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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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The ChT domain of Topo IIαis necessary for chromosome condensation and segregation in mitosis. (A and B) Still images taken from digital time-lapse imaging of live cells after depletion of endogenous Topo IIα and induced with doxycycline to express mCherry–Topo IIα or mCherry–Topo IIα ΔChT. Cells were assayed after a double thymidine synchrony and release protocol in conjunction with shRNA-mediated depletion of the endogenous Topo IIα and DNA observed via imaging of Hoechst. Red boxed regions are enlarged in B to highlight the differences in metaphase chromatin morphology. Frames were taken at 5-min intervals. Time is indicated (hours:minutes) in each movie frame. Bars, 10 µm. (C) Distribution of phenotypes seen in live single cells described in A and B. Induction of mCherry–Topo IIα rescues chromosome condensation and segregation defects, whereas Topo IIα ΔChT fails to rescue. WT − Dox, n = 96; WT + Dox, n = 100; ΔChT − Dox, n = 61; ΔChT + Dox, n = 82.
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fig9: The ChT domain of Topo IIαis necessary for chromosome condensation and segregation in mitosis. (A and B) Still images taken from digital time-lapse imaging of live cells after depletion of endogenous Topo IIα and induced with doxycycline to express mCherry–Topo IIα or mCherry–Topo IIα ΔChT. Cells were assayed after a double thymidine synchrony and release protocol in conjunction with shRNA-mediated depletion of the endogenous Topo IIα and DNA observed via imaging of Hoechst. Red boxed regions are enlarged in B to highlight the differences in metaphase chromatin morphology. Frames were taken at 5-min intervals. Time is indicated (hours:minutes) in each movie frame. Bars, 10 µm. (C) Distribution of phenotypes seen in live single cells described in A and B. Induction of mCherry–Topo IIα rescues chromosome condensation and segregation defects, whereas Topo IIα ΔChT fails to rescue. WT − Dox, n = 96; WT + Dox, n = 100; ΔChT − Dox, n = 61; ΔChT + Dox, n = 82.

Mentions: We next investigated the biological consequences of the altered dynamics of mCherry–Topo IIα ΔChT in terms of mitotic chromosome formation. Because previous studies have shown that Topo IIβ can weakly compensate for the lack of Topo IIα (Linka et al., 2007), we codepleted both isoforms, using shRNA lentiviral transduction, to achieve a minimum base level of type II topoisomerase activity (Fig. S3). We simultaneously induced expression of mCherry–Topo IIα, mCherry–Topo IIα ΔChT, or mCherry–Topo IIα Y804F (as a negative control) in the isogenic cell lines and monitored changes in metaphase chromosome morphology when cells reached mitosis after release from a double thymidine synchrony. Giemsa staining of Carnoy’s fixed cells allowed fine-scale examination of changes in chromosome morphology (Giménez-Abián et al., 1995; Giménez-Abián and Clarke, 2009; Fig. 8, C and D). In addition, we filmed cells, treated in the same manner, by digital time-lapse microscopy to observe single live cells as they entered and progressed through mitosis (Fig. 9).


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 ChT domain of Topo IIαis necessary for chromosome condensation and segregation in mitosis. (A and B) Still images taken from digital time-lapse imaging of live cells after depletion of endogenous Topo IIα and induced with doxycycline to express mCherry–Topo IIα or mCherry–Topo IIα ΔChT. Cells were assayed after a double thymidine synchrony and release protocol in conjunction with shRNA-mediated depletion of the endogenous Topo IIα and DNA observed via imaging of Hoechst. Red boxed regions are enlarged in B to highlight the differences in metaphase chromatin morphology. Frames were taken at 5-min intervals. Time is indicated (hours:minutes) in each movie frame. Bars, 10 µm. (C) Distribution of phenotypes seen in live single cells described in A and B. Induction of mCherry–Topo IIα rescues chromosome condensation and segregation defects, whereas Topo IIα ΔChT fails to rescue. WT − Dox, n = 96; WT + Dox, n = 100; ΔChT − Dox, n = 61; ΔChT + Dox, n = 82.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3824022&req=5

fig9: The ChT domain of Topo IIαis necessary for chromosome condensation and segregation in mitosis. (A and B) Still images taken from digital time-lapse imaging of live cells after depletion of endogenous Topo IIα and induced with doxycycline to express mCherry–Topo IIα or mCherry–Topo IIα ΔChT. Cells were assayed after a double thymidine synchrony and release protocol in conjunction with shRNA-mediated depletion of the endogenous Topo IIα and DNA observed via imaging of Hoechst. Red boxed regions are enlarged in B to highlight the differences in metaphase chromatin morphology. Frames were taken at 5-min intervals. Time is indicated (hours:minutes) in each movie frame. Bars, 10 µm. (C) Distribution of phenotypes seen in live single cells described in A and B. Induction of mCherry–Topo IIα rescues chromosome condensation and segregation defects, whereas Topo IIα ΔChT fails to rescue. WT − Dox, n = 96; WT + Dox, n = 100; ΔChT − Dox, n = 61; ΔChT + Dox, n = 82.
Mentions: We next investigated the biological consequences of the altered dynamics of mCherry–Topo IIα ΔChT in terms of mitotic chromosome formation. Because previous studies have shown that Topo IIβ can weakly compensate for the lack of Topo IIα (Linka et al., 2007), we codepleted both isoforms, using shRNA lentiviral transduction, to achieve a minimum base level of type II topoisomerase activity (Fig. S3). We simultaneously induced expression of mCherry–Topo IIα, mCherry–Topo IIα ΔChT, or mCherry–Topo IIα Y804F (as a negative control) in the isogenic cell lines and monitored changes in metaphase chromosome morphology when cells reached mitosis after release from a double thymidine synchrony. Giemsa staining of Carnoy’s fixed cells allowed fine-scale examination of changes in chromosome morphology (Giménez-Abián et al., 1995; Giménez-Abián and Clarke, 2009; Fig. 8, C and D). In addition, we filmed cells, treated in the same manner, by digital time-lapse microscopy to observe single live cells as they entered and progressed through mitosis (Fig. 9).

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