Immunoblots: Mitotic chromosomes are self-entangled and disentangle through a Topoisomerase II-dependent two stage exit from mitosis

Description

The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations and multi-contact 3C, and propose that, in contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a Topoisomerase II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias Topoisomerase II activity towards decatenation. At the second stage, the loops are released, and formation of new entanglements is prevented by lower Topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed, in experiment and models, a normal interphase state cannot be acquired. Original western blot images supporting this dataset are included in this repository.

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Protein immunoblots were performed using standard methods. Protein lysates were made by harvesting the same number of cells (generally 0.5x10^6 each) for each sample in an experiment and lysing in 2x Laemmli Buffer (.07M Tris pH6.8, 10% sucrose, 3% SDS, 50mM DTT) by boiling for 10 minutes. Western blots were performed by running protein lysates on 4-12% NuPage Bis-Tris gels using 1x MES running buffer for 45 minutes at 175V using an Invitrogen XCell Sure-Lock minigel and blotting system. Gels were transferred to 0.2um nitrocellulose membrane in Pierce™ 10X Western Blot Transfer Buffer, Methanol-free (Thermo Fisher Scientific, 35040), by running for 1 hour at 30V at 4 degrees C. The membranes were blocked with 4% milk in PBS-T (1x PBS and 0.1% Tween-20) for 30 minutes at room temperature. The membranes were then incubated with the specified antibodies diluted in 4% milk/PBS-T either for 2 hours at room temp or overnight at 4 degrees C, and washed three times with PBS-T for 10 minutes at room temperature. Membranes were then incubated with secondary antibodies (anti-rabbit IgG or anti-mouse IgG HRP linked) diluted 1:4000 in 4% milk/PBS-T for 30 minutes to 1 hr at room temperature, then washed three times with PBS-T for 10 minutes at room temperature. The membranes were then developed and imaged using SuperSignal West Dura Extended Duration Substrate (Thermo, 34076) and a Bio-Rad ChemiDoc. Primary antibodies used: mouse anti-Topoisomerase II (1:500, Santa Cruz sc-166934), rabbit anti-DNA Topoisomerase II and DNA Topoisomerase II (1:10,000, Abcam ab109524), rabbit anti-GFP (1:5,000, abcam ab290), mouse anti--actin (1:2,000, Cell Signaling 8H10D10), mouse anti-cyclin B1 (1:500, Cell Signaling 4135), rabbit anti GAPDH (1:1000, Cell Signaling 14C10). Secondary antibodies used: goat anti-rabbit IgG-HRP (1:4,000, Cell Signaling 7074), goat anti-mouse IgG-HRP (1:4,000, Cell Signaling 7076).

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