Visualizing Transcription Factor Binding on Mitotic Chromosomes Using Single-Molecule Live-Cell Imaging
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.
Key wordsTranscription factors Mouse embryonic stem cells Mitotic chromosomes Single-molecule live-cell imaging Dynamics Residence time Diffusion
- 6.John S, Workman JL (1998) Bookmarking genes for activation in condensed mitotic chromosomes. BioEssays: news and reviews in molecular, cellular and developmental biology 20:275–279. https://doi.org/10.1002/(SICI)1521-1878(199804)20:4<275::AID-BIES1>3.0.CO;2-PCrossRefGoogle Scholar
- 11.Coralie Pallier, Paola Scaffidi, Stéphanie Chopineau-Proust, Alessandra Agresti, Patrice Nordmann, Marco E. Bianchi, Vincent Marechal, (2003) Association of Chromatin Proteins High Mobility Group Box (HMGB) 1 and HMGB2 with Mitotic Chromosomes. Molecular Biology of the Cell 14 (8):3414-3426CrossRefPubMedGoogle Scholar
- 13.Los GV, Wood K (2007) The HaloTag: a novel technology for cell imaging and protein analysis. Methods Mol Biol (Clifton, NJ) 356:195–208Google Scholar
- 17.Teves SS, An L, Bhargava-Shah A, et al (2018) A stable mode of bookmarking by TBP recruits RNA polymerase II to mitotic chromosomes. eLife. https://elifesciences.org/articles/35621. Accessed 30 Aug 2018
- 20.Hansen AS, Woringer M, Grimm JB et al (2018) Robust model-based analysis of single-particle tracking experiments with Spot-On. eLife 7. https://doi.org/10.7554/elife.33125