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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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The mitotic chromosomal association of PRC1 fusion proteins Ring1b, Phc1, and Mel18 in Cbx2−/− ES cells. (A) Confocal fluorescence images of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins expressed in Cbx2−/− ES cells in metaphase (top) and anaphase (bottom). Scale bar, 5 μm. (B) Quantitative comparison of mitotic chromosomal association of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins in Cbx2+/+ and Cbx2−/− ES cells. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (C) Western blots of cell extracts from Cbx2+/+ and Cbx2−/− ES cells. Ponceau S staining indicates the loading control.
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Figure 3: The mitotic chromosomal association of PRC1 fusion proteins Ring1b, Phc1, and Mel18 in Cbx2−/− ES cells. (A) Confocal fluorescence images of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins expressed in Cbx2−/− ES cells in metaphase (top) and anaphase (bottom). Scale bar, 5 μm. (B) Quantitative comparison of mitotic chromosomal association of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins in Cbx2+/+ and Cbx2−/− ES cells. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (C) Western blots of cell extracts from Cbx2+/+ and Cbx2−/− ES cells. Ponceau S staining indicates the loading control.

Mentions: Because 1) Cbx2 is the primary Cbx-family protein accumulated at mitotic chromosomes, and 2) mitotic chromosomal accumulation of Cbx2 protein is independent of PRC1 or PRC2 complex proteins, we reasoned that Cbx2 protein is required for recruiting canonical PRC1 proteins to mitotic chromosomes. To this end, we inducibly expressed Cerulean-PRC1 protein (Cerulean-Ring1b, Cerulean-Phc1, or Cerulean-Mel18) and mCherry-H2A fusion protein in Cbx2−/− ES cell lines. Z-scan imaging of live cells by using confocal microscopy revealed that fluorescence intensities of Cerulean-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins at mitotic chromosomes in Cbx2 KO ES cells were greatly reduced in comparison to that seen in wild-type ES cells (compare Figures 1, F–H, and 3A). Quantitative analysis of Z-stack images revealed that (29 ± 8)% of Cerulean-Ring1b protein associated with mitotic chromosomes, (17 ± 7)% of Cerulean-Phc1 protein did, and (27 ± 5)% of Cerulean-Mel18 protein did (Figure 3B). To test whether Cbx2 gene knockout affects the level of Phc1, Mel18, and Ring1b proteins, we performed immunoblotting, using extracts from WT and Cbx2−/− ES cells. Western blots showed that the level of Phc1, Mel18, and Ring1b proteins in Cbx2−/− ES cells was similar to that seen in WT cells (Figure 3C). Thus these data suggest that Cbx2 protein is required for the accumulation of canonical PRC1 proteins Ring1b, Phc1, and Mel18 at 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)

The mitotic chromosomal association of PRC1 fusion proteins Ring1b, Phc1, and Mel18 in Cbx2−/− ES cells. (A) Confocal fluorescence images of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins expressed in Cbx2−/− ES cells in metaphase (top) and anaphase (bottom). Scale bar, 5 μm. (B) Quantitative comparison of mitotic chromosomal association of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins in Cbx2+/+ and Cbx2−/− ES cells. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (C) Western blots of cell extracts from Cbx2+/+ and Cbx2−/− ES cells. Ponceau S staining indicates the loading control.
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Figure 3: The mitotic chromosomal association of PRC1 fusion proteins Ring1b, Phc1, and Mel18 in Cbx2−/− ES cells. (A) Confocal fluorescence images of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins expressed in Cbx2−/− ES cells in metaphase (top) and anaphase (bottom). Scale bar, 5 μm. (B) Quantitative comparison of mitotic chromosomal association of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins in Cbx2+/+ and Cbx2−/− ES cells. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (C) Western blots of cell extracts from Cbx2+/+ and Cbx2−/− ES cells. Ponceau S staining indicates the loading control.
Mentions: Because 1) Cbx2 is the primary Cbx-family protein accumulated at mitotic chromosomes, and 2) mitotic chromosomal accumulation of Cbx2 protein is independent of PRC1 or PRC2 complex proteins, we reasoned that Cbx2 protein is required for recruiting canonical PRC1 proteins to mitotic chromosomes. To this end, we inducibly expressed Cerulean-PRC1 protein (Cerulean-Ring1b, Cerulean-Phc1, or Cerulean-Mel18) and mCherry-H2A fusion protein in Cbx2−/− ES cell lines. Z-scan imaging of live cells by using confocal microscopy revealed that fluorescence intensities of Cerulean-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins at mitotic chromosomes in Cbx2 KO ES cells were greatly reduced in comparison to that seen in wild-type ES cells (compare Figures 1, F–H, and 3A). Quantitative analysis of Z-stack images revealed that (29 ± 8)% of Cerulean-Ring1b protein associated with mitotic chromosomes, (17 ± 7)% of Cerulean-Phc1 protein did, and (27 ± 5)% of Cerulean-Mel18 protein did (Figure 3B). To test whether Cbx2 gene knockout affects the level of Phc1, Mel18, and Ring1b proteins, we performed immunoblotting, using extracts from WT and Cbx2−/− ES cells. Western blots showed that the level of Phc1, Mel18, and Ring1b proteins in Cbx2−/− ES cells was similar to that seen in WT cells (Figure 3C). Thus these data suggest that Cbx2 protein is required for the accumulation of canonical PRC1 proteins Ring1b, Phc1, and Mel18 at 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