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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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Nuclear localization of Topo IIα during interphase is not necessary for localization to chromosomes in mitosis. (A, top) Sequence of the extreme CTR of Topo IIα. Red residues indicate putative constituents of the bipartite NLS. Blue residues indicate the ChT domain described herein. (A, bottom) Schematic showing GFP- and mCherry-tagged WT Topo IIα and mutants and truncation constructs used herein. GFP–Topo IIα K3R comprised of K1489, K1490, and K1492 mutated to Arg. (B) M. muntjak cells transfected with GFP–Topo IIα or GFP–Topo IIα K3R imaged in live interphase cells. The WT protein is predominantly nuclear; the K3R mutant is predominantly cytoplasmic. Bars, 10 µm. (C) GFP–Topo IIα K3R localizes similarly to WT GFP–Topo IIα in live mitotic M. muntjak cells. Lines indicate locations of line profile plots in Fig 2 D. See also Video 2. Bars, 10 µm. (D) Quantification of WT Topo IIα and Topo IIα K3R abundance at mitotic chromosome cores in live M. muntjak cells. Both localize to the chromosome core with equal intensity. Shown are the mean of maximum intensities across n = 73 (K3R) and n = 53 (WT) core regions after background subtraction. P-value indicates two-tailed unpaired samples t test. Error bars indicate SD.
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fig2: Nuclear localization of Topo IIα during interphase is not necessary for localization to chromosomes in mitosis. (A, top) Sequence of the extreme CTR of Topo IIα. Red residues indicate putative constituents of the bipartite NLS. Blue residues indicate the ChT domain described herein. (A, bottom) Schematic showing GFP- and mCherry-tagged WT Topo IIα and mutants and truncation constructs used herein. GFP–Topo IIα K3R comprised of K1489, K1490, and K1492 mutated to Arg. (B) M. muntjak cells transfected with GFP–Topo IIα or GFP–Topo IIα K3R imaged in live interphase cells. The WT protein is predominantly nuclear; the K3R mutant is predominantly cytoplasmic. Bars, 10 µm. (C) GFP–Topo IIα K3R localizes similarly to WT GFP–Topo IIα in live mitotic M. muntjak cells. Lines indicate locations of line profile plots in Fig 2 D. See also Video 2. Bars, 10 µm. (D) Quantification of WT Topo IIα and Topo IIα K3R abundance at mitotic chromosome cores in live M. muntjak cells. Both localize to the chromosome core with equal intensity. Shown are the mean of maximum intensities across n = 73 (K3R) and n = 53 (WT) core regions after background subtraction. P-value indicates two-tailed unpaired samples t test. Error bars indicate SD.

Mentions: Near the C terminus of Topo IIα, a cluster of basic residues (aa 1,490–1,492) has been reported to be part of a functional bipartite NLS (Mirski et al., 1997; Wessel et al., 1997; Fig. 2 A, red text). Because NLS sequences can rely on stretches of positively charged residues (Conti et al., 1998; Cokol et al., 2000; Nitiss, 2009), we generated a triple mutant at lysines 1,489, 1,490, and 1,492, substituting with the conservative change to arginine that presumably minimizes changes to the overall structure of the protein. This mutant (Topo IIα K3R) was indeed defective for nuclear import, remaining predominantly cytosolic in interphase (Fig. 2 B).


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

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

Nuclear localization of Topo IIα during interphase is not necessary for localization to chromosomes in mitosis. (A, top) Sequence of the extreme CTR of Topo IIα. Red residues indicate putative constituents of the bipartite NLS. Blue residues indicate the ChT domain described herein. (A, bottom) Schematic showing GFP- and mCherry-tagged WT Topo IIα and mutants and truncation constructs used herein. GFP–Topo IIα K3R comprised of K1489, K1490, and K1492 mutated to Arg. (B) M. muntjak cells transfected with GFP–Topo IIα or GFP–Topo IIα K3R imaged in live interphase cells. The WT protein is predominantly nuclear; the K3R mutant is predominantly cytoplasmic. Bars, 10 µm. (C) GFP–Topo IIα K3R localizes similarly to WT GFP–Topo IIα in live mitotic M. muntjak cells. Lines indicate locations of line profile plots in Fig 2 D. See also Video 2. Bars, 10 µm. (D) Quantification of WT Topo IIα and Topo IIα K3R abundance at mitotic chromosome cores in live M. muntjak cells. Both localize to the chromosome core with equal intensity. Shown are the mean of maximum intensities across n = 73 (K3R) and n = 53 (WT) core regions after background subtraction. P-value indicates two-tailed unpaired samples t test. Error bars indicate SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3824022&req=5

fig2: Nuclear localization of Topo IIα during interphase is not necessary for localization to chromosomes in mitosis. (A, top) Sequence of the extreme CTR of Topo IIα. Red residues indicate putative constituents of the bipartite NLS. Blue residues indicate the ChT domain described herein. (A, bottom) Schematic showing GFP- and mCherry-tagged WT Topo IIα and mutants and truncation constructs used herein. GFP–Topo IIα K3R comprised of K1489, K1490, and K1492 mutated to Arg. (B) M. muntjak cells transfected with GFP–Topo IIα or GFP–Topo IIα K3R imaged in live interphase cells. The WT protein is predominantly nuclear; the K3R mutant is predominantly cytoplasmic. Bars, 10 µm. (C) GFP–Topo IIα K3R localizes similarly to WT GFP–Topo IIα in live mitotic M. muntjak cells. Lines indicate locations of line profile plots in Fig 2 D. See also Video 2. Bars, 10 µm. (D) Quantification of WT Topo IIα and Topo IIα K3R abundance at mitotic chromosome cores in live M. muntjak cells. Both localize to the chromosome core with equal intensity. Shown are the mean of maximum intensities across n = 73 (K3R) and n = 53 (WT) core regions after background subtraction. P-value indicates two-tailed unpaired samples t test. Error bars indicate SD.
Mentions: Near the C terminus of Topo IIα, a cluster of basic residues (aa 1,490–1,492) has been reported to be part of a functional bipartite NLS (Mirski et al., 1997; Wessel et al., 1997; Fig. 2 A, red text). Because NLS sequences can rely on stretches of positively charged residues (Conti et al., 1998; Cokol et al., 2000; Nitiss, 2009), we generated a triple mutant at lysines 1,489, 1,490, and 1,492, substituting with the conservative change to arginine that presumably minimizes changes to the overall structure of the protein. This mutant (Topo IIα K3R) was indeed defective for nuclear import, remaining predominantly cytosolic in interphase (Fig. 2 B).

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