A Promoter Competition Hub Orchestrates Runx1 Alternative Promoter Usage during Skeletal Muscle Stem Cell Activation

Alternative promoter (AP) usage profoundly expands transcriptomic and proteomic diversity, yet the regulatory principles governing promoter choice remain poorly understood. Here, using the dual-promoter Runx1 locus as a paradigm in skeletal muscle stem cells (MuSCs), we uncover a promoter competition hub that orchestrates AP selection during MuSC fate transition from activation to proliferation. We find antagonistic expression dynamics between the two Runx1 promoters, with the primary promoter (PP) active in the early activating stage and the secondary promoter (SP) induced later in proliferation. Combinatorial genetic perturbations in vitro and in vivo establish the non-redundant roles of PP- and SP-derived isoforms in MuSC lineage progression. Mechanistically, we find that PP and SP engage in reciprocal competition in a multi-connected enhancer-promoter (E-P) loop hub. Further dissection identifies a cohort of key enhancers that dynamically interact with PP/SP to orchestrate the competition. Moreover, we establish the transcription factor USF1 as a key factor driving the dynamic E-P interactions and PP/SP competition. Beyond Runx1, high-resolution Micro-C reveals promoter competition as a general mechanism regulating a subset of AP choices in MuSC activation/proliferation. Collectively, our study uncovers a dynamic promoter competition hub that governs AP selection in MuSC lineage progression, offering fundamental insights into how 3D genome architecture coordinates precise, stage-specific gene expression programs during cell fate transitions.