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Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes.

Zhen CY, Duc HN, Kokotovic M, Phiel CJ, Ren X - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Colorado Denver, Denver, CO 80217-3364.

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Directly recruiting PRC1 fusion proteins to mitotic chromosomes by YFP-Cbx2 but not by YFP-Cbx21-498. (A) Confocal fluorescence images of Cerulean-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins coexpressed with YFP-Cbx2 and mCherry-H2A fusion proteins in Cbx2−/− ES cells in metaphase. Scale bar, 5 μm. (B) Confocal fluorescence images of Cerulean-Ring1b fusion protein coexpressed with YFP-Cbx21-498 and mCherry-H2A fusion protein in Cbx2−/− ES cells in metaphase. Scale bar, 5 μm. (C) Quantification of mitotic chromosomal association of YFP-Cbx2 and YFP-Cbx21-498 fusion proteins in Cbx2−/− ES cells. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (D) Quantitative analysis of mitotic chromosomal association of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins coexpressed with YFP-Cbx2 and of Cerulea-Ring1b coexpressed with YFP-Cbx21-498 in Cbx2−/− ES cells in metaphase. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (E) Photobleaching FRET images of Cerulean-Ring1b interaction with YFP-Cbx2 and YFP-Cbx21-498 at mitotic chromosomes. The YFP-Cbx2, YFP-Cbx21-498, and Cerulean-Ring1b fusion proteins as indicated above images expressed in Cbx2−/− ES cells. Half-area of fluorescence of YFP-Cbx2 or YFP-Cbx21-498 fusion proteins at mitotic chromosomes was photobleached. Z-scan imaging of live cells by confocal laser microscope was performed before (top) and after (bottom) photobleaching. The arrowheads indicate the bleaching areas.
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Figure 4: Directly recruiting PRC1 fusion proteins to mitotic chromosomes by YFP-Cbx2 but not by YFP-Cbx21-498. (A) Confocal fluorescence images of Cerulean-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins coexpressed with YFP-Cbx2 and mCherry-H2A fusion proteins in Cbx2−/− ES cells in metaphase. Scale bar, 5 μm. (B) Confocal fluorescence images of Cerulean-Ring1b fusion protein coexpressed with YFP-Cbx21-498 and mCherry-H2A fusion protein in Cbx2−/− ES cells in metaphase. Scale bar, 5 μm. (C) Quantification of mitotic chromosomal association of YFP-Cbx2 and YFP-Cbx21-498 fusion proteins in Cbx2−/− ES cells. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (D) Quantitative analysis of mitotic chromosomal association of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins coexpressed with YFP-Cbx2 and of Cerulea-Ring1b coexpressed with YFP-Cbx21-498 in Cbx2−/− ES cells in metaphase. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (E) Photobleaching FRET images of Cerulean-Ring1b interaction with YFP-Cbx2 and YFP-Cbx21-498 at mitotic chromosomes. The YFP-Cbx2, YFP-Cbx21-498, and Cerulean-Ring1b fusion proteins as indicated above images expressed in Cbx2−/− ES cells. Half-area of fluorescence of YFP-Cbx2 or YFP-Cbx21-498 fusion proteins at mitotic chromosomes was photobleached. Z-scan imaging of live cells by confocal laser microscope was performed before (top) and after (bottom) photobleaching. The arrowheads indicate the bleaching areas.

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.


Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes.

Zhen CY, Duc HN, Kokotovic M, Phiel CJ, Ren X - Mol. Biol. Cell (2014)

Directly recruiting PRC1 fusion proteins to mitotic chromosomes by YFP-Cbx2 but not by YFP-Cbx21-498. (A) Confocal fluorescence images of Cerulean-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins coexpressed with YFP-Cbx2 and mCherry-H2A fusion proteins in Cbx2−/− ES cells in metaphase. Scale bar, 5 μm. (B) Confocal fluorescence images of Cerulean-Ring1b fusion protein coexpressed with YFP-Cbx21-498 and mCherry-H2A fusion protein in Cbx2−/− ES cells in metaphase. Scale bar, 5 μm. (C) Quantification of mitotic chromosomal association of YFP-Cbx2 and YFP-Cbx21-498 fusion proteins in Cbx2−/− ES cells. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (D) Quantitative analysis of mitotic chromosomal association of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins coexpressed with YFP-Cbx2 and of Cerulea-Ring1b coexpressed with YFP-Cbx21-498 in Cbx2−/− ES cells in metaphase. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (E) Photobleaching FRET images of Cerulean-Ring1b interaction with YFP-Cbx2 and YFP-Cbx21-498 at mitotic chromosomes. The YFP-Cbx2, YFP-Cbx21-498, and Cerulean-Ring1b fusion proteins as indicated above images expressed in Cbx2−/− ES cells. Half-area of fluorescence of YFP-Cbx2 or YFP-Cbx21-498 fusion proteins at mitotic chromosomes was photobleached. Z-scan imaging of live cells by confocal laser microscope was performed before (top) and after (bottom) photobleaching. The arrowheads indicate the bleaching areas.
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Figure 4: Directly recruiting PRC1 fusion proteins to mitotic chromosomes by YFP-Cbx2 but not by YFP-Cbx21-498. (A) Confocal fluorescence images of Cerulean-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins coexpressed with YFP-Cbx2 and mCherry-H2A fusion proteins in Cbx2−/− ES cells in metaphase. Scale bar, 5 μm. (B) Confocal fluorescence images of Cerulean-Ring1b fusion protein coexpressed with YFP-Cbx21-498 and mCherry-H2A fusion protein in Cbx2−/− ES cells in metaphase. Scale bar, 5 μm. (C) Quantification of mitotic chromosomal association of YFP-Cbx2 and YFP-Cbx21-498 fusion proteins in Cbx2−/− ES cells. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (D) Quantitative analysis of mitotic chromosomal association of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins coexpressed with YFP-Cbx2 and of Cerulea-Ring1b coexpressed with YFP-Cbx21-498 in Cbx2−/− ES cells in metaphase. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (E) Photobleaching FRET images of Cerulean-Ring1b interaction with YFP-Cbx2 and YFP-Cbx21-498 at mitotic chromosomes. The YFP-Cbx2, YFP-Cbx21-498, and Cerulean-Ring1b fusion proteins as indicated above images expressed in Cbx2−/− ES cells. Half-area of fluorescence of YFP-Cbx2 or YFP-Cbx21-498 fusion proteins at mitotic chromosomes was photobleached. Z-scan imaging of live cells by confocal laser microscope was performed before (top) and after (bottom) photobleaching. The arrowheads indicate the bleaching areas.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Colorado Denver, Denver, CO 80217-3364.

Show MeSH
Related in: MedlinePlus