Oral Presentation 46th Lorne Genome Conference 2025

Evolutionarily conserved cis regulatory elements in cardiac development and congenital heart disease (115191)

Mengyi Song 1 2 3 4 , Esra Erkut 1 4 , Casey Carlisle 1 4 , Simon Monis 1 2 , Anna Prentice 2 , Xuefei Yuan 1 2 4 5 , Huayun Hou 2 , Rebekah Jobling 6 , Raymond Kim 6 , Ian C Scott 1 4 , Michael D Wilson 1 2
  1. Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
  2. Program in Genetics and Genome Biology, Hospital for Sick Children, Toronto, Ontario, Canada
  3. The Eli and Edythe Broad Centre of Regeneration Medicine and Stem Cell Research, University of San Francisco, San Francisco, California, USA
  4. Program in Cell and Developmental Biology, Hospital for Sick Children, Toronto, Ontario, Canada
  5. Center for Molecular Biology (ZMBH), Universität Heidelberg, Heidelberg, Baden-Württemberg, Germany
  6. Department of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, Ontario, Canada

Congenital heart disease (CHD) affects ~1% of live births, yet the causes are largely unknown. Variants disrupting transcription factors (TFs) contribute to CHD, however the impact of genetic variation within the cis-regulatory elements bound by these TFs remains unclear. Using comparative epigenomics, we identified ~6,000 accessible chromatin regions (aCNEs) in early zebrafish embryos orthologous to ~9,000 regions in the human genome. These aCNEs showed hallmarks of poised developmental enhancers. ~200 aCNEs possessed enhanced chromatin accessibility in zebrafish cardiac progenitor cells and were enriched for cardiac developmental processes and Gata TF binding sites. Using stable zebrafish reporter gene expression lines, we found that 16/18 aCNEs drove expression in the heart. Four showed specificity for the outflow tract (OFT). The aCNEs driving OFT-expression were enriched for Fox motifs. Removing Fox motifs from these aCNEs broadened reporter expression to the entire heart. Conversely, replacing a Gata motif with a Fox motif in a pan-cardiac aCNE restricted expression to the OFT. Since gata5/gata6 are required for zebrafish heart development, we used gata5/6 knockdowns to identify gata-dependent aCNEs. 47 aCNEs showed reduced chromatin accessibility upon gata5/6 knockdown. Remarkably, orthologs of three of these aCNEs also showed reduced accessibility upon the knockdown of GATA4 in human cardiomyocytes. One of these gata-sensitive aCNEs forms long-range interactions with the promoter of hand2/HAND2 in zebrafish/human. Its deletion in vivo led to reduced hand2 expression and laterality defects. Two other aCNEs coincided with ultra-rare human CHD variants in patients enrolled in the SickKids Cardiac Genome Clinic. One variant fell in an aCNE associated with tbx20/TBX20. The zebrafish and human orthologs of tbx20-aCNE replicate tbx20 expression patterns and one patient variant weakened its expression. Deletion of this aCNE partially replicates the tbx20 loss-of-function phenotype. This study highlights the importance of aCNEs in vertebrate development and potentially human genetic disease.