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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.

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.

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The C-terminal 31 residues of Topo IIα are important for CTR chromosomal association and association with DNA in vitro. (A) M. muntjak cells transfected with mCherry–Topo IIα CTR (Topo IIα residues 1,321–1,531) or mCherry–Topo IIα CTRΔ31 (Topo IIα residues 1,321–1,500) imaged in live metaphase cells. mCherry–Topo IIα CTR localizes to mitotic chromosomes, whereas mCherry–Topo IIα CTRΔ31 is localized diffusely through the nucleoplasm. (A, right) A representative quantification (>3 experimental repeats) of mCherry signal across mitotic chromosomes (the broken lines in the images). Bars, 5 µm. (B) The CTR of Topo IIα binds to plasmid DNA in vitro. EMSA analysis of supercoiled, linear, and relaxed pUC19 DNA mixed with recombinant histone H1° or CTR fragments of Topo IIα (defined in A) and resolved on agarose gels. M = 1 kb ladder. The star indicates the lane where the H1°–DNA complex is assumed to be net positively charged and has reversed its migration direction. Brackets highlight the mobility shifts in the 12.5–25 pmol range for CTR and CTRΔ31. (C) Schematic showing recombinant Topo IIα fragments used in DNA–beads binding assays (D). (D, left) Anti-His tag immunoblot of Topo IIα fragment pull-downs using DNA-coated beads and purified HIS-tagged Topo IIα CTR fragments. (D, right) Quantification of pull-down efficiency when uncoated bead background is subtracted (percentage of input). Topo IIα CTRΔ31 and Topo IIα CTRΔ52 bind DNA with reduced efficiency. The CTR of Topo IIβ (βCTR, residues 1,359–1,621) has negligible DNA-binding activity. n = 3. Error bars indicate SD. n.s., not a statistically significant difference.
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fig3: The C-terminal 31 residues of Topo IIα are important for CTR chromosomal association and association with DNA in vitro. (A) M. muntjak cells transfected with mCherry–Topo IIα CTR (Topo IIα residues 1,321–1,531) or mCherry–Topo IIα CTRΔ31 (Topo IIα residues 1,321–1,500) imaged in live metaphase cells. mCherry–Topo IIα CTR localizes to mitotic chromosomes, whereas mCherry–Topo IIα CTRΔ31 is localized diffusely through the nucleoplasm. (A, right) A representative quantification (>3 experimental repeats) of mCherry signal across mitotic chromosomes (the broken lines in the images). Bars, 5 µm. (B) The CTR of Topo IIα binds to plasmid DNA in vitro. EMSA analysis of supercoiled, linear, and relaxed pUC19 DNA mixed with recombinant histone H1° or CTR fragments of Topo IIα (defined in A) and resolved on agarose gels. M = 1 kb ladder. The star indicates the lane where the H1°–DNA complex is assumed to be net positively charged and has reversed its migration direction. Brackets highlight the mobility shifts in the 12.5–25 pmol range for CTR and CTRΔ31. (C) Schematic showing recombinant Topo IIα fragments used in DNA–beads binding assays (D). (D, left) Anti-His tag immunoblot of Topo IIα fragment pull-downs using DNA-coated beads and purified HIS-tagged Topo IIα CTR fragments. (D, right) Quantification of pull-down efficiency when uncoated bead background is subtracted (percentage of input). Topo IIα CTRΔ31 and Topo IIα CTRΔ52 bind DNA with reduced efficiency. The CTR of Topo IIβ (βCTR, residues 1,359–1,621) has negligible DNA-binding activity. n = 3. Error bars indicate SD. n.s., not a statistically significant difference.

Mentions: Linka et al. (2007) have shown that the C-terminal 211 residues of hTopo IIα (1,321–1,531) are sufficient to direct localization of a fluorescent protein to mitotic human chromosomes. However, it is not known if this fragment can bind to the endogenous full-length human Topo IIα and be carried to chromosomes passively. We fused this region, referred to as the CTR of hTopo IIα, to mCherry and expressed it in M. muntjak cells. We found that mCherry-CTR localized efficiently to mitotic M. muntjak chromosomes (Fig. 3 A). Although the CTR was enriched at the centromere regions, like full-length Topo IIα, the CTR occupied a broader localization on the chromosome arms, rather than being enriched at the axial core. This indicates that the CTR mediates a more broad interaction with chromosomes, similar to the observed localization of full-length Topo IIα seen in the absence of condensin (Coelho et al., 2003). Together, the data suggest that the CTR could provide a condensin-independent mechanism of chromatin association. Strikingly, a truncated CTR protein lacking only the extreme C-terminal 31 residues of the Topo IIα CTR (CTRΔ31) showed only minimal enrichment on mitotic chromosomes, which indicates that these are key residues that facilitate chromatin association (Fig. 3 A).


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

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

The C-terminal 31 residues of Topo IIα are important for CTR chromosomal association and association with DNA in vitro. (A) M. muntjak cells transfected with mCherry–Topo IIα CTR (Topo IIα residues 1,321–1,531) or mCherry–Topo IIα CTRΔ31 (Topo IIα residues 1,321–1,500) imaged in live metaphase cells. mCherry–Topo IIα CTR localizes to mitotic chromosomes, whereas mCherry–Topo IIα CTRΔ31 is localized diffusely through the nucleoplasm. (A, right) A representative quantification (>3 experimental repeats) of mCherry signal across mitotic chromosomes (the broken lines in the images). Bars, 5 µm. (B) The CTR of Topo IIα binds to plasmid DNA in vitro. EMSA analysis of supercoiled, linear, and relaxed pUC19 DNA mixed with recombinant histone H1° or CTR fragments of Topo IIα (defined in A) and resolved on agarose gels. M = 1 kb ladder. The star indicates the lane where the H1°–DNA complex is assumed to be net positively charged and has reversed its migration direction. Brackets highlight the mobility shifts in the 12.5–25 pmol range for CTR and CTRΔ31. (C) Schematic showing recombinant Topo IIα fragments used in DNA–beads binding assays (D). (D, left) Anti-His tag immunoblot of Topo IIα fragment pull-downs using DNA-coated beads and purified HIS-tagged Topo IIα CTR fragments. (D, right) Quantification of pull-down efficiency when uncoated bead background is subtracted (percentage of input). Topo IIα CTRΔ31 and Topo IIα CTRΔ52 bind DNA with reduced efficiency. The CTR of Topo IIβ (βCTR, residues 1,359–1,621) has negligible DNA-binding activity. n = 3. Error bars indicate SD. n.s., not a statistically significant difference.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3824022&req=5

fig3: The C-terminal 31 residues of Topo IIα are important for CTR chromosomal association and association with DNA in vitro. (A) M. muntjak cells transfected with mCherry–Topo IIα CTR (Topo IIα residues 1,321–1,531) or mCherry–Topo IIα CTRΔ31 (Topo IIα residues 1,321–1,500) imaged in live metaphase cells. mCherry–Topo IIα CTR localizes to mitotic chromosomes, whereas mCherry–Topo IIα CTRΔ31 is localized diffusely through the nucleoplasm. (A, right) A representative quantification (>3 experimental repeats) of mCherry signal across mitotic chromosomes (the broken lines in the images). Bars, 5 µm. (B) The CTR of Topo IIα binds to plasmid DNA in vitro. EMSA analysis of supercoiled, linear, and relaxed pUC19 DNA mixed with recombinant histone H1° or CTR fragments of Topo IIα (defined in A) and resolved on agarose gels. M = 1 kb ladder. The star indicates the lane where the H1°–DNA complex is assumed to be net positively charged and has reversed its migration direction. Brackets highlight the mobility shifts in the 12.5–25 pmol range for CTR and CTRΔ31. (C) Schematic showing recombinant Topo IIα fragments used in DNA–beads binding assays (D). (D, left) Anti-His tag immunoblot of Topo IIα fragment pull-downs using DNA-coated beads and purified HIS-tagged Topo IIα CTR fragments. (D, right) Quantification of pull-down efficiency when uncoated bead background is subtracted (percentage of input). Topo IIα CTRΔ31 and Topo IIα CTRΔ52 bind DNA with reduced efficiency. The CTR of Topo IIβ (βCTR, residues 1,359–1,621) has negligible DNA-binding activity. n = 3. Error bars indicate SD. n.s., not a statistically significant difference.
Mentions: Linka et al. (2007) have shown that the C-terminal 211 residues of hTopo IIα (1,321–1,531) are sufficient to direct localization of a fluorescent protein to mitotic human chromosomes. However, it is not known if this fragment can bind to the endogenous full-length human Topo IIα and be carried to chromosomes passively. We fused this region, referred to as the CTR of hTopo IIα, to mCherry and expressed it in M. muntjak cells. We found that mCherry-CTR localized efficiently to mitotic M. muntjak chromosomes (Fig. 3 A). Although the CTR was enriched at the centromere regions, like full-length Topo IIα, the CTR occupied a broader localization on the chromosome arms, rather than being enriched at the axial core. This indicates that the CTR mediates a more broad interaction with chromosomes, similar to the observed localization of full-length Topo IIα seen in the absence of condensin (Coelho et al., 2003). Together, the data suggest that the CTR could provide a condensin-independent mechanism of chromatin association. Strikingly, a truncated CTR protein lacking only the extreme C-terminal 31 residues of the Topo IIα CTR (CTRΔ31) showed only minimal enrichment on mitotic chromosomes, which indicates that these are key residues that facilitate chromatin association (Fig. 3 A).

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