The Related Transcriptional Enhancer Factor-1 Isoform, TEAD4216, Can Repress Vascular Endothelial Growth Factor Expression in Mammalian Cells Binoy Appukuttan*, Trevor J. McFarland, Andrew Stempel, Jean B. Kassem, Matthew Hartzell, Yi Zhang, Derek Bond, Kelsey West, Reid Wilson, Andrew Stout, Yuzhen Pan, Hoda Ilias, Kathryn Robertson, Michael L. Klein, David Wilson, Justine R. Smith, J. Timothy Stout Casey Eye Institute, Oregon Health and Science University, Portland, Oregon, United States of America Abstract Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4216, which represses VEGF promoter activity. The TEAD4216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4216 isoform can competitively repress the stimulatory activity of the TEAD4434 and TEAD4148 enhancers. Synthesis of the native VEGF165 protein and cellular proliferation is suppressed by the TEAD4216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases. Citation: Appukuttan B, McFarland TJ, Stempel A, Kassem JB, Hartzell M, et al. (2012) The Related Transcriptional Enhancer Factor-1 Isoform, TEAD4216, Can Repress Vascular Endothelial Growth Factor Expression in Mammalian Cells. PLoS ONE 7(6): e31260. doi:10.1371/journal.pone.0031260 Editor: Emanuele Buratti, International Centre for Genetic Engineering and Biotechnology, Italy Received August 4, 2011; Accepted January 4, 2012; Published June 22, 2012 Copyright: 2012 Appukuttan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this work was provided by: National Institutes of Health (R01 EY019042); Clayton Foundation for Research; Research to Prevent Blindness; Collins Medical Trust; and the American Health Assistance Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts: Authors BA, TJM and JTS have submitted a patent for the use of RTEF-1 variants as therapy for ocular neovascular disease. Title of patent is: ‘‘RTEF-1 variants and the use thereof for inhibition of angiogenesis.’’ Application number: 12/134,626 Publication number: US 2009/0117119 A1 Filing date: Jun 6, 2008 A link to the patent application is given below: http://www.google. com/patents?id=ZUXGAAAAEBAJ&printsec=frontcover&dq=appukuttan&hl=en&ei=24Q4ToxDzNaIAuOGvPIO&sa=X&oi=book_result&ct=result&resnum=3&sqi= 2&ved=0CCwQ6AEwAg. The authors state that this does not alter their adherence to all the PLoS ONE policies on sharing data and materials, as detailed in the guide to authors. In addition, the authors verify that the majority of the experiments/data presented in this manuscript was discovered prior to the filing of the patent and that no personal financial interest will be gained from the publication of the data presented in this manuscript. The authors have obtained funding from the National Institutes of Health to continue experiments on RTEF isoforms and verify that they are presenting the data in an unbiased fashion for peer review. All other authors declare that no competing interests exist. * E-mail: appukutt@ohsu.edu Introduction Alternate splicing to generate multiple mRNAs and subsequent protein products from one gene is exploited in many organisms, and in different tissues within the same organism, to augment the functional complexity of the translated genome. [1–3] Transcrip- tional enhancer factor 1 (TEF-1) is a member of the TEA DNA- binding family (TEAD), in which tissue and disease specific isoforms, generated by alternate splicing, have been observed. [4,5] The TEAD family of proteins is remarkably conserved from yeast to humans and, depending upon interaction with other proteins, can either activate or repress gene expression. [6] Four TEAD genes exist in mammals (TEAD 1 to 4) and expression of these genes has been characterized in various mammalian tissues and cell types.[7–9] Transcriptional enhancer factor 1-related (RTEF-1 or TEAD4) protein was originally reported to regulate muscle-specific genes in cardiac and smooth muscle cells. [8] The TEAD4 transcription factor requires the presence of myocyte- specific (M-CAT) sequences and muscle-specific cofactors to facilitate cell-specific gene regulation. Human and mouse TEAD4 transcripts exhibit dissimilar tissue specific expression profiles, where highest expression in humans is observed in pancreas and skeletal muscle with low levels in heart and kidney, whereas murine lung tissue has the most abundant message with low levels in heart and skeletal muscle. [7,10] The mouse embryo expresses high levels of TEAD4 message within skeletal muscle as well as an alternatively spliced isoform that lacks exon 5 relative to the full- length gene, indicating that within the mouse, TEAD4 is regulated developmentally and at least one isoform exists. [10] Alternative PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e31260