Lili Zhu1,2, Krishna Choudhary1, Barbara Gonzalez-Teran1,2, Yen-Sin Ang1,2, Reuben Thomas1, Nicole R Stone1,2, Lei Liu1,2, Ping Zhou1,2, Chenchen Zhu3,4, Hongmei Ruan5,6,7,8, Yu Huang1,2, Shibo Jin9, Angelo Pelonero1,2, Frances Koback1,2, Arun Padmanabhan1,2, Nandhini Sadagopan1,2, Austin Hsu1, Mauro W Costa1,2, Casey A Gifford1,2, Joke G van Bemmel1,2, Ruth Hüttenhain1, Vasanth Vedantham5,6, Bruce R Conklin1,2,5,7, Brian L Black6, Benoit G Bruneau1,2,6,10, Lars Steinmetz3,4,11, Nevan J Krogan1,7,8, Katherine S Pollard1,12,13, Deepak Srivastava1,2,10,14. 1. Gladstone Institutes, San Francisco, CA (L.Z., K.C., B.G-T., Y-S.A., R.T., N.R.S., L.L., P.Z., Y.H., A.P., F.K., A.P., N.S., A.H., M.W.C., C.A.G., J.G.v.B., R.H., B.R.C., B.G.B., N.J.K., K.S.P., D.S.). 2. Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, CA (L.Z., B.G-T., Y-S.A., N.R.S., L.L., P.Z., Y.H., A.P., F.K., A.P., N.S., M.W.C., C.A.G., J.G.v.B., B.R.C., B.G.B., D.S.). 3. Department of Genetics, Stanford University School of Medicine, Stanford, CA (C.Z., L.S.). 4. Stanford Genome Technology Center, Palo Alto, CA (C.Z., L.S.). 5. Department of Medicine (H.R., V.V., B.R.C.);, University of California, San Francisco. 6. Cardiovascular Research Institute (H.R., V.V., B.L.B., B.G.B.);, University of California, San Francisco. 7. Department of Cellular and Molecular Pharmacology (R.H., B.R.C., N.J.K.);, University of California, San Francisco. 8. Quantitative Biosciences Institute (R.H., N.J.K.);, University of California, San Francisco. 9. Division of Cellular and Developmental Biology, Molecular and Cell Biology Department, University of California at Berkeley (S.J.). 10. Department of Pediatrics (B.G.B., D.S.);, University of California, San Francisco. 11. European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany (L.S.). 12. Department of Epidemiology and Biostatistics, Institute for Computational Health Sciences and Institute for Human Genetics (K.S.P.);, University of California, San Francisco. 13. Chan Zuckerberg Biohub, San Francisco, CA (K.S.P). 14. Department of Biochemistry and Biophysics (D.S.), University of California, San Francisco.
Abstract
BACKGROUND: GATA4 (GATA-binding protein 4), a zinc finger-containing, DNA-binding transcription factor, is essential for normal cardiac development and homeostasis in mice and humans, and mutations in this gene have been reported in human heart defects. Defects in alternative splicing are associated with many heart diseases, yet relatively little is known about how cell type- or cell state-specific alternative splicing is achieved in the heart. Here, we show that GATA4 regulates cell type-specific splicing through direct interaction with RNA and the spliceosome in human induced pluripotent stem cell-derived cardiac progenitors. METHODS: We leveraged a combination of unbiased approaches including affinity purification of GATA4 and mass spectrometry, enhanced cross-linking with immunoprecipitation, electrophoretic mobility shift assays, in vitro splicing assays, and unbiased transcriptomic analysis to uncover GATA4's novel function as a splicing regulator in human induced pluripotent stem cell-derived cardiac progenitors. RESULTS: We found that GATA4 interacts with many members of the spliceosome complex in human induced pluripotent stem cell-derived cardiac progenitors. Enhanced cross-linking with immunoprecipitation demonstrated that GATA4 also directly binds to a large number of mRNAs through defined RNA motifs in a sequence-specific manner. In vitro splicing assays indicated that GATA4 regulates alternative splicing through direct RNA binding, resulting in functionally distinct protein products. Correspondingly, knockdown of GATA4 in human induced pluripotent stem cell-derived cardiac progenitors resulted in differential alternative splicing of genes involved in cytoskeleton organization and calcium ion import, with functional consequences associated with the protein isoforms. CONCLUSIONS: This study shows that in addition to its well described transcriptional function, GATA4 interacts with members of the spliceosome complex and regulates cell type-specific alternative splicing via sequence-specific interactions with RNA. Several genes that have splicing regulated by GATA4 have functional consequences and many are associated with dilated cardiomyopathy, suggesting a novel role for GATA4 in achieving the necessary cardiac proteome in normal and stress-responsive conditions.
BACKGROUND: GATA4 (GATA-binding protein 4), a zinc finger-containing, DNA-binding transcription factor, is essential for normal cardiac development and homeostasis in mice and humans, and mutations in this gene have been reported in human heart defects. Defects in alternative splicing are associated with many heart diseases, yet relatively little is known about how cell type- or cell state-specific alternative splicing is achieved in the heart. Here, we show that GATA4 regulates cell type-specific splicing through direct interaction with RNA and the spliceosome in human induced pluripotent stem cell-derived cardiac progenitors. METHODS: We leveraged a combination of unbiased approaches including affinity purification of GATA4 and mass spectrometry, enhanced cross-linking with immunoprecipitation, electrophoretic mobility shift assays, in vitro splicing assays, and unbiased transcriptomic analysis to uncover GATA4's novel function as a splicing regulator in human induced pluripotent stem cell-derived cardiac progenitors. RESULTS: We found that GATA4 interacts with many members of the spliceosome complex in human induced pluripotent stem cell-derived cardiac progenitors. Enhanced cross-linking with immunoprecipitation demonstrated that GATA4 also directly binds to a large number of mRNAs through defined RNA motifs in a sequence-specific manner. In vitro splicing assays indicated that GATA4 regulates alternative splicing through direct RNA binding, resulting in functionally distinct protein products. Correspondingly, knockdown of GATA4 in human induced pluripotent stem cell-derived cardiac progenitors resulted in differential alternative splicing of genes involved in cytoskeleton organization and calcium ion import, with functional consequences associated with the protein isoforms. CONCLUSIONS: This study shows that in addition to its well described transcriptional function, GATA4 interacts with members of the spliceosome complex and regulates cell type-specific alternative splicing via sequence-specific interactions with RNA. Several genes that have splicing regulated by GATA4 have functional consequences and many are associated with dilated cardiomyopathy, suggesting a novel role for GATA4 in achieving the necessary cardiac proteome in normal and stress-responsive conditions.
Authors: Zhenyu Hu; Jiong-Wei Wang; Dejie Yu; Jia Lin Soon; Dominique P V de Kleijn; Roger Foo; Ping Liao; Henry M Colecraft; Tuck Wah Soong Journal: Sci Rep Date: 2016-10-12 Impact factor: 4.379
Authors: Sunil K Verma; Vaibhav Deshmukh; Curtis A Nutter; Elizabeth Jaworski; Wenhao Jin; Lalita Wadhwa; Joshua Abata; Marco Ricci; Joy Lincoln; James F Martin; Gene W Yeo; Muge N Kuyumcu-Martinez Journal: Sci Rep Date: 2016-08-03 Impact factor: 4.379
Authors: Matthias Heinig; Michiel E Adriaens; Sebastian Schafer; Hanneke W M van Deutekom; Elisabeth M Lodder; James S Ware; Valentin Schneider; Leanne E Felkin; Esther E Creemers; Benjamin Meder; Hugo A Katus; Frank Rühle; Monika Stoll; François Cambien; Eric Villard; Philippe Charron; Andras Varro; Nanette H Bishopric; Alfred L George; Cristobal Dos Remedios; Aida Moreno-Moral; Francesco Pesce; Anja Bauerfeind; Franz Rüschendorf; Carola Rintisch; Enrico Petretto; Paul J Barton; Stuart A Cook; Yigal M Pinto; Connie R Bezzina; Norbert Hubner Journal: Genome Biol Date: 2017-09-14 Impact factor: 13.583