Tbx20 regulates a genetic program essential to adult mouse cardiomyocyte function.

Human mutations in or variants of TBX20 are associated with congenital heart disease, cardiomyopathy, and arrhythmias. To investigate whether cardiac disease in patients with these conditions results from an embryonic or ongoing requirement for Tbx20 in myocardium, we ablated Tbx20 specifically in adult cardiomyocytes in mice. This ablation resulted in the onset of severe cardiomyopathy accompanied by arrhythmias, with death ensuing within 1 to 2 weeks of Tbx20 ablation. Accounting for this dramatic phenotype, we identified molecular signatures that posit Tbx20 as a central integrator of a genetic program that maintains cardiomyocyte function in the adult heart. Expression of a number of genes encoding critical transcription factors, ion channels, and cytoskeletal/myofibrillar proteins was downregulated consequent to loss of Tbx20. Genome-wide ChIP analysis of Tbx20-binding regions in the adult heart revealed that many of these genes were direct downstream targets of Tbx20 and uncovered a previously undescribed DNA-binding site for Tbx20. Bioinformatics and in vivo functional analyses revealed a cohort of transcription factors that, working with Tbx20, integrated multiple environmental signals to maintain ion channel gene expression in the adult heart. Our data provide insight into the mechanisms by which mutations in TBX20 cause adult heart disease in humans.

[1]  E R Behr,et al.  Inherited Cardiomyopathies , 2012, BMJ.

[2]  V. Giguère,et al.  Functional and physiological genomics of estrogen-related receptors (ERRs) in health and disease. , 2011, Biochimica et biophysica acta.

[3]  W. Miller,et al.  Genome-wide identification of conserved regulatory function in diverged sequences. , 2011, Genome research.

[4]  Randy L. Johnson,et al.  Hippo Pathway Inhibits Wnt Signaling to Restrain Cardiomyocyte Proliferation and Heart Size , 2011, Science.

[5]  Aibin He,et al.  Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in heart , 2011, Proceedings of the National Academy of Sciences.

[6]  Jingyuan Fu,et al.  Common variants in 22 loci are associated with QRS duration and cardiac ventricular conduction , 2010, Nature Genetics.

[7]  N. Lakdawala,et al.  Dilated cardiomyopathy with conduction disease and arrhythmia. , 2010, Circulation.

[8]  M. Gollob,et al.  The genetic and clinical features of cardiac channelopathies. , 2010, Future cardiology.

[9]  Elizabeth M. Mandel,et al.  The BMP pathway acts to directly regulate Tbx20 in the developing heart , 2010, Development.

[10]  A. Visel,et al.  Homotypic clusters of transcription factor binding sites are a key component of human promoters and enhancers. , 2010, Genome research.

[11]  V. Garg,et al.  Genetics of Congenital Heart Disease , 2010, Current cardiology reviews.

[12]  John M Westlund,et al.  Genome-wide discovery of human heart enhancers. , 2010, Genome research.

[13]  Christian Gieger,et al.  Genome-wide association study of PR interval , 2010, Nature Genetics.

[14]  A. Rauch,et al.  Comprehensive genotype–phenotype analysis in 230 patients with tetralogy of Fallot , 2009, Journal of Medical Genetics.

[15]  A. Mégarbané,et al.  A gain-of-function TBX20 mutation causes congenital atrial septal defects, patent foramen ovale and cardiac valve defects , 2009, Journal of Medical Genetics.

[16]  Mauro W. Costa,et al.  Conformational stability and DNA binding specificity of the cardiac T-box transcription factor Tbx20. , 2009, Journal of molecular biology.

[17]  Ju Chen,et al.  Cardiac-specific ablation of Cypher leads to a severe form of dilated cardiomyopathy with premature death. , 2009, Human molecular genetics.

[18]  A. Visel,et al.  ChIP-seq accurately predicts tissue-specific activity of enhancers , 2009, Nature.

[19]  Ivan Ovcharenko,et al.  Variable locus length in the human genome leads to ascertainment bias in functional inference for non-coding elements , 2009, Bioinform..

[20]  K. Ocorr,et al.  Transcription factor neuromancer/TBX20 is required for cardiac function in Drosophila with implications for human heart disease , 2008, Proceedings of the National Academy of Sciences.

[21]  Xiaofeng Li,et al.  T-box transcription factor TBX20 mutations in Chinese patients with congenital heart disease. , 2008, European journal of medical genetics.

[22]  Daniel E. Newburger,et al.  UniPROBE: an online database of protein binding microarray data on protein–DNA interactions , 2008, Nucleic Acids Res..

[23]  S. Batzoglou,et al.  Genome-Wide Analysis of Transcription Factor Binding Sites Based on ChIP-Seq Data , 2008, Nature Methods.

[24]  Jun Hyoung Lee,et al.  Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli , 2008, Nucleic acids research.

[25]  R. Hetzer,et al.  Characterization of TBX20 in human hearts and its regulation by TFAP2 , 2008, Journal of cellular biochemistry.

[26]  H. Kiyonari,et al.  Redundant Roles of Tead1 and Tead2 in Notochord Development and the Regulation of Cell Proliferation and Survival , 2008, Molecular and Cellular Biology.

[27]  E. Olson,et al.  MEF2: a central regulator of diverse developmental programs , 2007, Development.

[28]  Ole Winther,et al.  JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update , 2007, Nucleic Acids Res..

[29]  Michael J Ackerman,et al.  Inherited Arrhythmias: A National Heart, Lung, and Blood Institute and Office of Rare Diseases Workshop Consensus Report About the Diagnosis, Phenotyping, Molecular Mechanisms, and Therapeutic Approaches for Primary Cardiomyopathies of Gene Mutations Affecting Ion Channel Function , 2007, Circulation.

[30]  J. Seidman,et al.  Corrigendum to “Tbx5-dependent rheostatic control of cardiac gene expression and morphogenesis” [Dev. Biol. 297 (2006) 566–586] , 2007 .

[31]  Mauro W. Costa,et al.  Mutations in cardiac T-box factor gene TBX20 are associated with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy. , 2007, American journal of human genetics.

[32]  Brian J. Wilson,et al.  Genome-wide orchestration of cardiac functions by the orphan nuclear receptors ERRalpha and gamma. , 2007, Cell metabolism.

[33]  Panayiotis V. Benos,et al.  STAMP: a web tool for exploring DNA-binding motif similarities , 2007, Nucleic Acids Res..

[34]  K. Devriendt,et al.  A novel CSX/NKX2-5 mutation causes autosomal-dominant AV block: are atrial fibrillation and syncopes part of the phenotype? , 2006, European Journal of Human Genetics.

[35]  S. Fisher,et al.  Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish , 2006, Nature Protocols.

[36]  S. Ikeda,et al.  Gata4 is required for maintenance of postnatal cardiac function and protection from pressure overload-induced heart failure , 2006, Proceedings of the National Academy of Sciences.

[37]  Kazuko Koshiba-Takeuchi,et al.  Tbx5-dependent rheostatic control of cardiac gene expression and morphogenesis. , 2006, Developmental biology.

[38]  Linh Vong,et al.  MEF2C is required for the normal allocation of cells between the ventricular and sinoatrial precursors of the primary heart field , 2006, Developmental dynamics : an official publication of the American Association of Anatomists.

[39]  S. Fisher,et al.  Conservation of RET Regulatory Function from Human to Zebrafish Without Sequence Similarity , 2006, Science.

[40]  V. Stoppioni,et al.  Missense mutation in the transcription factor NKX2-5: a novel molecular event in the pathogenesis of thyroid dysgenesis. , 2006, The Journal of clinical endocrinology and metabolism.

[41]  R. Schwartz,et al.  Cardiac-Specific Deletion of Gata4 Reveals Its Requirement for Hypertrophy, Compensation, and Myocyte Viability , 2006, Circulation research.

[42]  S. Priori,et al.  Role of Genetic Analyses in Cardiology: Part I: Mendelian Diseases: Cardiac Channelopathies , 2006, Circulation.

[43]  J. Nerbonne,et al.  Targeted Deletion of Kv4.2 Eliminates Ito,f and Results in Electrical and Molecular Remodeling, With No Evidence of Ventricular Hypertrophy or Myocardial Dysfunction , 2005, Circulation research.

[44]  D. Haussler,et al.  Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. , 2005, Genome research.

[45]  J. Borlak,et al.  Functional dissection of sequence-specific NKX2-5 DNA binding domain mutations associated with human heart septation defects using a yeast-based system. , 2005, Human molecular genetics.

[46]  A. Kispert,et al.  Tbx20 is essential for cardiac chamber differentiation and repression of Tbx2 , 2005, Development.

[47]  W. Pu,et al.  Morphogenesis of the right ventricle requires myocardial expression of Gata4. , 2005, The Journal of clinical investigation.

[48]  B. Bruneau,et al.  Tbx20 dose-dependently regulates transcription factor networks required for mouse heart and motoneuron development , 2005, Development.

[49]  Milena B. Furtado,et al.  Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation , 2005, Development.

[50]  M. Rosenfeld,et al.  T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis , 2005, Development.

[51]  M. Furutani,et al.  Phenotypes with GATA4 or NKX2.5 mutations in familial atrial septal defect , 2005, American journal of medical genetics. Part A.

[52]  F. Conlon,et al.  Tbx5 and Tbx20 act synergistically to control vertebrate heart morphogenesis , 2005, Development.

[53]  J. Borlak,et al.  Somatic NKX2-5 mutations as a novel mechanism of disease in complex congenital heart disease , 2004, Journal of Medical Genetics.

[54]  K. Kawakami,et al.  A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. , 2004, Developmental cell.

[55]  W. Giles,et al.  Nkx2-5 Pathways and Congenital Heart Disease Loss of Ventricular Myocyte Lineage Specification Leads to Progressive Cardiomyopathy and Complete Heart Block , 2004, Cell.

[56]  Z. Weng,et al.  Detection of functional DNA motifs via statistical over-representation. , 2004, Nucleic acids research.

[57]  Mauro W. Costa,et al.  Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the developing heart. , 2003, Developmental biology.

[58]  Jonathan C. Cohen,et al.  GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5 , 2003, Nature.

[59]  B. De Moor,et al.  Toucan: deciphering the cis-regulatory logic of coregulated genes. , 2003, Nucleic acids research.

[60]  K. Wickman,et al.  Contribution of the Kir3.1 Subunit to the Muscarinic-gated Atrial Potassium Channel IKACh * , 2002, The Journal of Biological Chemistry.

[61]  B. Black,et al.  Mitochondrial deficiency and cardiac sudden death in mice lacking the MEF2A transcription factor , 2002, Nature Medicine.

[62]  Tom H. Pringle,et al.  The human genome browser at UCSC. , 2002, Genome research.

[63]  E. Goldmuntz,et al.  NKX2.5 mutations in patients with tetralogy of fallot. , 2002 .

[64]  J. Ross,et al.  A Defect in the Kv Channel-Interacting Protein 2 (KChIP2) Gene Leads to a Complete Loss of I to and Confers Susceptibility to Ventricular Tachycardia , 2001, Cell.

[65]  Qiang Zhou,et al.  Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy , 2001, The Journal of cell biology.

[66]  J. Schmitt,et al.  A Murine Model of Holt-Oram Syndrome Defines Roles of the T-Box Transcription Factor Tbx5 in Cardiogenesis and Disease , 2001, Cell.

[67]  G. Fishman,et al.  Heterogeneous Expression of Gap Junction Channels in the Heart Leads to Conduction Defects and Ventricular Dysfunction , 2001, Circulation.

[68]  M. Crackower,et al.  Temporally Regulated and Tissue-Specific Gene Manipulations in the Adult and Embryonic Heart Using a Tamoxifen-Inducible Cre Protein , 2001, Circulation research.

[69]  T. Schwarz,et al.  The consequences of disrupting cardiac inwardly rectifying K+ current (IK1) as revealed by the targeted deletion of the murine Kir2.1 and Kir2.2 genes , 2001, The Journal of physiology.

[70]  Michael D. Schneider,et al.  Conduction Slowing and Sudden Arrhythmic Death in Mice With Cardiac-Restricted Inactivation of Connexin43 , 2001, Circulation research.

[71]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[72]  J. Seidman,et al.  Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways. , 1999, The Journal of clinical investigation.

[73]  T. Doetschman,et al.  Impaired Cardiac Performance in Heterozygous Mice with a Null Mutation in the Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2 (SERCA2) Gene* , 1999, The Journal of Biological Chemistry.

[74]  J. Nerbonne,et al.  Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit. , 1998, Circulation research.

[75]  U. Frey,et al.  Deficits in memory tasks of mice with CREB mutations depend on gene dosage. , 1998, Learning & memory.

[76]  J. Seidman,et al.  Congenital heart disease caused by mutations in the transcription factor NKX2-5. , 1998, Science.

[77]  M. Iino,et al.  Embryonic lethality and abnormal cardiac myocytes in mice lacking ryanodine receptor type 2 , 1998, The EMBO journal.

[78]  P. Gass,et al.  Impaired fetal T cell development and perinatal lethality in mice lacking the cAMP response element binding protein. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[79]  C. Bucana,et al.  Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. , 1997, Science.

[80]  L A Herzenberg,et al.  Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[81]  Philippe Soriano,et al.  Transcriptional enhancer factor 1 disruption by a retroviral gene trap leads to heart defects and embryonic lethality in mice. , 1994, Genes & development.

[82]  Wolfgang Schmid,et al.  Targeted mutation of the CREB gene: compensation within the CREB/ATF family of transcription factors. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[83]  H. Watkins,et al.  Inherited cardiomyopathies. , 2011, The New England journal of medicine.

[84]  Af Smit,et al.  RepeatMasker software program (computer program), ver. 3.1.8. Seattle: Institute for Systems Biology. , 2007 .

[85]  E. Wingender,et al.  TRANSFAC®: transcriptional regulation, from patterns to profiles , 2003, Nucleic Acids Res..

[86]  Alex E. Lash,et al.  Gene Expression Omnibus: NCBI gene expression and hybridization array data repository , 2002, Nucleic Acids Res..

[87]  E. Goldmuntz,et al.  NKX 2 . 5 Mutations in Patients With Tetralogy of Fallot , 2001 .

[88]  N. Klugbauer,et al.  Functional embryonic cardiomyocytes after disruption of the L-type alpha1C (Cav1.2) calcium channel gene in the mouse. , 2000, The Journal of biological chemistry.

[89]  J. Seidman,et al.  Mutations in human TBX5 [corrected] cause limb and cardiac malformation in Holt-Oram syndrome. , 1997, Nature genetics.

[90]  David I. Wilson,et al.  Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family , 1997, Nature Genetics.

[91]  R. Kucherlapati,et al.  Mutations in human cause limb and cardiac malformation in Holt-Oram syndrome , 1997, Nature Genetics.

[92]  J. Seidman,et al.  Erratum: Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome , 1997, Nature Genetics.

[93]  Charles Elkan,et al.  Fitting a Mixture Model By Expectation Maximization To Discover Motifs In Biopolymer , 1994, ISMB.