Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes.
Bottom Line: Depletion of PRC1 or PRC2 protein has no effect on the immobilization of Cbx2 on mitotic chromosomes.We find that the N-terminus of Cbx2 is needed for its recruitment to mitotic chromosomes, whereas the C-terminus is required for its immobilization.Thus these results provide fundamental insights into the molecular mechanisms of epigenetic inheritance.
Affiliation: Department of Chemistry, University of Colorado Denver, Denver, CO 80217-3364.Show MeSH
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Mentions: Because Cbx2 protein affects the accumulation of canonical PRC1 proteins at mitotic chromosomes, we reasoned that Cbx2 directly recruits canonical PRC1 proteins to mitotic chromosomes. To this end, we coexpressed the three fusion proteins in Cbx2 KO ES cell lines: YFP-Cbx2, Cerulean-PRC1 subunit (Cerulean-Ring1b, or Cerulean-Phc1, or Cerulean-Mel18), and mCherry-H2A. We expected that the introduction of an YFP-Cbx2 fusion protein to the Cbx2- background ES cells would restore the mitotic chromosomal association of the three PRC1 proteins (Cerulean-Ring1b, Cerulean-Phc1, and Cerulean-Mel18). We performed three-color Z-scan imaging of live cells by using confocal microscope. Quantitative analysis of Z-stack images from three ES cell lines expressing Ring1b, Phc1, and Mel18 fusion proteins showed an average of (95 ± 7)% of YFP-Cbx2 associated with mitotic chromosomes, consistent with YFP-Cbx2 localization in wild-type ES cells (Figure 4, A and C), indicating that mitotic chromosomal association of YFP-Cbx2 fusion protein is independent of endogenous Cbx2 protein. Of note, quantitative image analysis revealed that (63 ± 8)% of Cerulean-Ring1b, (71 ± 12)% of Cerulean-Phc1, and (74 ± 10)% of Cerulean-Mel18 also associated with mitotic chromosomes (Figure 4, A and D). The fraction of retention of the three fusion proteins at mitotic chromosomes in Cbx2 KO ES cell lines complemented with YFP-Cbx2 was similar to that seen in wild-type ES cells, indicating that YFP-Cbx2 fusion protein recruits the three canonical PRC1 fusion proteins to mitotic chromosomes.
Affiliation: Department of Chemistry, University of Colorado Denver, Denver, CO 80217-3364.