Analysis of the role of CTCF and GATA4 on chromatin remodelling during cardiac fate specification
Reorganization of three-dimensional (3D) chromatin architecture is a critical feature of human embryonic development. Yet, the mechanisms regulating the coordinated remodelling of local structures (e.g., loops) and global architecture (e.g., A/B compartmentalization) remain unclear. Here, we investigate this process during cardiomyogenesis, characterized by pronounced B-to-A remodelling of several cardiac-specific genes such as \textit{TTN}. We focus on the roles of the pioneer transcription factor GATA4 and the architectural protein CTCF. Using an inducible knockdown system during human induced pluripotent stem cell differentiation, we show that GATA4 is essential for timely topological and transcriptional activation of key cardiac genes, while partial depletion of CTCF, anticipating its physiological downregulation during development, enhances cardiac gene activation and is accompanied by \textit{TTN} decompaction. Precise disruption of a single CTCF binding site at \textit{TTN} leads to modest transcriptional activation despite limited changes in chromatin folding. Bulk and single-cell RNA sequencing of chamber-specific cardiac organoids reveals that loss of GATA4 delays differentiation and sustains proliferation of early cardiomyocytes, whereas premature CTCF depletion accelerates yet \textcolor{magenta}{alters} cardiomyocyte maturation. These findings suggest that CTCF and GATA4 have antagonistic roles in regulating gene expression and 3D chromatin dynamics during cardiomyogenesis, forming a rheostat that maintains accurate developmental tempo. Disruption of this mechanism may contribute to congenital heart defects caused by mutations in these factors.
Reorganization of three-dimensional (3D) chromatin architecture is a critical feature of human embryonic development. Yet, the mechanisms regulating the coordinated remodelling of local structures (e.g., loops) and global architecture (e.g., A/B compartmentalization) remain unclear. Here, we investigate this process during cardiomyogenesis, characterized by pronounced B-to-A remodelling of several cardiac-specific genes such as \textit{TTN}. We focus on the roles of the pioneer transcription factor GATA4 and the architectural protein CTCF. Using an inducible knockdown system during human induced pluripotent stem cell differentiation, we show that GATA4 is essential for timely topological and transcriptional activation of key cardiac genes, while partial depletion of CTCF, anticipating its physiological downregulation during development, enhances cardiac gene activation and is accompanied by \textit{TTN} decompaction. Precise disruption of a single CTCF binding site at \textit{TTN} leads to modest transcriptional activation despite limited changes in chromatin folding. Bulk and single-cell RNA sequencing of chamber-specific cardiac organoids reveals that loss of GATA4 delays differentiation and sustains proliferation of early cardiomyocytes, whereas premature CTCF depletion accelerates yet \textcolor{magenta}{alters} cardiomyocyte maturation. These findings suggest that CTCF and GATA4 have antagonistic roles in regulating gene expression and 3D chromatin dynamics during cardiomyogenesis, forming a rheostat that maintains accurate developmental tempo. Disruption of this mechanism may contribute to congenital heart defects caused by mutations in these factors.