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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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An inducible system for analysis of Topo IIα mutants. (A, left) Schematic describing the strategy to derive HeLa EM2-11ht cells expressing RNAi-resistant mCherry–Topo IIα at close to endogenous levels. Single copy insertion of Topo IIα alleles at locus 5q31.3 was achieved using asymmetric FLP (F/F3) recombinase sites (see Materials and methods and Weidenfeld et al., 2009). (A, right) Immunoblot detecting endogenous Topo IIα and exogenously expressed mCherry–Topo IIα. HeLa EM2-11 cells were infected with lentiviral particles encoding knockdown shRNA constructs at day 0 against Topo IIα and Topo IIβ simultaneously with the addition of 250 ng/ml doxycycline to induce expression of the exogenous allele. Endogenous Topo IIα is effectively depleted, whereas the exogenous protein is expressed to approximately endogenous levels within 24 h. (B) Localization of mCherry–Topo IIα and mCherry–Topo IIα ΔChT after 48 h of induction with doxycycline in live HeLa EM2-11 cells (top) or after fixation with methanol (bottom). Topo IIα ΔChT is labile upon fixation. Bars, 10 µm.
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fig7: An inducible system for analysis of Topo IIα mutants. (A, left) Schematic describing the strategy to derive HeLa EM2-11ht cells expressing RNAi-resistant mCherry–Topo IIα at close to endogenous levels. Single copy insertion of Topo IIα alleles at locus 5q31.3 was achieved using asymmetric FLP (F/F3) recombinase sites (see Materials and methods and Weidenfeld et al., 2009). (A, right) Immunoblot detecting endogenous Topo IIα and exogenously expressed mCherry–Topo IIα. HeLa EM2-11 cells were infected with lentiviral particles encoding knockdown shRNA constructs at day 0 against Topo IIα and Topo IIβ simultaneously with the addition of 250 ng/ml doxycycline to induce expression of the exogenous allele. Endogenous Topo IIα is effectively depleted, whereas the exogenous protein is expressed to approximately endogenous levels within 24 h. (B) Localization of mCherry–Topo IIα and mCherry–Topo IIα ΔChT after 48 h of induction with doxycycline in live HeLa EM2-11 cells (top) or after fixation with methanol (bottom). Topo IIα ΔChT is labile upon fixation. Bars, 10 µm.

Mentions: The system employs a HeLa cell line (EM2-11ht) in which a gene of interest is integrated stably at the well-characterized 5q31.3 “silent-but-activatable” genomic locus using the FLP recombinase (Fig. 7 A, left; Weidenfeld et al., 2009). This avoids detectable expression in the absence of doxycycline (Fig. 7 A, right). Constructs were inserted encoding WT mCherry–Topo IIα, mCherry–Topo IIα ΔChT, or mCherry–Topo IIα Y804F (a catalytic mutant of the active site tyrosine, serving as a negative control), and clonal lines were isolated. Because the locus of integration was the same for all cell lines generated, the induction characteristics were similar, permitting a direct comparison of independently derived transgenic cell lines. This enabled us to determine a concentration of doxycycline at which the level of transgenic mCherry–Topo IIα was similar to that of the endogenous protein (Fig. 7 A, right). Lentivirus-mediated shRNA resulted in depletion of endogenous Topo IIα to levels undetectable by immunoblotting (Fig. 7 A, right). Transgenic mCherry–Topo IIα and mutants of interest were rendered insensitive to the shRNA construct by introducing silent mutations before integration.


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

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

An inducible system for analysis of Topo IIα mutants. (A, left) Schematic describing the strategy to derive HeLa EM2-11ht cells expressing RNAi-resistant mCherry–Topo IIα at close to endogenous levels. Single copy insertion of Topo IIα alleles at locus 5q31.3 was achieved using asymmetric FLP (F/F3) recombinase sites (see Materials and methods and Weidenfeld et al., 2009). (A, right) Immunoblot detecting endogenous Topo IIα and exogenously expressed mCherry–Topo IIα. HeLa EM2-11 cells were infected with lentiviral particles encoding knockdown shRNA constructs at day 0 against Topo IIα and Topo IIβ simultaneously with the addition of 250 ng/ml doxycycline to induce expression of the exogenous allele. Endogenous Topo IIα is effectively depleted, whereas the exogenous protein is expressed to approximately endogenous levels within 24 h. (B) Localization of mCherry–Topo IIα and mCherry–Topo IIα ΔChT after 48 h of induction with doxycycline in live HeLa EM2-11 cells (top) or after fixation with methanol (bottom). Topo IIα ΔChT is labile upon fixation. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3824022&req=5

fig7: An inducible system for analysis of Topo IIα mutants. (A, left) Schematic describing the strategy to derive HeLa EM2-11ht cells expressing RNAi-resistant mCherry–Topo IIα at close to endogenous levels. Single copy insertion of Topo IIα alleles at locus 5q31.3 was achieved using asymmetric FLP (F/F3) recombinase sites (see Materials and methods and Weidenfeld et al., 2009). (A, right) Immunoblot detecting endogenous Topo IIα and exogenously expressed mCherry–Topo IIα. HeLa EM2-11 cells were infected with lentiviral particles encoding knockdown shRNA constructs at day 0 against Topo IIα and Topo IIβ simultaneously with the addition of 250 ng/ml doxycycline to induce expression of the exogenous allele. Endogenous Topo IIα is effectively depleted, whereas the exogenous protein is expressed to approximately endogenous levels within 24 h. (B) Localization of mCherry–Topo IIα and mCherry–Topo IIα ΔChT after 48 h of induction with doxycycline in live HeLa EM2-11 cells (top) or after fixation with methanol (bottom). Topo IIα ΔChT is labile upon fixation. Bars, 10 µm.
Mentions: The system employs a HeLa cell line (EM2-11ht) in which a gene of interest is integrated stably at the well-characterized 5q31.3 “silent-but-activatable” genomic locus using the FLP recombinase (Fig. 7 A, left; Weidenfeld et al., 2009). This avoids detectable expression in the absence of doxycycline (Fig. 7 A, right). Constructs were inserted encoding WT mCherry–Topo IIα, mCherry–Topo IIα ΔChT, or mCherry–Topo IIα Y804F (a catalytic mutant of the active site tyrosine, serving as a negative control), and clonal lines were isolated. Because the locus of integration was the same for all cell lines generated, the induction characteristics were similar, permitting a direct comparison of independently derived transgenic cell lines. This enabled us to determine a concentration of doxycycline at which the level of transgenic mCherry–Topo IIα was similar to that of the endogenous protein (Fig. 7 A, right). Lentivirus-mediated shRNA resulted in depletion of endogenous Topo IIα to levels undetectable by immunoblotting (Fig. 7 A, right). Transgenic mCherry–Topo IIα and mutants of interest were rendered insensitive to the shRNA construct by introducing silent mutations before integration.

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