Opposing CTCF and GATA4 activities set the pace of chromatin topology remodeling during cardiomyogenesis

SUMMARY Reorganization of the three-dimensional chromatin structure is a critical feature of human embryonic development. Yet, the mechanisms regulating integrative remodelling of local structures (e.g., loops) and global architecture (e.g., A/B compartmentalization) remain un-clear. Here, we investigate this aspect in the context of cardiomyogenesis, characterized by pronounced B-to-A remodelling of several cardiac-specific genes such as 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 differ-entiation, we show that GATA4 is essential for timely topological activation of key cardiac genes, while partial depletion of CTCF, anticipating physiological downregulation during de-velopment, enhances this process. Deletion of a single CTCF binding site on TTN leads to modest gene decompaction and transcriptional activation. Bulk and single-cell RNA se-quencing of chamber-specific cardiac organoids reveals that loss of GATA4 delays differ-entiation and sustains proliferation of early cardiomyocytes, whereas premature CTCF de-pletion accelerates yet alters cardiomyocyte maturation. These findings suggest that CTCF and GATA4 have antagonistic roles on chromatin dynamics during cardiomyogenesis, form-ing a rheostat that maintains accurate developmental tempo. Disruption of this mecha-nism may contribute to congenital heart defects caused by mutations in these factors. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵5 Lead contact This is a preliminary revision in response to the peer review performed at Review Commons. All changes are described in detail in our point-by-point responses to the reviewer's comments.