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A novel chromatin tether domain controls topoisomerase IIα dynamics and mitotic chromosome formation.

Lane AB, Giménez-Abián JF, Clarke DJ - J. Cell Biol. (2013)

Bottom Line: Here we describe a critical mechanism of chromatin recruitment and exchange that relies on a novel chromatin tether (ChT) domain and mediates interaction with histone H3 and DNA.We show that the ChT domain controls the residence time of Topo IIα on chromatin in mitosis and is necessary for the formation of mitotic chromosomes.Our data suggest that the dynamics of Topo IIα on chromosomes are important for successful mitosis and implicate histone tail posttranslational modifications in regulating Topo IIα.

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Affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455.

ABSTRACT
DNA topoisomerase IIα (Topo IIα) is the target of an important class of anticancer drugs, but tumor cells can become resistant by reducing the association of the enzyme with chromosomes. Here we describe a critical mechanism of chromatin recruitment and exchange that relies on a novel chromatin tether (ChT) domain and mediates interaction with histone H3 and DNA. We show that the ChT domain controls the residence time of Topo IIα on chromatin in mitosis and is necessary for the formation of mitotic chromosomes. Our data suggest that the dynamics of Topo IIα on chromosomes are important for successful mitosis and implicate histone tail posttranslational modifications in regulating Topo IIα.

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Topo IIα localizes to the axial core of M. muntjak mitotic chromosomes. (A) GFP–human Topo IIα transfected into M. muntjak cells does not coimmunoprecipitate with endogenous M. muntjak Topo IIα when precipitated using an anti-GFP antibody (right). GFP–human Topo IIα ΔCTR (i.e., amino acids 1–1,321) transfected into HeLa cells and immunoprecipitated in the same way coprecipitates the endogenous, untagged protein (left). (The anti–Topo IIα antibody used for Western blotting recognizes only WT human or M. muntjak Topo IIα and not Topo IIα ΔCTR.) (B) Human Topo IIα localization in M. muntjak cells transfected with a construct encoding GFP–Topo IIα, fixed and stained with DAPI, demonstrating densely punctate localization of GFP–Topo IIα to the axial core of mitotic chromosomes (bottom) and nuclear localization during interphase (top). Bars, 10 µm. (See also Video 1.) (C, left) Enlarged portion of region marked by the broken-line box in B to show apparent coiling of the axial core. (C, right) A representative (more than three experimental repeats) plot across mitotic chromosome arms showing that the GFP signal (Topo IIα) occupies a narrower region than the DNA (DAPI) signal (corresponds to the solid boxed region in B). Bars, 1 µm.
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fig1: Topo IIα localizes to the axial core of M. muntjak mitotic chromosomes. (A) GFP–human Topo IIα transfected into M. muntjak cells does not coimmunoprecipitate with endogenous M. muntjak Topo IIα when precipitated using an anti-GFP antibody (right). GFP–human Topo IIα ΔCTR (i.e., amino acids 1–1,321) transfected into HeLa cells and immunoprecipitated in the same way coprecipitates the endogenous, untagged protein (left). (The anti–Topo IIα antibody used for Western blotting recognizes only WT human or M. muntjak Topo IIα and not Topo IIα ΔCTR.) (B) Human Topo IIα localization in M. muntjak cells transfected with a construct encoding GFP–Topo IIα, fixed and stained with DAPI, demonstrating densely punctate localization of GFP–Topo IIα to the axial core of mitotic chromosomes (bottom) and nuclear localization during interphase (top). Bars, 10 µm. (See also Video 1.) (C, left) Enlarged portion of region marked by the broken-line box in B to show apparent coiling of the axial core. (C, right) A representative (more than three experimental repeats) plot across mitotic chromosome arms showing that the GFP signal (Topo IIα) occupies a narrower region than the DNA (DAPI) signal (corresponds to the solid boxed region in B). Bars, 1 µm.

Mentions: To determine the mechanism of Topo IIα localization to mitotic chromosomes in intact mammalian cells, we used a previously characterized human fusion protein (GFP-hTopo IIα; Tavormina et al., 2002) and expressed it in M. muntjak cells. These cells are well suited to this study because M. muntjak has the largest mammalian chromosomes and the lowest diploid chromosomal number in mammals (2n = 6, females), facilitating detailed visualization of individual arms and centromeres. Further, we found that human Topo IIα cannot be immunoprecipitated with the M. muntjak orthologue, which indicates that these interspecies isoforms cannot dimerize (Fig. 1 A). This was important because type II topoisomerases function as stable homodimeric enzymes (Tennyson and Lindsley, 1997), raising the concern that a transfected mutant Topo IIα would dimerize with the endogenous wild-type (WT) protein and localize passively, masking effects of mutations. We thus analyzed mutant transfected forms of GFP-hTopo IIα that apparently localized independently of the WT endogenous M. muntjak protein.


A novel chromatin tether domain controls topoisomerase IIα dynamics and mitotic chromosome formation.

Lane AB, Giménez-Abián JF, Clarke DJ - J. Cell Biol. (2013)

Topo IIα localizes to the axial core of M. muntjak mitotic chromosomes. (A) GFP–human Topo IIα transfected into M. muntjak cells does not coimmunoprecipitate with endogenous M. muntjak Topo IIα when precipitated using an anti-GFP antibody (right). GFP–human Topo IIα ΔCTR (i.e., amino acids 1–1,321) transfected into HeLa cells and immunoprecipitated in the same way coprecipitates the endogenous, untagged protein (left). (The anti–Topo IIα antibody used for Western blotting recognizes only WT human or M. muntjak Topo IIα and not Topo IIα ΔCTR.) (B) Human Topo IIα localization in M. muntjak cells transfected with a construct encoding GFP–Topo IIα, fixed and stained with DAPI, demonstrating densely punctate localization of GFP–Topo IIα to the axial core of mitotic chromosomes (bottom) and nuclear localization during interphase (top). Bars, 10 µm. (See also Video 1.) (C, left) Enlarged portion of region marked by the broken-line box in B to show apparent coiling of the axial core. (C, right) A representative (more than three experimental repeats) plot across mitotic chromosome arms showing that the GFP signal (Topo IIα) occupies a narrower region than the DNA (DAPI) signal (corresponds to the solid boxed region in B). Bars, 1 µm.
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fig1: Topo IIα localizes to the axial core of M. muntjak mitotic chromosomes. (A) GFP–human Topo IIα transfected into M. muntjak cells does not coimmunoprecipitate with endogenous M. muntjak Topo IIα when precipitated using an anti-GFP antibody (right). GFP–human Topo IIα ΔCTR (i.e., amino acids 1–1,321) transfected into HeLa cells and immunoprecipitated in the same way coprecipitates the endogenous, untagged protein (left). (The anti–Topo IIα antibody used for Western blotting recognizes only WT human or M. muntjak Topo IIα and not Topo IIα ΔCTR.) (B) Human Topo IIα localization in M. muntjak cells transfected with a construct encoding GFP–Topo IIα, fixed and stained with DAPI, demonstrating densely punctate localization of GFP–Topo IIα to the axial core of mitotic chromosomes (bottom) and nuclear localization during interphase (top). Bars, 10 µm. (See also Video 1.) (C, left) Enlarged portion of region marked by the broken-line box in B to show apparent coiling of the axial core. (C, right) A representative (more than three experimental repeats) plot across mitotic chromosome arms showing that the GFP signal (Topo IIα) occupies a narrower region than the DNA (DAPI) signal (corresponds to the solid boxed region in B). Bars, 1 µm.
Mentions: To determine the mechanism of Topo IIα localization to mitotic chromosomes in intact mammalian cells, we used a previously characterized human fusion protein (GFP-hTopo IIα; Tavormina et al., 2002) and expressed it in M. muntjak cells. These cells are well suited to this study because M. muntjak has the largest mammalian chromosomes and the lowest diploid chromosomal number in mammals (2n = 6, females), facilitating detailed visualization of individual arms and centromeres. Further, we found that human Topo IIα cannot be immunoprecipitated with the M. muntjak orthologue, which indicates that these interspecies isoforms cannot dimerize (Fig. 1 A). This was important because type II topoisomerases function as stable homodimeric enzymes (Tennyson and Lindsley, 1997), raising the concern that a transfected mutant Topo IIα would dimerize with the endogenous wild-type (WT) protein and localize passively, masking effects of mutations. We thus analyzed mutant transfected forms of GFP-hTopo IIα that apparently localized independently of the WT endogenous M. muntjak protein.

Bottom Line: Here we describe a critical mechanism of chromatin recruitment and exchange that relies on a novel chromatin tether (ChT) domain and mediates interaction with histone H3 and DNA.We show that the ChT domain controls the residence time of Topo IIα on chromatin in mitosis and is necessary for the formation of mitotic chromosomes.Our data suggest that the dynamics of Topo IIα on chromosomes are important for successful mitosis and implicate histone tail posttranslational modifications in regulating Topo IIα.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455.

ABSTRACT
DNA topoisomerase IIα (Topo IIα) is the target of an important class of anticancer drugs, but tumor cells can become resistant by reducing the association of the enzyme with chromosomes. Here we describe a critical mechanism of chromatin recruitment and exchange that relies on a novel chromatin tether (ChT) domain and mediates interaction with histone H3 and DNA. We show that the ChT domain controls the residence time of Topo IIα on chromatin in mitosis and is necessary for the formation of mitotic chromosomes. Our data suggest that the dynamics of Topo IIα on chromosomes are important for successful mitosis and implicate histone tail posttranslational modifications in regulating Topo IIα.

Show MeSH
Related in: MedlinePlus