DNase Footprint Signatures Are Dictated by Factor Dynamics and DNA Sequence

Genomic footprinting has emerged as an unbiased discovery method for transcription factor (TF) occupancy at cognate DNA in vivo. A basic premise of footprinting is that sequence-specific TF-DNA interactions are associated with localized resistance to nucleases, leaving observable signatures of cleavage within accessible chromatin. This phenomenon is interpreted to imply protection of the critical nucleotides by the stably bound protein factor. However, this model conflicts with previous reports of many TFs exchanging with specific binding sites in living cells on a timescale of seconds. We show that TFs with short DNA residence times have no footprints at bound motif elements. Moreover, the nuclease cleavage profile within a footprint originates from the DNA sequence in the factor-binding site, rather than from the protein occupying specific nucleotides. These findings suggest a revised understanding of TF footprinting and reveal limitations in comprehensive reconstruction of the TF regulatory network using this approach. [Display omitted] •Many factor-binding events detected by ChIP-seq do not produce DNase footprints•DNA cleavage signatures at these sites are present in the absence of the factor•The cleavage signatures are observed at the cognate binding site in naked DNA•Depth of footprint is related to the residence time for the factor in living cells “Footprints” in chromatin imply protein binding. Sung et al. show that the primary structure of DNA largely influences the nuclease cleavage profile within a footprint, and the footprint’s depth reflects how long a given protein binds DNA.