1. Live cell imaging of the cancer-related transcription factor RUNX2 during mitotic progression.
- Author
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Pockwinse SM, Kota KP, Quaresma AJ, Imbalzano AN, Lian JB, van Wijnen AJ, Stein JL, Stein GS, and Nickerson JA
- Subjects
- Binding Sites, Cell Line, Tumor, Cell Nucleolus metabolism, Core Binding Factor Alpha 1 Subunit genetics, Fluorescence Recovery After Photobleaching, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Histones genetics, Histones metabolism, Humans, Kinetics, Protein Binding, RNA Polymerase I metabolism, RNA Polymerase II metabolism, Recombinant Fusion Proteins metabolism, Transfection, Video Recording, Cell Nucleus metabolism, Chromosomes, Human metabolism, Core Binding Factor Alpha 1 Subunit metabolism, Microscopy, Fluorescence, Mitosis
- Abstract
The nuclear matrix bound transcription factor RUNX2 is a lineage-specific developmental regulator that is linked to cancer. We have previously shown that RUNX2 controls transcription of both RNA polymerase II genes and RNA polymerase I-dependent ribosomal RNA genes. RUNX2 is epigenetically retained through mitosis on both classes of target genes in condensed chromosomes. We have used fluorescence recovery after photobleaching to measure the relative binding kinetics of enhanced green fluorescent protein (EGFP)-RUNX2 at transcription sites in the nucleus and nucleoli during interphase, as well as on mitotic chromosomes. RUNX2 becomes more strongly bound as cells go from interphase through prophase, with a doubling of the most tightly bound "immobile fraction." RUNX2 exchange then becomes much more facile during metaphase to telophase. During interphase the less tightly bound pool of RUNX2 exchanges more slowly at nucleoli than at subnuclear foci, and the non-exchanging immobile fraction is greater in nucleoli. These results are consistent with a model in which the molecular mechanism of RUNX2 binding is different at protein-coding and ribosomal RNA genes. The binding interactions of RUNX2 change as cells go through mitosis, with binding affinity increasing as chromosomes condense and then decreasing through subsequent mitotic phases. The increased binding affinity of RUNX2 at mitotic chromosomes may reflect its epigenetic function in "bookmarking" of target genes in cancer cells., (Copyright © 2010 Wiley-Liss, Inc.)
- Published
- 2011
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