Efficient Depletion of Fission Yeast Condensin by Combined Transcriptional Repression and Auxin-Induced Degradation

Yasutaka Kakui, Frank Uhlmann

Published: 2021-09-03 DOI: 10.17504/protocols.io.bnx7mfrn

Abstract

Structural maintenance of chromosomes (SMC) complexes play pivotal roles in controlling chromatin organization. Condensin is an essential SMC complex that compacts chromatin to form condensed chromosomes in mitosis. Complete condensin inactivation is necessary to reveal how condensin converts interphase chromatin into mitotic chromosomes. Here, we have developed a condensin depletion system in fission yeast that combines transcriptional repression with auxin-inducible protein degradation. This achieves efficient condensin depletion without need for a temperature shift. Our system is useful when studying how condensin contributes to chromosome architecture and is applicable to the study of other SMC complexes.

Steps

Depletion of the Condensin SMC2/Cut14 Subunit

Culture cells in PGM at 25°C until OD₅₉₅ reaches 0.2–0.4 (4–8 × 10⁶ cells/mL).

Note

To prepare a culture with suitable density in the next morning, an inoculation at OD₅₉₅ = 0.05 (approximately 1 × 10⁶ cells/mL) and 0h 0m 0s growth is recommended.

Add 1/2000 culture volume of thiamine solution.

Note

When comparing nmt1 -derived promoters of different strengths, we found that an attenuated variant, nmt81 , yields Cut14 levels similar to the endogenous cut14 promoter (Fig.3a). Addition of thiamine led to only weak depletion of Cut14 protein after 3h 0m 0s (Fig.3b).

Fig. 3 Cut14 protein levels under the indicated conditions. Protein extracts were prepared as described and analyzed by SDS-PAGE and western blotting. Cut14 and α-tubulin were detected using anti-aid tag (IAA17) and anti-TAT1 antibodies, respectively. α-tubulin serves as a loading control.(a) Cut14 protein levels expressed from different promoters, in the absence of thiamine. +: endogenous cut14 promoter, 41:nmt41 promoter, 81:nmt81 promoter. The Cut14 expression level under nmt81 promoter control is comparable to endogenous levels.(b) Time course analysis of Cut14 depletion under the indicated conditions. Samples were taken every hour after addition of either thiamine (+thi), IAA (+IAA) or both thiamine and IAA (+thi & IAA). Time is indicated in hours. Cut14 protein is hardly detectable 2 h after addition of both thiamine and IAA

Add 1/1000 culture volume IAA stock solution to the culture.

Note

An aid tag fused to Cut14 destabilizes condensin within 60 min, although Cut14 is still detected even after 3 h if the nmt81 promoter remains active (Fig.3b). Simultaneous addition of thiamine and auxin leads to almost complete condensin depletion in less than 2h 0m 0s (Fig.3b).The timing of IAA addition can be adjusted, for example, to accommodate arrest at a certain cell cycle stage. To minimize chromosome segregation defects in mitosis prior to a cell cycle arrest, thiamine and auxin can be added 3h 0m 0s and 1h 30m 0s before the arrest endpoint, respectively [20].

Incubate for 3h 0m 0s at 25°C.

Collect cells.

Confirmation of Condensin Depletion by Western Blotting

Harvest 2.5 OD₅₉₅ units of cells (5 × 10⁷ cells) in 15 mL tubes.

Centrifuge at 3000rpm,4°C.

Discard the supernatant.

Suspend cells in 1mL.

10.

Transfer cells to screw cap 2 mL tube. As required, samples can be stored 25On ice at this stage.

11.

Centrifuge 13000rpm,4°C.

12.

Discard supernatant.

13.

Suspend cells in 1mL.

14.

Centrifuge 13000rpm,4°C.

15.

Discard supernatant. Remove all the liquid carefully.

16.

Suspend cells in 100µL.

17.

Boil at 95°C for 0h 2m 0s.

18.

Add 200µL to the screw cap 2 mL tubes.

Note

Use a 0.2 mL PCR tube that can be glued to an inoculation loop as a handle for ease of use. One scoop of glass beads is 200 μL.

19.

Boil at 95°C for 0h 2m 0s.

20.

Break cells using a Multibead shocker (6.0 m/s for 0h 0m 40s, or until cells are broken).

21.

Boil at 95°C for 0h 2m 0s.

22.

Puncture the bottom of the screw cap tubes using a 23 G needle.

Note

Spin down briefly, then loosen the screw cap to release the pressure and close again tightly to avoid spillage while puncturing the tube.

23.

Place the screw cap tube onto a 1.5 mL tube (Fig. 2a).

Fig. 2 Setup to recover cell extracts from screw cap tubes after cell breaking. (a) A punctured screw cap tube is firmly placed onto a 1.5 mL tube. (b) The tubes prepared in (a) are placed into a 50 mL tube for centrifugation. If handling multiple samples, two sets of tubes can be placed into one 50 mL tube

24.

Place both tubes into a 50 mL tube (Fig. 2b).

25.

Centrifuge 50 mL tubes (from previous step) at 1000rpm.

26.

Discard screw cap tubes, recover the 1.5 mL tubes that contain the protein extract.

Note

These 50 mL tubes can be reused.

27.

Boil at 95°C for 0h 2m 0s.

28.

Spin at 10000rpm to remove cell debris.

29.

Load 5µL–10µL for analysis by SDS-PAGE.

30.

Transfer proteins to a nitrocellulose membrane.

31.

Blocking: Incubate the membrane with 5% skim milk in PBST at 25Room temperature for 0h 30m 0s.

32.

Incubate the membrane with Primary antibody.

Note

Anti-aid tag (IAA17) antibody, Cosmobio, CAC-APC004AM. Use at 1:5000 dilution in 5% skim milk. We found this anti-aid antibody to be weak but specific. 3h 0m 0s incubation at 4°C is recommended.Anti-Tat1 antibody: Anti-Tat1 antibodies are comparatively strong. Incubation at 25Room temperaturefor 1h 0m 0s is recommended.

33.

Wash the membrane with PBST at 25Room temperature for 0h 5m 0s. (1/4)

33.1.

Wash the membrane with PBST at 25Room temperature for 0h 5m 0s. (2/4)

33.2.

Wash the membrane with PBST at 25Room temperature for 0h 5m 0s. (3/4)

33.3.

Wash the membrane with PBST at 25Room temperature for 0h 5m 0s. (3/4)

34.

Incubate the membrane with Secondary antibody.

35.

Wash the membrane with PBST at 25Room temperature for 0h 5m 0s. (1/3)

35.1.

Wash the membrane with PBST at 25Room temperature for 0h 5m 0s. (2/3)

35.2.

Wash the membrane with PBST at 25Room temperature for 0h 5m 0s. (3/3)

36.

Detection of the protein. Follow the manufacturer’s instruction for using the ECL reagents.

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Efficient Depletion of Fission Yeast Condensin by Combined Transcriptional Repression and Auxin-Induced Degradation

Abstract

Steps

Depletion of the Condensin SMC2/Cut14 Subunit

Confirmation of Condensin Depletion by Western Blotting

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