Essential role of NONO-HOXA1-Wnt axis in cardiomyocyte differentiation [ChIP-seq]

NONO is recognized as a critical molecular scaffold involved in both transcriptional and posttranscriptional regulation. Mutations in NONO are frequently linked to congenital heart diseases (CHDs) in humans. However, the mechanisms by which NONO regulates cardiac development remain elusive. Here, we identified NONO as a pivotal dual-function regulator of cardiomyocyte differentiation in human induced pluripotent stem cells (hiPSCs). NONO deficiency in hiPSCs results in a distinct defect in early cardiomyocyte differentiation. Mechanistically, NONO interacts with HOXA1 and regulates the dynamic expression of key genes during early cardiomyocyte differentiation. ChIP-seq analysis reveals that NONO loss reduces HOXA1 occupancy at target genes, compromising its transcriptional regulation. Additionally, NONO and HOXA1 cooperatively activate the Wnt signaling. Taken together, these findings establish the NONO-HOXA1-Wnt axis as a key molecular mechanism in cardiomyocyte differentiation and provid insights into the etiology of CHDs associated with NONO mutations. Overall design: We performed ChIP-seq of NONO in Day2-differentiated cells. We performed ChIP-seq of HOXA1 in WT and NONO-KO Day2-differentiated cells overexpressing Dox-induced HOXA1-HA using anti-HA tag antibody, Input of ChIP-seq in Day2-differentiated cells, mRNA-seq of WT, NONO-KO Day2, 5, 7, 9-differentiated cells and HOXA1-KO Day2-differentiated cells. We performed reChIP-seq of NONO in Day2-differentiated cells overexpressing Dox-induced HOXA1-HA using anti-HA for primary ChIP and anti-NONO for re-ChIP. We performed ChIP-seq of HOXA1 in WT and NONO-KO Day2-differentiated cells using anti-H3K27AC antibody, Input of ChIP-seq in Day2-differentiated cells. We performed ATAC-seq in Day2-differentiated cells.