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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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Analysis of structural elements of YFP-Cbx2 fusion protein required for its targeting and immobilizing. (A) Diagram of structural domains of Cbx2. The dashed rectangles indicate the region required for targeting Cbx2 to mitotic chromosomes (left) and the region required for immobilizing Cbx2 at mitotic chromosomes (right). ATH, AT-hook domain; Cbox, chromobox domain; CHD, chromodomain domain. The numbers in parentheses indicate the starting and ending of amino acid sequence. (B) Confocal images of YFP-Cbx2 and its variant fusion proteins in metaphase of HeLa cells. YFP-Cbx2 mutant and mCherry-H2A fusion proteins were stably expressed in HeLa cells. The mCherry-H2A was used to mark mitotic chromosomes. Scale bar, 5 μm. (C) FRAP curves of interphases and metaphases of YFP-Cbx2 and its variant fusion proteins expressed in HeLa cells. FRAP analysis is described in Figure 5. (D) Residence time and immobile fraction of YFP-Cbx2 variant fusion proteins at interphasic and mitotic chromatins. The residence time and immobile fraction were calculated from FRAP curves by fitting a first-order kinetic model.
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Figure 7: Analysis of structural elements of YFP-Cbx2 fusion protein required for its targeting and immobilizing. (A) Diagram of structural domains of Cbx2. The dashed rectangles indicate the region required for targeting Cbx2 to mitotic chromosomes (left) and the region required for immobilizing Cbx2 at mitotic chromosomes (right). ATH, AT-hook domain; Cbox, chromobox domain; CHD, chromodomain domain. The numbers in parentheses indicate the starting and ending of amino acid sequence. (B) Confocal images of YFP-Cbx2 and its variant fusion proteins in metaphase of HeLa cells. YFP-Cbx2 mutant and mCherry-H2A fusion proteins were stably expressed in HeLa cells. The mCherry-H2A was used to mark mitotic chromosomes. Scale bar, 5 μm. (C) FRAP curves of interphases and metaphases of YFP-Cbx2 and its variant fusion proteins expressed in HeLa cells. FRAP analysis is described in Figure 5. (D) Residence time and immobile fraction of YFP-Cbx2 variant fusion proteins at interphasic and mitotic chromatins. The residence time and immobile fraction were calculated from FRAP curves by fitting a first-order kinetic model.

Mentions: To test whether the Cbx2 interaction with Ring1b is required for the recruitment of Ring1b protein to mitotic chromosomes, the three fusion proteins mCherry-H2A, Cerulean-Ring1b, and YFP-Cbx21-498 were expressed in Cbx2 KO ES cells. The YFP-Cbx21-498 fusion protein lacks the chromobox (Cbox) domain required for interaction with Ring1b (Satijn et al., 1997; Schoorlemmer et al., 1997; Bardos et al., 2000). We expected that the Cbx2 mutant fusion protein would not be able to recruit Cerulean-Ring1b to mitotic chromosomes. Quantitative image analysis showed that (92 ± 7)% of YFP-Cbx21-498 fusion protein accumulated at mitotic chromosomes, indicating the deletion of the Cbox domain of Cbx2 protein does not affect its mitotic chromosomal association (Figure 4, B and C; also see later discussion of Figure 7B). However, only (32 ± 8)% of Cerulean-Ring1b fusion protein associated with mitotic chromosomes (Figure 4, B and D). The fraction of mitotic retention of Ringb1b fusion protein in Cbx2 KO ES cell lines complemented with Cbx21-498 was similar to that observed in Cbx2 KO ES cells. These data indicate that the Cbx2 interaction with Ring1b is required for the recruitment of Cerulean-Ring1b fusion protein 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)

Analysis of structural elements of YFP-Cbx2 fusion protein required for its targeting and immobilizing. (A) Diagram of structural domains of Cbx2. The dashed rectangles indicate the region required for targeting Cbx2 to mitotic chromosomes (left) and the region required for immobilizing Cbx2 at mitotic chromosomes (right). ATH, AT-hook domain; Cbox, chromobox domain; CHD, chromodomain domain. The numbers in parentheses indicate the starting and ending of amino acid sequence. (B) Confocal images of YFP-Cbx2 and its variant fusion proteins in metaphase of HeLa cells. YFP-Cbx2 mutant and mCherry-H2A fusion proteins were stably expressed in HeLa cells. The mCherry-H2A was used to mark mitotic chromosomes. Scale bar, 5 μm. (C) FRAP curves of interphases and metaphases of YFP-Cbx2 and its variant fusion proteins expressed in HeLa cells. FRAP analysis is described in Figure 5. (D) Residence time and immobile fraction of YFP-Cbx2 variant fusion proteins at interphasic and mitotic chromatins. The residence time and immobile fraction were calculated from FRAP curves by fitting a first-order kinetic model.
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Figure 7: Analysis of structural elements of YFP-Cbx2 fusion protein required for its targeting and immobilizing. (A) Diagram of structural domains of Cbx2. The dashed rectangles indicate the region required for targeting Cbx2 to mitotic chromosomes (left) and the region required for immobilizing Cbx2 at mitotic chromosomes (right). ATH, AT-hook domain; Cbox, chromobox domain; CHD, chromodomain domain. The numbers in parentheses indicate the starting and ending of amino acid sequence. (B) Confocal images of YFP-Cbx2 and its variant fusion proteins in metaphase of HeLa cells. YFP-Cbx2 mutant and mCherry-H2A fusion proteins were stably expressed in HeLa cells. The mCherry-H2A was used to mark mitotic chromosomes. Scale bar, 5 μm. (C) FRAP curves of interphases and metaphases of YFP-Cbx2 and its variant fusion proteins expressed in HeLa cells. FRAP analysis is described in Figure 5. (D) Residence time and immobile fraction of YFP-Cbx2 variant fusion proteins at interphasic and mitotic chromatins. The residence time and immobile fraction were calculated from FRAP curves by fitting a first-order kinetic model.
Mentions: To test whether the Cbx2 interaction with Ring1b is required for the recruitment of Ring1b protein to mitotic chromosomes, the three fusion proteins mCherry-H2A, Cerulean-Ring1b, and YFP-Cbx21-498 were expressed in Cbx2 KO ES cells. The YFP-Cbx21-498 fusion protein lacks the chromobox (Cbox) domain required for interaction with Ring1b (Satijn et al., 1997; Schoorlemmer et al., 1997; Bardos et al., 2000). We expected that the Cbx2 mutant fusion protein would not be able to recruit Cerulean-Ring1b to mitotic chromosomes. Quantitative image analysis showed that (92 ± 7)% of YFP-Cbx21-498 fusion protein accumulated at mitotic chromosomes, indicating the deletion of the Cbox domain of Cbx2 protein does not affect its mitotic chromosomal association (Figure 4, B and C; also see later discussion of Figure 7B). However, only (32 ± 8)% of Cerulean-Ring1b fusion protein associated with mitotic chromosomes (Figure 4, B and D). The fraction of mitotic retention of Ringb1b fusion protein in Cbx2 KO ES cell lines complemented with Cbx21-498 was similar to that observed in Cbx2 KO ES cells. These data indicate that the Cbx2 interaction with Ring1b is required for the recruitment of Cerulean-Ring1b fusion protein 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