In Australia, every year more than 2000 children are born with a heart malformation, referred to as a Congenital Heart Defect (CHD). However, in the majority of cases the underlying genetic causes are unknown. There is growing evidence that mutations not only in protein-coding DNA but also in non-coding DNA, functioning as cis-regulatory elements (CREs) controlling gene expression, can cause CHDs. Consequently, we hypothesise that heart defects result from disrupted cardiac gene expression, which are triggered by variants in cis-regulatory domains.
To address this, we sought to identify CREs essential for human cardiac function. First, we identified the genome-wide binding locations of the cardiac transcription factor NKX2-5, using chromatin immunoprecipitation followed by deep-sequencing (ChIP-seq) in cardiomyocytes derived from human embryonic stem cells. This experiment provided an initial set of cardiac CREs, that were mined using an in-house bioinformatics comparative genomics pipeline to provide an extensive atlas of regulatory regions potentially controlling gene expression in the human heart.
In order to validate the cardiac activity of these CREs in vivo, we performed transgenesis assays in medaka (Oryzias latipes). In this study, we demonstrate that the function of primate specific, human cardiac-specific regulatory elements can be systematically and rapidly characterised in vivo in medaka. Subsequently, the most promising candidates will be functionally tested in vivo using the CRISPR/Cas9 system and in vitro using cardiomyocytes derived from human embryonic stem cells.
By deciphering the conserved cis-regulatory landscape driving cardiogenesis and functionally analysing them in human and fish, we hope to determine the cis-regulatory elements vital in the formation of the heart and identify novel genetic mechanisms of human heart disease.