AbstractMammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding. How these properties affect their ability to occupy specific genomic sites and modify the epigenetic landscape is unclear. The association of TFs with mitotic chromosomes observed by fluorescence microscopy is largely mediated by non-specific DNA interactions and differs broadly between TFs. Here we combine quantitative measurements of mitotic chromosome binding (MCB) of 501 TFs, TF mobility measurements by fluorescence recovery after photobleaching, single molecule imaging of DNA binding, and mapping of TF binding and chromatin accessibility. TFs associating to mitotic chromosomes are enriched in DNA-rich compartments in interphase and display slower mobility in interphase and mitosis. Remarkably, MCB correlates with relative TF on-rates and genome-wide specific site occupancy, but not with TF residence times. This suggests that non-specific DNA binding properties of TFs regulate their search efficiency and occupancy of specific genomic sites.