1. Developmental Biology
Download icon

NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets

  1. Romaric Bouveret  Is a corresponding author
  2. Ashley J Waardenberg
  3. Nicole Schonrock
  4. Mirana Ramialison
  5. Tram Doan
  6. Danielle de Jong
  7. Antoine Bondue
  8. Gurpreet Kaur
  9. Stephanie Mohamed
  10. Hananeh Fonoudi
  11. Chiann-mun Chen
  12. Merridee Wouters
  13. Shoumo Bhattacharya
  14. Nicolas Plachta
  15. Sally L Dunwoodie
  16. Gavin Chapman
  17. Cédric Blanpain
  18. Richard P Harvey
  1. Victor Chang Cardiac Research Institute, Australia
  2. Université Libre de Bruxelles, Belgium
  3. Monash University, Australia
  4. University of Oxford, United Kingdom
Research Article
  • Cited 36
  • Views 3,062
  • Annotations
Cite this article as: eLife 2015;4:e06942 doi: 10.7554/eLife.06942

Abstract

To model cardiac gene regulatory networks in health and disease we used DamID to establish robust target gene sets for the cardiac homeodomain factor NKX2-5 and two congenital heart disease-associated mutants carrying a crippled homeodomain, which normally functions as DNA- and protein-binding interface. Despite compromised direct DNA-binding, NKX2-5 mutants retained partial functionality and bound hundreds of targets, including NKX2-5 wild type targets and unique sets of 'off-targets'. NKX2-5∆HD, which lacks the entire homeodomain, could still dimerise with wild type NKX2-5 and its cofactors, including newly-discovered cofactors of the ETS family, through the conserved tyrosine-rich domain (YRD). NKX2-5∆HD off-targets showed overrepresentation of many binding motifs, including ETS motifs, the majority co-occupied by ETS proteins as determined by DamID. Off-targets of an NKX2-5 YRD mutant were not enriched in ETS targets. Our study reveals off-target binding and transcriptional activity for NKX2-5 mutations driven in part by cofactor interactions, suggesting a novel type of gain-of-function in congenital heart disease.

Article and author information

Author details

  1. Romaric Bouveret

    Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    For correspondence
    r.bouveret@victorchang.edu.au
    Competing interests
    The authors declare that no competing interests exist.
  2. Ashley J Waardenberg

    Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.
  3. Nicole Schonrock

    Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.
  4. Mirana Ramialison

    Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.
  5. Tram Doan

    Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.
  6. Danielle de Jong

    Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.
  7. Antoine Bondue

    Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, Brussels, Belgium
    Competing interests
    The authors declare that no competing interests exist.
  8. Gurpreet Kaur

    European Molecular Biology Laboratory, Australian Regenerative Medicine Institute, Monash University, Clayton, Australia
    Competing interests
    The authors declare that no competing interests exist.
  9. Stephanie Mohamed

    Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.
  10. Hananeh Fonoudi

    Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.
  11. Chiann-mun Chen

    Department of Cardiovascular Medicine, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  12. Merridee Wouters

    Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.
  13. Shoumo Bhattacharya

    Department of Cardiovascular Medicine, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  14. Nicolas Plachta

    European Molecular Biology Laboratory, Australian Regenerative Medicine Institute, Monash University, Clayton, Australia
    Competing interests
    The authors declare that no competing interests exist.
  15. Sally L Dunwoodie

    Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.
  16. Gavin Chapman

    Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.
  17. Cédric Blanpain

    Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, Brussels, Belgium
    Competing interests
    The authors declare that no competing interests exist.
  18. Richard P Harvey

    Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.

Ethics

Animal experimentation: Animal experimentation was performed with approval of the Garvan Institute/St Vincent's Hospital Animal Ethics Committee (Project numbers 10/19 and 10/01).

Reviewing Editor

  1. Margaret Buckingham, Institut Pasteur, France

Publication history

  1. Received: February 10, 2015
  2. Accepted: July 5, 2015
  3. Accepted Manuscript published: July 6, 2015 (version 1)
  4. Version of Record published: August 25, 2015 (version 2)

Copyright

© 2015, Bouveret et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 3,062
    Page views
  • 645
    Downloads
  • 36
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

  1. Further reading

Further reading

    1. Developmental Biology
    2. Evolutionary Biology
    Periklis Paganos et al.
    Research Article

    Identifying the molecular fingerprint of organismal cell types is key for understanding their function and evolution. Here, we use single cell RNA sequencing (scRNA-seq) to survey the cell types of the sea urchin early pluteus larva, representing an important developmental transition from non-feeding to feeding larva. We identify 21 distinct cell clusters, representing cells of the digestive, skeletal, immune, and nervous systems. Further subclustering of these reveal a highly detailed portrait of cell diversity across the larva, including the identification of neuronal cell types. We then validate important gene regulatory networks driving sea urchin development and reveal new domains of activity within the larval body. Focusing on neurons that co-express Pdx-1 and Brn1/2/4, we identify an unprecedented number of genes shared by this population of neurons in sea urchin and vertebrate endocrine pancreatic cells. Using differential expression results from Pdx-1 knockdown experiments, we show that Pdx1 is necessary for the acquisition of the neuronal identity of these cells. We hypothesize that a network similar to the one orchestrated by Pdx1 in the sea urchin neurons was active in an ancestral cell type and then inherited by neuronal and pancreatic developmental lineages in sea urchins and vertebrates.

    1. Developmental Biology
    Jody A Summers, Elizabeth Martinez
    Research Article Updated

    Postnatal ocular growth is regulated by a vision-dependent mechanism that acts to minimize refractive error through coordinated growth of the ocular tissues. Of great interest is the identification of the chemical signals that control visually guided ocular growth. Here, we provide evidence that the pro-inflammatory cytokine, interleukin-6 (IL-6), may play a pivotal role in the control of ocular growth using a chicken model of myopia. Microarray, real-time RT-qPCR, and ELISA analyses identified IL-6 upregulation in the choroids of chick eyes under two visual conditions that introduce myopic defocus and slow the rate of ocular elongation (recovery from induced myopia and compensation for positive lenses). Intraocular administration of atropine, an agent known to slow ocular elongation, also resulted in an increase in choroidal IL-6 gene expression. Nitric oxide appears to directly or indirectly upregulate choroidal IL-6 gene expression, as administration of the non-specific nitric oxide synthase inhibitor, L-NAME, inhibited choroidal IL-6 gene expression, and application of a nitric oxide donor stimulated IL-6 gene and protein expression in isolated chick choroids. Considering the pleiotropic nature of IL-6 and its involvement in many biological processes, these results suggest that IL-6 may mediate many aspects of the choroidal response in the control of ocular growth.