Studying transcription factor function in the genome at molecular resolution

About 7% of the human genome encodes cis-regulatory elements (CREs) that function as regulatory switches to modulate the expression of genes. These short genetic sequences control the complex transcriptional changes necessary for organismal development. A topical challenge in the field is to understand how transcription factors (TFs) read and translate this information into gene expression patterns. Here, I review how the development of single-molecule footprinting (SMF) that resolves the genome occupancy of TFs on individual DNA molecules resolution contributes to our ability to establish how the regulatory genetic information is interpreted at the mechanistic level. I further discuss how future developments in the nascent field of single-molecule genomics (SMG) could impact our understanding of gene regulation mechanisms. Single-molecule footprinting (SMF) resolves the heterogeneity of transcription factor (TF) occupancy in a cellular population.Detecting multiple TFs on individual DNA molecules can quantify co-occupancy and infer dependencies.SMF allows studies of how large collections of genetic variants affect TF binding and transcription activation.Future development of single-molecule methods will allow integrative studies of the multiple processes controlled by TFs.