Abstract
For over two decades, scientists have observed that most transcription factors (TFs) become excluded from mitotic chromosomes of mammalian cells undergoing cell division. The few TFs that were observed to remain bound to chromosomes have been termed mitotic bookmarkers and were predicted to play important roles in reestablishing transcription after mitosis. Using live-cell imaging of endogenous TFs in mouse embryonic stem cells, we discovered that the observed exclusion from mitotic chromosomes is largely a result of formaldehyde cross-linking and that in fact, most TFs bind to mitotic chromosomes throughout mitosis. Here, we describe the single-molecule live-cell imaging and analytical tools we used to characterize and quantify TF diffusion and binding as mouse embryonic stem cells proceed through mitosis.