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July 18th, 2025
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Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of the Ministry of Education, Department of Orthopedics, Tongji Hospital, School of Life
molecular biology
biorxiv

Genome-Wide Investigation of Transcription Factor Occupancy and Dynamics Using cFOOT-seq

Wang, H.Open in Google Scholar•Wu, A.Open in Google Scholar•Yang, M.-C.Open in Google Scholar•Zhou, D.Open in Google Scholar•Chen, X.Open in Google Scholar•Shi, Z.Open in Google Scholar•Zhang, Y.Open in Google Scholar•Liu, Y.-X.Open in Google Scholar•Chen, K.Open in Google Scholar•Wang, X.Open in Google Scholaret al.

Gene regulation relies on the precise binding of transcription factors (TFs) at regulatory elements, but simultaneously detecting hundreds of TFs on chromatin is challenging. We developed cFOOT-seq, a cytosine deaminase-based TF footprinting assay, for high-resolution, quantitative genome-wide assessment of TF binding in both open and closed chromatin, even with small cell numbers. By utilizing the dsDNA deaminase SsdAtox, cFOOT-seq converts accessible cytosines to uracil while preserving genomic integrity, making it compatible with techniques like ATAC-seq for sensitive and cost-effective detection of TF occupancy at single-molecule and single-cell level. Our approach enables the delineation of TF footprints, quantification of occupancy, and examination of chromatin influences. Notably, cFOOT-seq, combined with FootTrack analysis, predicts TF occupancy dynamics. We demonstrate its application in capturing cell type-specific TFs, analyzing TF dynamics during reprogramming, and revealing TF dependencies on chromatin remodelers. Overall, cFOOT-seq represents a robust approach for investigating the genome-wide dynamics of TF occupancy and elucidating the cis-regulatory architecture underlying gene regulation.

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