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Serum response factor (SRF) mediates immediate early gene (IEG) and cytoskeletal gene expression programs in almost any cell type. So far, SRF transcriptional dynamics have not been investigated at single-molecule resolution. We provide a study of single Halo-tagged SRF molecules in fibroblasts and primary neurons. In both cell types, individual binding events of SRF molecules segregated into three chromatin residence time regimes, short, intermediate, and long binding, indicating a cell type-independent SRF property. The chromatin residence time of the long bound fraction was up to 1 min in quiescent cells and significantly increased upon stimulation. Stimulation also enhanced the long bound SRF fraction at specific timepoints (20 and 60 min) in both cell types. These peaks correlated with activation of the SRF cofactors MRTF-A and MRTF-B (myocardin-related transcription factors). Interference with signaling pathways and cofactors demonstrated modulation of SRF chromatin occupancy by actin signaling, MAP kinases, and MRTFs.

Single-molecule imaging of the transcription factor SRF reveals prolonged chromatin-binding kinetics upon cell stimulation