9p21.3 Coronary Artery Disease Risk Variants Disrupt TEAD Transcription Factor-Dependent Transforming Growth Factor β Regulation of p16 Expression in Human Aortic Smooth Muscle Cells.
BACKGROUND
The mechanism whereby the 9p21.3 locus confers risk for coronary artery disease remains incompletely understood. Risk alleles are associated with reduced expression of the cell cycle suppressor genes CDKN2A (p16 and p14) and CDKN2B (p15) and increased vascular smooth muscle cell proliferation. We asked whether risk alleles disrupt transcription factor binding to account for this effect.


RESULTS
A bioinformatic screen was used to predict which of 59 single nucleotide polymorphisms at the 9p21.3 locus disrupt (or create) transcription factor binding sites. Electrophoretic mobility shift and luciferase reporter assays examined the binding and functionality of the predicted regulatory sequences. Primary human aortic smooth muscle cells (HAoSMCs) were genotyped for 9p21.3, and HAoSMCs homozygous for the risk allele showed reduced p15 and p16 levels and increased proliferation. rs10811656 and rs4977757 disrupted functional TEF-1 TEC1 AbaA domain (TEAD) transcription factor binding sites. TEAD3 and TEAD4 overexpression induced p16 in HAoSMCs homozygous for the nonrisk allele, but not for the risk allele. Transforming growth factor β, known to activate p16 and also to interact with TEAD factors, failed to induce p16 or to inhibit proliferation of HAoSMCs homozygous for the risk allele. Knockdown of TEAD3 blocked transforming growth factor β-induced p16 mRNA and protein expression, and dual knockdown of TEAD3 and TEAD4 markedly reduced p16 expression in heterozygous HAoSMCs.


CONCLUSIONS
Here, we identify a novel mechanism whereby sequences at the 9p21.3 risk locus disrupt TEAD factor binding and TEAD3-dependent transforming growth factor β induction of p16 in HAoSMCs. This mechanism accounts, in part, for the 9p21.3 coronary artery disease risk.
From Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ontario, Canada (N.A.M.A., D.A., R.O.V., S.X.Z., K.N.H., A.F.R.S.); Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ontario, Canada (N.A.M.A., D.A., R.O.V., A.F.R.S.); Center for Genetics and Inherited Diseases, Taibah University, Almadina, Saudi Arabia (N.A.M.A.); Ottawa Hospital Research Institute, Ontario, Canada (X.Z.); and Department of Medicine, University of Ottawa, Ontario, Canada (H.-H.C., A.F.R.S.).
Naif A M
NA
Almontashiri
From Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ontario, Canada (N.A.M.A., D.A., R.O.V., S.X.Z., K.N.H., A.F.R.S.); Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ontario, Canada (N.A.M.A., D.A., R.O.V., A.F.R.S.); Center for Genetics and Inherited Diseases, Taibah University, Almadina, Saudi Arabia (N.A.M.A.); Ottawa Hospital Research Institute, Ontario, Canada (X.Z.); and Department of Medicine, University of Ottawa, Ontario, Canada (H.-H.C., A.F.R.S.).
Darlène
D
Antoine
From Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ontario, Canada (N.A.M.A., D.A., R.O.V., S.X.Z., K.N.H., A.F.R.S.); Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ontario, Canada (N.A.M.A., D.A., R.O.V., A.F.R.S.); Center for Genetics and Inherited Diseases, Taibah University, Almadina, Saudi Arabia (N.A.M.A.); Ottawa Hospital Research Institute, Ontario, Canada (X.Z.); and Department of Medicine, University of Ottawa, Ontario, Canada (H.-H.C., A.F.R.S.).
Xun
X
Zhou
From Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ontario, Canada (N.A.M.A., D.A., R.O.V., S.X.Z., K.N.H., A.F.R.S.); Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ontario, Canada (N.A.M.A., D.A., R.O.V., A.F.R.S.); Center for Genetics and Inherited Diseases, Taibah University, Almadina, Saudi Arabia (N.A.M.A.); Ottawa Hospital Research Institute, Ontario, Canada (X.Z.); and Department of Medicine, University of Ottawa, Ontario, Canada (H.-H.C., A.F.R.S.).
Ragnar O
RO
Vilmundarson
From Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ontario, Canada (N.A.M.A., D.A., R.O.V., S.X.Z., K.N.H., A.F.R.S.); Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ontario, Canada (N.A.M.A., D.A., R.O.V., A.F.R.S.); Center for Genetics and Inherited Diseases, Taibah University, Almadina, Saudi Arabia (N.A.M.A.); Ottawa Hospital Research Institute, Ontario, Canada (X.Z.); and Department of Medicine, University of Ottawa, Ontario, Canada (H.-H.C., A.F.R.S.).
Sean X
SX
Zhang
From Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ontario, Canada (N.A.M.A., D.A., R.O.V., S.X.Z., K.N.H., A.F.R.S.); Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ontario, Canada (N.A.M.A., D.A., R.O.V., A.F.R.S.); Center for Genetics and Inherited Diseases, Taibah University, Almadina, Saudi Arabia (N.A.M.A.); Ottawa Hospital Research Institute, Ontario, Canada (X.Z.); and Department of Medicine, University of Ottawa, Ontario, Canada (H.-H.C., A.F.R.S.).
Kennedy N
KN
Hao
From Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ontario, Canada (N.A.M.A., D.A., R.O.V., S.X.Z., K.N.H., A.F.R.S.); Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ontario, Canada (N.A.M.A., D.A., R.O.V., A.F.R.S.); Center for Genetics and Inherited Diseases, Taibah University, Almadina, Saudi Arabia (N.A.M.A.); Ottawa Hospital Research Institute, Ontario, Canada (X.Z.); and Department of Medicine, University of Ottawa, Ontario, Canada (H.-H.C., A.F.R.S.). astewart@ottawaheart.ca hchen@uottawa.ca.
Hsiao-Huei
HH
Chen
From Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ontario, Canada (N.A.M.A., D.A., R.O.V., S.X.Z., K.N.H., A.F.R.S.); Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ontario, Canada (N.A.M.A., D.A., R.O.V., A.F.R.S.); Center for Genetics and Inherited Diseases, Taibah University, Almadina, Saudi Arabia (N.A.M.A.); Ottawa Hospital Research Institute, Ontario, Canada (X.Z.); and Department of Medicine, University of Ottawa, Ontario, Canada (H.-H.C., A.F.R.S.). astewart@ottawaheart.ca hchen@uottawa.ca.
Alexandre F R
AF
Stewart
D000293:Adolescent; D000328:Adult; D000483:Alleles; D001011:Aorta / Q000166:cytology; D002478:Cells, Cultured; D002899:Chromosomes, Human, Pair 9 / Q000235:genetics*; D003327:Coronary Disease / Q000235:genetics*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D005260:Female; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D019942:Genes, p16; D006801:Humans; D008297:Male; D008875:Middle Aged; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D020641:Polymorphism, Single Nucleotide; D011994:Recombinant Proteins / Q000378:metabolism; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000502:physiology*; D055815:Young Adult
D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; D009124:Muscle Proteins; D011994:Recombinant Proteins; D000091262:TEA Domain Transcription Factors; C106169:TEAD3 protein, human; C486309:TEAD4 protein, human; D014157:Transcription Factors; D016212:Transforming Growth Factor beta
atherosclerosis; coronary disease; genetics; molecular biology; smooth muscle cells