TITLE: Efficient Depletion of Fission Yeast Condensin by Combined Transcriptional Repression and Auxin-Induced Degradation
AUTHORS: Yasutaka Kakui, Frank Uhlmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Depletion of the Condensin SMC2/Cut14 Subunit]
Culture cells in PGM at  until OD595 reaches 0.2–0.4 (4–8 × 106 cells/mL).
25 °C
To prepare a culture with suitable density in the next morning, an inoculation at OD595 = 0.05 (approximately 1 × 106 cells/mL) and   growth is recommended.
?. [Depletion of the Condensin SMC2/Cut14 Subunit]
Add 1/2000 culture volume of thiamine solution.
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   (Fig.3b).
?. [Depletion of the Condensin SMC2/Cut14 Subunit]
Add 1/1000 culture volume IAA stock solution to the culture.
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   (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   and   before the arrest endpoint, respectively [20].
?. [Depletion of the Condensin SMC2/Cut14 Subunit]
Incubate for  at .
25 °C
?. [Depletion of the Condensin SMC2/Cut14 Subunit]
Collect cells.
?. [Confirmation of Condensin Depletion by Western Blotting]
Harvest 2.5 OD595 units of cells (5 × 107 cells) in 15 mL tubes.
?. [Confirmation of Condensin Depletion by Western Blotting]
Centrifuge at .
Centrifuge: 3000 33, 5 min, 4 10
?. [Confirmation of Condensin Depletion by Western Blotting]
Discard the supernatant.
?. [Confirmation of Condensin Depletion by Western Blotting]
Suspend cells in .
[20% TCA solution]
?. [Confirmation of Condensin Depletion by Western Blotting]
Transfer cells to screw cap 2 mL tube. As required, samples can be stored  at this stage.
on ice
?. [Confirmation of Condensin Depletion by Western Blotting]
Centrifuge .
Centrifuge: 13000 33, 1 min, 4 10
?. [Confirmation of Condensin Depletion by Western Blotting]
Discard supernatant.
?. [Confirmation of Condensin Depletion by Western Blotting]
Suspend cells in .
[1 M Tris]
?. [Confirmation of Condensin Depletion by Western Blotting]
Centrifuge .
Centrifuge: 13000 33, 1 min, 4 10
?. [Confirmation of Condensin Depletion by Western Blotting]
Discard supernatant. Remove all the liquid carefully.
?. [Confirmation of Condensin Depletion by Western Blotting]
Suspend cells in .
[SDS buffer]
?. [Confirmation of Condensin Depletion by Western Blotting]
Boil at  for .
95 °C
?. [Confirmation of Condensin Depletion by Western Blotting]
Add  to the screw cap 2 mL tubes.
[glass beads]
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.
?. [Confirmation of Condensin Depletion by Western Blotting]
Boil at  for .
95 °C
?. [Confirmation of Condensin Depletion by Western Blotting]
Break cells using a Multibead shocker (6.0 m/s for , or until cells are broken).
?. [Confirmation of Condensin Depletion by Western Blotting]
Boil at  for .
95 °C
?. [Confirmation of Condensin Depletion by Western Blotting]
Puncture the bottom of the screw cap tubes using a 23 G needle.
Spin down briefly, then loosen the screw cap to release the pressure and close again tightly to avoid spillage while puncturing the tube.
?. [Confirmation of Condensin Depletion by Western Blotting]
Place the screw cap tube onto a 1.5 mL tube (Fig. 2a).
?. [Confirmation of Condensin Depletion by Western Blotting]
Place both tubes into a 50 mL tube (Fig. 2b).
?. [Confirmation of Condensin Depletion by Western Blotting]
Centrifuge 50 mL tubes (from previous step) at .
Centrifuge: 1000 33, 2 min
?. [Confirmation of Condensin Depletion by Western Blotting]
Discard screw cap tubes, recover the 1.5 mL tubes that contain the protein extract.
These 50 mL tubes can be reused.
?. [Confirmation of Condensin Depletion by Western Blotting]
Boil at  for .
95 °C
?. [Confirmation of Condensin Depletion by Western Blotting]
Spin at  to remove cell debris.
Centrifuge: 10000 33, 2 min
?. [Confirmation of Condensin Depletion by Western Blotting]
Load – for analysis by SDS-PAGE.
5 µl
10 µl
?. [Confirmation of Condensin Depletion by Western Blotting]
Transfer proteins to a nitrocellulose membrane.
?. [Confirmation of Condensin Depletion by Western Blotting]
Blocking: Incubate the membrane with 5% skim milk in PBST at  for .
25 Room temperature
?. [Confirmation of Condensin Depletion by Western Blotting]
Incubate the membrane with Primary antibody.
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.   incubation at   is recommended.Anti-Tat1 antibody: Anti-Tat1 antibodies are comparatively strong. Incubation at  for   is recommended.
?. [Confirmation of Condensin Depletion by Western Blotting]
Wash the membrane with PBST at  for . (1/4)
25 Room temperature
?. [Confirmation of Condensin Depletion by Western Blotting]
Incubate the membrane with Secondary antibody.
?. [Confirmation of Condensin Depletion by Western Blotting]
Wash the membrane with PBST at  for . (1/3)
25 Room temperature
?. [Confirmation of Condensin Depletion by Western Blotting]
Detection of the protein. Follow the manufacturer’s instruction for using the ECL reagents. Open image in new window.
?. [Confirmation of Condensin Depletion by Western Blotting]
Wash the membrane with PBST at  for . (2/4)
25 Room temperature
?. [Confirmation of Condensin Depletion by Western Blotting]
Wash the membrane with PBST at  for . (3/4)
25 Room temperature
?. [Confirmation of Condensin Depletion by Western Blotting]
Wash the membrane with PBST at  for . (2/3)
25 Room temperature
?. [Confirmation of Condensin Depletion by Western Blotting]
Wash the membrane with PBST at  for . (3/3)
25 Room temperature
?. [Confirmation of Condensin Depletion by Western Blotting]
Wash the membrane with PBST at  for . (3/4)
25 Room temperature