Genome-wide investigation of TF dynamics on chromatin by cFOOT-seq

Gene regulation is precisely orchestrated by the binding of specific transcription factors (TFs) at regulatory elements, yet systematically investigating the occupancy of hundreds of TFs on chromatin remains challenging. We have developed a cytosine deaminase-based genomic footprinting assay for the occupancy and organization of transcription factors on chromatin by sequencing (cFOOT-seq), which enables high-resolution, sensitive, and quantitative genome-wide assessment of TF binding dynamics for hundreds of TFs simultaneously. cFOOT-seq leverages nonspecific dsDNA cytosine deaminase-mediated C to U DNA conversion in open chromatin regions, thereby encoding chromatin state information into the DNA sequence. This technique captures chromatin accessibility, nucleosome positioning, and the occupancy of DNA-binding proteins at nucleotide resolution. Through cFOOT-seq, we are able to delineate genomic footprints of TFs, quantify TF binding affinity via TF depth, and analyze TF coordination. Our findings demonstrate that cFOOT-seq can detect TF binding dynamics during OSKM-mediated reprogramming of mouse embryonic fibroblasts (MEFs), identify TFs potentially involved in the transcriptional response to heat shock, and elucidate the dependencies of TF binding on the chromatin remodeling complex SWI-SNF. In summary, cFOOT-seq represents a robust approach for investigating the genome-wide dynamics of TFs and elucidating the cis-regulatory architecture underlying gene regulation.