-M1), RPE-19 and vascular endothelial cells derived from primary human iris, choroid and retina. Prediction of protein-protein binding efficiencies was obtained with computer modeling methods (CN3D NCBI) using the 3D crystal structure of the human YAP1 and TEAD1 Complex (TEAD1 and TEAD4 share 89% identity). Expression vectors containing the GFP gene fused to 1305, 651 or 447 TEAD4 isoforms were cotransfected with a YAP1 expression vector into 293T cells. Cells were cultured for 36 hours, washed in ice cold PBS and lysed using a non-denaturing buffer. Pull-down assays were performed using the GFP-Trap Kit (Allele Biotechnology). Western blots were probed with antibodies for both TEAD4 and YAP1 using samples obtained from input, non-bound and bound pull-down fractions.
All cell lines were RT-PCR positive for TEAD4 and YAP1, with the exception of MIO-M1, which was positive for TEAD4 only. An additional isoform of YAP was found in all endothelial cell types. 3D crystal structure analysis revealed that the 1305 TEAD4 possesses 100% of the YAP1 binding domain, while the 651 and 447 isoforms possess 57% and 5% of the binding domain, respectively. Protein pull-down assays indicate that the 1305 and 651 bind to YAP1. It appeared that the 447 isoform was incapable of binding YAP1 in this study.
Differential binding of TEAD4 isoforms to co-factors such as YAP1 may explain the disparity previously observed between isoforms and gene promoter activity. How the 447 isoform is able to enhance VEGF promoter activity several fold higher then the other TEAD4 isoforms without binding YAP1, a key cofactor, is intriguing. The 447 isoform may require other ocular cell-specific cofactors to function.
This PDF is available to Subscribers Only