A Transcription Factor Collective Defines Cardiac Cell Fate and Reflects Lineage History

Cell fate decisions are driven through the integration of inductive signals and tissue-specific transcription factors (TFs), although the details on how this information converges in cis remain unclear. Here, we demonstrate that the five genetic components essential for cardiac specification in Drosophila, including the effectors of Wg and Dpp signaling, act as a collective unit to cooperatively regulate heart enhancer activity, both in vivo and in vitro. Their combinatorial binding does not require any specific motif orientation or spacing, suggesting an alternative mode of enhancer function whereby cooperative activity occurs with extensive motif flexibility. A fraction of enhancers co-occupied by cardiogenic TFs had unexpected activity in the neighboring visceral mesoderm but could be rendered active in heart through single-site mutations. Given that cardiac and visceral cells are both derived from the dorsal mesoderm, this dormant TF binding signature may represent a molecular footprint of these cells' developmental lineage. º Collective binding of five transcription factors promotes cardiac enhancer activity º Enhancer co-occupancy does not require specific spacing or orientation of binding sites º Some cardiac enhancers are active in another tissue, the visceral mesoderm º The dormant cardiac binding signature reflects the developmental history of the tissue The five transcription factors that specify cardiac fate in Drosophila bind cooperatively to cardiac enhancers. Surprisingly, the arrangement of binding sites along the enhancer is flexible, but all five must be present for maximal enhancer activity.