title: NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets.
abstract: We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of "off-targets", and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease.
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, Australia.
authors.forename: Romaric
authors.initials: R
authors.lastname: Bouveret
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, Australia.
authors.forename: Ashley J
authors.initials: AJ
authors.lastname: Waardenberg
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, Australia.
authors.forename: Nicole
authors.initials: N
authors.lastname: Schonrock
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, Australia.
authors.forename: Mirana
authors.initials: M
authors.lastname: Ramialison
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, Australia.
authors.forename: Tram
authors.initials: T
authors.lastname: Doan
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, Australia.
authors.forename: Danielle
authors.initials: D
authors.lastname: de Jong
authors.affiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, Brussels, Belgium.
authors.forename: Antoine
authors.initials: A
authors.lastname: Bondue
authors.affiliation: European Molecular Biology Laboratory, Australian Regenerative Medicine Institute, Monash University, Clayton, Australia.
authors.forename: Gurpreet
authors.initials: G
authors.lastname: Kaur
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, Australia.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Mohamed
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, Australia.
authors.forename: Hananeh
authors.initials: H
authors.lastname: Fonoudi
authors.affiliation: Department of Cardiovascular Medicine, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Chiann-Mun
authors.initials: CM
authors.lastname: Chen
authors.affiliation: Bioinformatics, Olivia Newton-John Cancer Research Institute, Melbourne, Australia.
authors.forename: Merridee A
authors.identifier: http://orcid.org/0000-0002-2121-912X
authors.initials: MA
authors.lastname: Wouters
authors.affiliation: Department of Cardiovascular Medicine, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Shoumo
authors.initials: S
authors.lastname: Bhattacharya
authors.affiliation: European Molecular Biology Laboratory, Australian Regenerative Medicine Institute, Monash University, Clayton, Australia.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Plachta
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, Australia.
authors.forename: Sally L
authors.initials: SL
authors.lastname: Dunwoodie
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, Australia.
authors.forename: Gavin
authors.initials: G
authors.lastname: Chapman
authors.affiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, Brussels, Belgium.
authors.forename: Cédric
authors.initials: C
authors.lastname: Blanpain
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, Australia.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Harvey
mesh_terms: D000818:Animals; D053263:Gene Regulatory Networks; D006331:Heart Diseases / Q000151:congenital* / Q000235:genetics*; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D018085:Regulon; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D050505:Mutant Proteins; C484469:Nkx2-5 protein, mouse; D014157:Transcription Factors
keywords: cardiogenesis; developmental biology; genetic diseases; mouse; stem cells; transcription factors