An Orthologous Epigenetic Gene Expression Signature Derived from Differentiating Embryonic Stem Cells Identifies Regulators of Cardiogenesis

<div><p>Here we used predictive gene expression signatures within a multi-species framework to identify the genes that underlie cardiac cell fate decisions in differentiating embryonic stem cells. We show that the overlapping orthologous mouse and human genes are the most accurate candidate cardiogenic genes as these genes identified the most conserved developmental pathways that characterize the cardiac lineage. An RNAi-based screen of the candidate genes in <i>Drosophila</i> uncovered numerous novel cardiogenic genes. shRNA knockdown combined with transcriptome profiling of the newly-identified transcription factors zinc finger protein 503 and zinc finger E-box binding homeobox 2 and the well-known cardiac regulatory factor NK2 homeobox 5 revealed that zinc finger E-box binding homeobox 2 activates terminal differentiation genes required for cardiomyocyte structure and function whereas zinc finger protein 503 and NK2 homeobox 5 are required for specification of the cardiac lineage. We further demonstrated that an essential role of NK2 homeobox 5 and zinc finger protein 503 in specification of the cardiac lineage is the repression of gene expression programs characteristic of alternative cell fates. Collectively, these results show that orthologous gene expression signatures can be used to identify conserved cardiogenic pathways.</p></div

ZNF503, ZEB2 and NKX2-5 are necessary for differentiation of CMs from human ESCs.

<p>(A) Flow cytometric analysis of cTnT expression on human ESCs after 10 days of differentiation along the cardiac lineage following shRNA knockdown of the indicated genes. Representative results of five experiments. WT cells were infected with a shRNA targeting GFP or empty vector, which yielded identical results as non-infected cells. (B) Hierarchical clustering of genes showing significant gene expression changes following shRNA knockdown of the indicated genes at the indicated time points. WT = wild-type; ZE = ZEB2 shRNA; ZN = ZNF503 shRNA; NK = NKX2-5 shRNA.</p

A multi-species epigenetic and gene expression signature identifies candidate cardiogenic genes.

<p>(A) The distribution of the indicated histone modifications to the genomic region of the mouse and human NKX2-5 gene at the ESC or CP state. Genomic coordinates are indicated for human chromosome 5 (hg19) and mouse chromosome 17 (mm9). Brackets indicate scale of peak. (B) Percentage of indicated genes known to regulate mammalian cardiogenesis. (C) Percentage of indicated cardiogenic genes conserved in <i>Drosophila</i>. (D) Enriched GO categories associated with the indicated genes. * p < 0.0001; **p < 0.0005.</p