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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 association of Cbx2 fusion protein with mitotic chromosomes in PRC1 and PRC2 gene-knockout ES cells. (A) Confocal fluorescence images of YFP-Cbx2 fusion protein expressed in PGK12.1 ES cells (WT-ES). The different version of YFP-Cbx2 of Figure 1A is presented here in order to compare with B and C. Scale bar, 5 μm. (B) Confocal fluorescence images of YFP-Cbx2 fusion protein expressed in Eed−/− ES cells in various phases of mitosis. Prometaphase (top), metaphase (middle), and anaphase/telophase (bottom). Scale bar, 5 μm. (C) Confocal fluorescence images of YFP-Cbx2 fusion protein expressed in Ring1a−/−/Ring1bfl/fl and Bmi1−/−/Mel18−/− ES cell lines in various phases of mitosis. Prometaphase (top), metaphase (middle), and anaphase/telophase (bottom). The Ring1a−/−/Ring1bfl/fl was treated with OHT to induce depletion of Ring1b for 3 d before imaging. The protein level of Ring1b in Ring1a−/−/Ring1bfl/fl cells with or without OHT is presented in Supplemental Figure S5. Scale bar, 5 μm. (D) Quantitative analysis of mitotic binding fractions of YFP-Cbx2 fusion protein at mitotic chromosomes. The data represent at least 10 cells analyzed. Error bars, SD of the mean.
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Figure 2: The association of Cbx2 fusion protein with mitotic chromosomes in PRC1 and PRC2 gene-knockout ES cells. (A) Confocal fluorescence images of YFP-Cbx2 fusion protein expressed in PGK12.1 ES cells (WT-ES). The different version of YFP-Cbx2 of Figure 1A is presented here in order to compare with B and C. Scale bar, 5 μm. (B) Confocal fluorescence images of YFP-Cbx2 fusion protein expressed in Eed−/− ES cells in various phases of mitosis. Prometaphase (top), metaphase (middle), and anaphase/telophase (bottom). Scale bar, 5 μm. (C) Confocal fluorescence images of YFP-Cbx2 fusion protein expressed in Ring1a−/−/Ring1bfl/fl and Bmi1−/−/Mel18−/− ES cell lines in various phases of mitosis. Prometaphase (top), metaphase (middle), and anaphase/telophase (bottom). The Ring1a−/−/Ring1bfl/fl was treated with OHT to induce depletion of Ring1b for 3 d before imaging. The protein level of Ring1b in Ring1a−/−/Ring1bfl/fl cells with or without OHT is presented in Supplemental Figure S5. Scale bar, 5 μm. (D) Quantitative analysis of mitotic binding fractions of YFP-Cbx2 fusion protein at mitotic chromosomes. The data represent at least 10 cells analyzed. Error bars, SD of the mean.

Mentions: The Cbx-containing PRC1 complexes are recruited to interphasic chromatin via Cbx-family protein interactions with H3K27me3 mediated by PRC2 (Cao et al., 2002; Wang et al., 2004b; Bernstein et al., 2006; Tavares et al., 2012; Morey et al., 2013). Therefore we asked whether PRC2 is required for the accumulation of Cbx2 protein at mitotic chromosomes. To test the hypothesis, we stably and inducibly expressed both YFP-Cbx2 and Cerulean-H2A fusion proteins in Eed knockout (KO) ES cell lines, which lack the H3K27me3 modification. Z-scan of confocal images showed that YFP-Cbx2 fusion protein was granularly distributed at mitotic chromosomes, consistent with its distribution in wild-type ES cells. Quantitative analysis of Z-stack images showed that (97 ± 5)% of Cbx2 protein was enriched at mitotic chromosomes in Eed KO ES cell lines (Figure 2, B and D), consistent with YFP-Cbx2 associating with mitotic chromosomes in wild-type ES cells (Figure 2, A and D), indicating that a core component of the PRC2 complex, Eed, is not required for accumulation of Cbx2 protein 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 association of Cbx2 fusion protein with mitotic chromosomes in PRC1 and PRC2 gene-knockout ES cells. (A) Confocal fluorescence images of YFP-Cbx2 fusion protein expressed in PGK12.1 ES cells (WT-ES). The different version of YFP-Cbx2 of Figure 1A is presented here in order to compare with B and C. Scale bar, 5 μm. (B) Confocal fluorescence images of YFP-Cbx2 fusion protein expressed in Eed−/− ES cells in various phases of mitosis. Prometaphase (top), metaphase (middle), and anaphase/telophase (bottom). Scale bar, 5 μm. (C) Confocal fluorescence images of YFP-Cbx2 fusion protein expressed in Ring1a−/−/Ring1bfl/fl and Bmi1−/−/Mel18−/− ES cell lines in various phases of mitosis. Prometaphase (top), metaphase (middle), and anaphase/telophase (bottom). The Ring1a−/−/Ring1bfl/fl was treated with OHT to induce depletion of Ring1b for 3 d before imaging. The protein level of Ring1b in Ring1a−/−/Ring1bfl/fl cells with or without OHT is presented in Supplemental Figure S5. Scale bar, 5 μm. (D) Quantitative analysis of mitotic binding fractions of YFP-Cbx2 fusion protein at mitotic chromosomes. The data represent at least 10 cells analyzed. Error bars, SD of the mean.
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Figure 2: The association of Cbx2 fusion protein with mitotic chromosomes in PRC1 and PRC2 gene-knockout ES cells. (A) Confocal fluorescence images of YFP-Cbx2 fusion protein expressed in PGK12.1 ES cells (WT-ES). The different version of YFP-Cbx2 of Figure 1A is presented here in order to compare with B and C. Scale bar, 5 μm. (B) Confocal fluorescence images of YFP-Cbx2 fusion protein expressed in Eed−/− ES cells in various phases of mitosis. Prometaphase (top), metaphase (middle), and anaphase/telophase (bottom). Scale bar, 5 μm. (C) Confocal fluorescence images of YFP-Cbx2 fusion protein expressed in Ring1a−/−/Ring1bfl/fl and Bmi1−/−/Mel18−/− ES cell lines in various phases of mitosis. Prometaphase (top), metaphase (middle), and anaphase/telophase (bottom). The Ring1a−/−/Ring1bfl/fl was treated with OHT to induce depletion of Ring1b for 3 d before imaging. The protein level of Ring1b in Ring1a−/−/Ring1bfl/fl cells with or without OHT is presented in Supplemental Figure S5. Scale bar, 5 μm. (D) Quantitative analysis of mitotic binding fractions of YFP-Cbx2 fusion protein at mitotic chromosomes. The data represent at least 10 cells analyzed. Error bars, SD of the mean.
Mentions: The Cbx-containing PRC1 complexes are recruited to interphasic chromatin via Cbx-family protein interactions with H3K27me3 mediated by PRC2 (Cao et al., 2002; Wang et al., 2004b; Bernstein et al., 2006; Tavares et al., 2012; Morey et al., 2013). Therefore we asked whether PRC2 is required for the accumulation of Cbx2 protein at mitotic chromosomes. To test the hypothesis, we stably and inducibly expressed both YFP-Cbx2 and Cerulean-H2A fusion proteins in Eed knockout (KO) ES cell lines, which lack the H3K27me3 modification. Z-scan of confocal images showed that YFP-Cbx2 fusion protein was granularly distributed at mitotic chromosomes, consistent with its distribution in wild-type ES cells. Quantitative analysis of Z-stack images showed that (97 ± 5)% of Cbx2 protein was enriched at mitotic chromosomes in Eed KO ES cell lines (Figure 2, B and D), consistent with YFP-Cbx2 associating with mitotic chromosomes in wild-type ES cells (Figure 2, A and D), indicating that a core component of the PRC2 complex, Eed, is not required for accumulation of Cbx2 protein 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