Identification of IRX5 transcription factor regulatory mechanisms in human cardiomyocyte function

Abstract

In the heart, several transcription factors (TF) are powerful regulators of cardiac cell fate and organogenesis. Less well studied are the roles of TF in physiological functions. Several studies on animal models have shown that the Iroquois homeobox (Irx) TFs are key patterning and differentiation regulators that confer cell type-specific properties to specialized cardiac lineages. Importantly, studies in mice have shown that IRX5 is expressed very early during cardiogenesis and maintained in adult heart where it regulates ventricular electrophysiological properties, the repolarizing gradient. It has also been shown that IRX5 target-genes regulation occurs through recruitment of histone modifiers. In order to study IRX5 in human, we used cardiomyocytes differentiated from induced pluripotent stem cells (hiPS-CMs) generated from patients carrying loss of function mutations in IRX5 (IRX5Mut) and control individuals. As we detected IRX5 protein as early as in the pluripotent state, we also analyzed cells at hiPS stage in our experiments. High-throughput transcriptomic and epigenetic analyses were performed to obtain a global understanding of IRX5 function. The transcriptomic experiments allowed us to determine the entire set of differentially expressed genes in hiPS and hiPS-CMs IRX5Mut cells as compared to control cells. The epigenetic experiments, performed by ChIPseq, suggested that IRX5 binds to already open chromatin, and preferentially, to enhancer rich regions rather than proximal promoters. Combining these high-throughput experiments, we found direct target genes of IRX5 at hiPS-CM stage, and some of them are relevant to cardiac function. Notably, we showed that this TF regulates the expression of several actors of electrical conduction, such as SCN5A, at hiPS-CM stage. IRX5 seems to be an important factor for the regulation of human cardiac function, and more specifically of its electrical function.