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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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FRAP analysis of YFP-Cbx2 fusion protein binding to chromatins in Eed−/−, Ring1a−/−/Ring1b−/−, and Bmi1−/−/Mel18−/− ES cells. (A–C) FRAP curves of interphases and metaphases of YFP-Cbx2 fusion protein in Eed−/−, Ring1a−/−/Ring1b−/−, and Bmi1−/−/Mel18−/− ES cells. The FRAP curves were normalized and plotted as described in Figure 5. More than eight cells were analyzed. Error bars, SDs of means. (D) Residence time and immobile fraction of YFP-Cbx2 on chromatins. The residence time and immobile fraction were calculated from FRAP curves by fitting a first-order kinetic model. Bars with NA indicate that the residence time is not available due immobilization of YFP-Cbx2 on mitotic chromosomes. The dashed lines indicate the contrast between interphase and metaphase. WT-ES is denoted as PGK12.1 ES cells. The data are an average of at least eight cells.
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Figure 6: FRAP analysis of YFP-Cbx2 fusion protein binding to chromatins in Eed−/−, Ring1a−/−/Ring1b−/−, and Bmi1−/−/Mel18−/− ES cells. (A–C) FRAP curves of interphases and metaphases of YFP-Cbx2 fusion protein in Eed−/−, Ring1a−/−/Ring1b−/−, and Bmi1−/−/Mel18−/− ES cells. The FRAP curves were normalized and plotted as described in Figure 5. More than eight cells were analyzed. Error bars, SDs of means. (D) Residence time and immobile fraction of YFP-Cbx2 on chromatins. The residence time and immobile fraction were calculated from FRAP curves by fitting a first-order kinetic model. Bars with NA indicate that the residence time is not available due immobilization of YFP-Cbx2 on mitotic chromosomes. The dashed lines indicate the contrast between interphase and metaphase. WT-ES is denoted as PGK12.1 ES cells. The data are an average of at least eight cells.

Mentions: Because Cbx2 protein is recruited to mitotic chromosomes by a PRC2-independent mechanism, we reasoned that depletion of PRC2 complex gene Eed would not affect the immobilization of Cbx2 on mitotic chromosomes. To test the hypothesis, we performed FRAP analysis of YFP-Cbx2 protein binding to both mitotic and interphasic chromosomes in Eed KO ES cells. Analysis of mitotic FRAP curves revealed that >85% of YFP-Cbx2 fusion protein showed no recovery of fluorescence within 120 s (Figure 6, A and D). Calculation of interphasic FRAP curves of YFP-Cbx2 fusion protein revealed that the residence time for the mobile fraction is 25 s, slightly higher than that observed in wild-type ES cells, whereas the immobile fraction is the same as seen in wild-type ES cells. Thus these data indicate that the dynamics of YFP-Cbx2 fusion protein binding to interphase and mitotic chromatin is independent of the PRC2 gene Eed.


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)

FRAP analysis of YFP-Cbx2 fusion protein binding to chromatins in Eed−/−, Ring1a−/−/Ring1b−/−, and Bmi1−/−/Mel18−/− ES cells. (A–C) FRAP curves of interphases and metaphases of YFP-Cbx2 fusion protein in Eed−/−, Ring1a−/−/Ring1b−/−, and Bmi1−/−/Mel18−/− ES cells. The FRAP curves were normalized and plotted as described in Figure 5. More than eight cells were analyzed. Error bars, SDs of means. (D) Residence time and immobile fraction of YFP-Cbx2 on chromatins. The residence time and immobile fraction were calculated from FRAP curves by fitting a first-order kinetic model. Bars with NA indicate that the residence time is not available due immobilization of YFP-Cbx2 on mitotic chromosomes. The dashed lines indicate the contrast between interphase and metaphase. WT-ES is denoted as PGK12.1 ES cells. The data are an average of at least eight cells.
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Related In: Results  -  Collection

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Figure 6: FRAP analysis of YFP-Cbx2 fusion protein binding to chromatins in Eed−/−, Ring1a−/−/Ring1b−/−, and Bmi1−/−/Mel18−/− ES cells. (A–C) FRAP curves of interphases and metaphases of YFP-Cbx2 fusion protein in Eed−/−, Ring1a−/−/Ring1b−/−, and Bmi1−/−/Mel18−/− ES cells. The FRAP curves were normalized and plotted as described in Figure 5. More than eight cells were analyzed. Error bars, SDs of means. (D) Residence time and immobile fraction of YFP-Cbx2 on chromatins. The residence time and immobile fraction were calculated from FRAP curves by fitting a first-order kinetic model. Bars with NA indicate that the residence time is not available due immobilization of YFP-Cbx2 on mitotic chromosomes. The dashed lines indicate the contrast between interphase and metaphase. WT-ES is denoted as PGK12.1 ES cells. The data are an average of at least eight cells.
Mentions: Because Cbx2 protein is recruited to mitotic chromosomes by a PRC2-independent mechanism, we reasoned that depletion of PRC2 complex gene Eed would not affect the immobilization of Cbx2 on mitotic chromosomes. To test the hypothesis, we performed FRAP analysis of YFP-Cbx2 protein binding to both mitotic and interphasic chromosomes in Eed KO ES cells. Analysis of mitotic FRAP curves revealed that >85% of YFP-Cbx2 fusion protein showed no recovery of fluorescence within 120 s (Figure 6, A and D). Calculation of interphasic FRAP curves of YFP-Cbx2 fusion protein revealed that the residence time for the mobile fraction is 25 s, slightly higher than that observed in wild-type ES cells, whereas the immobile fraction is the same as seen in wild-type ES cells. Thus these data indicate that the dynamics of YFP-Cbx2 fusion protein binding to interphase and mitotic chromatin is independent of the PRC2 gene Eed.

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