Sep 02, 2021

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

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DOI
dx.doi.org/10.17504/protocols.io.bnx7mfrn
  • Yasutaka Kakui1,
  • Frank Uhlmann1
  • 1Chromosome Segregation Laboratory, The Francis Crick Institute, London, UK
  • Springer Nature Books
Satyavati Kharde
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DOI: dx.doi.org/10.17504/protocols.io.bnx7mfrn
External link: https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_3
Protocol Citation: Yasutaka Kakui, Frank Uhlmann 2021. Efficient Depletion of Fission Yeast Condensin by Combined Transcriptional Repression and Auxin-Induced Degradation. protocols.io https://dx.doi.org/10.17504/protocols.io.bnx7mfrn
Manuscript citation:
Kakui Y., Uhlmann F. (2019) Efficient Depletion of Fission Yeast Condensin by Combined Transcriptional Repression and Auxin-Induced Degradation. In: Badrinarayanan A. (eds) SMC Complexes. Methods in Molecular Biology, vol 2004. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9520-2_3
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 25, 2020
Last Modified: September 02, 2021
Protocol Integer ID: 43743
Keywords: Condensin, SMC complex, Chromosome condensation, Auxin-inducible degron, Transcriptional repression, Fission yeast ,
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.
Guidelines
Introduction

Spatial chromatin organization by SMC complexes is at the heart of genome stability and faithful chromosome segregation. SMC complexes are evolutionary conserved, large proteinaceous rings that topologically entrap one or more DNAs to engage in higher order chromatin architecture [1]. The SMC family member, condensin, plays a crucial role in the compaction of interphase chromatin to form condensed chromosomes in mitosis [2]. It also plays roles in genome maintenance during interphase. Condensin consists of two SMC coiled-coil subunits, SMC2/Cut14 and SMC4/Cut3, and three non-SMC accessory subunits, CAP-D2/Cnd1, CAP-H/Cnd2, and CAP-G/Cnd3 (Fig.1a). How condensin accomplishes chromosome condensation is not yet understood.


Fig. 1
Schematic illustration of the cut14 shut off system. (a) A schematic of condensin. (b) Condensin depletion strategy. The endogenous promoter of the cut14 gene is replaced by weakened version of thiamine repressible nmt1 promoter, nmt81. The cut14 gene is also fused to an auxin-inducible degron (aid) tag. Addition of thiamine to the growth medium represses cut14 transcription. Auxin addition targets Cut14 for degradation through ubiquitination by the SCF (Cul1-Skp1-Tir1) complex.
To study condensin’s function in vivo, an important approach is to inactivate or deplete the complex. Historically, temperature sensitive mutants obtained in yeast genetic screens have been utilized to characterize protein function. In fission yeast, condensin temperature sensitive mutants have been isolated with a “cell untimely torn (cut)” phenotype [3]. A block to nuclear division, but not cytokinesis, results in chromosomes that are apparently “cut” during cell division. Cytological analyses of these mutants have revealed the importance of condensin for mitotic chromosome condensation [4]. These temperature-sensitive mutants provide a powerful tool but also come with limitations. It is difficult to know how quickly and how completely condensin is inactivated after temperature shift. Furthermore, the required temperature shift not only inactivates condensin but affects cell physiology in additional ways (e.g., eliciting a transcriptional heat shock response) that could impact on chromatin architecture.

Alternatives to temperature sensitive mutants have been developed. Protein function can be eliminated by forced localization away from its required site of action. In case of budding yeast condensin, cytoplasmic sequestration using the anchor-away approach successfully abolishes nuclear condensin function [5,6,7]. However rapamycin, the ligand used to sequester condensin to its cytoplasmic anchor, inhibits cell growth. Elaborate strain construction is required to circumvent this effect.

Condensin depletion in vertebrates has been achieved using RNA interference or promoter shut-off [8,9,10]. In these cases, depletion progresses slowly, typically over the duration of several cell divisions. Consequently, condensin depletion at the time of analysis is often incomplete. An alternative approach is the use of TEV protease to target and inactivate an engineered condensin complex more quickly [11]. Recently, efficient depletion of chicken DT40 cell condensin was reported using an auxin-inducible degron (aid) [12].

In fission yeast, the thiamine repressible nmt1 promoter and derivatives have been used to repress gene transcription [13,14]. Replacing endogenous gene promoters with the nmt1 promoter has allowed for efficient depletion of proteins that are intrinsically unstable, such as the APC/C activator Slp1 or DNA replication licensing factor Cdc18 [15,16]. Condensin depletion under control of the nmt1 promoter has been reported, but depletion remains incomplete even after longer periods [17]. Following transcriptional repression, protein degradation depends on physiological protein turnover. The stability of condensin prevents its acute depletion by transcriptional repression alone.

We therefore decided to combine transcriptional repression with conditional destabilization of condensin using an auxin-inducible degron. The aid approach relies on the SCF (Skp, Cullin, F-box containing complex)–proteasome pathway to degrade a target protein [18,19]. The plant-specific F-box protein Tir1 recognizes an aid degron tag, fused to condensin, only in the presence of the plant hormone auxin (Fig.1b). Together with transcriptional repression this leads to improved condensin depletion.

Here we document this condensin depletion protocol in fission yeast. We target the SMC2/Cut14 subunit for depletion, one of the two central coiled-coil subunits that are crucial for condensin complex assembly (Fig.1a). The endogenous cut14 promoter is replaced by the weaker nmt1 promoter, nmt81, and an aid tag is fused to the C-terminus of Cut14. Two copies of Tir1, derived from two plant species, are expressed for efficient targeting [19]. Addition of thiamine to represses cut14 expression and auxin to destabilize the Cut14 protein together lead to fast and efficient condensin depletion (see Fig.3, below). This approach facilitated the study of condensin’s contribution to chromosome formation in fission yeast [20] and should be applicable to the study of other SMC complex members.


Notes

  1. Expression levels of the Skp1-Tir1 fusion proteins are crucial for efficient target protein degradation [19].
  2. Prepare SDS buffer without 200 mM DTT and keep at room temperature. Add 1/5 volume of 1 M DTT to the SDS buffer just before use.
  3. 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. Overnight incubation at 4 °C is recommended.
  4. Anti-Tat1 antibody: Anti-Tat1 antibodies are comparatively strong. Incubation at room temperature for 1 h is recommended.
  5. To prepare a culture with suitable density in the next morning, an inoculation at OD595 = 0.05 (approximately 1 × 106 cells/mL) and overnight growth is recommended.
  6. 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 3 h (Fig.3b).
  7. 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 2 h (Fig.3b).
  8. 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 180 min and 90 min before the arrest endpoint, respectively [20].
  9. 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.
  10. Spin down briefly, then loosen the screw cap to release the pressure and close again tightly to avoid spillage while puncturing the tube.
  11. These 50 mL tubes can be reused.


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

Acknowledgments

We would like to thank Prof. Masukata for strains and plasmids. This work was supported by the European Research Council and the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001198), the UK Medical Research Council (FC001198), and the Wellcome Trust (FC001198). Y.K. was supported by the Japanese Society for the Promotion of Science (JSPS).


References

  1. Uhlmann F (2016) SMC complexes: from DNA to chromosomes. Nat Rev Mol Cell Biol 17:399–412
  2. Hirano T (2016) Condensin-based chromosome organization from bacteria to vertebrates. Cell 164:847–857
  3. Hirano T, Funahashi S, Uemura T, Yanagida M (1986) Isolation and characterization of Schizosaccharomyces pombe cut mutants that block nuclear division but not cytokinesis. EMBO J 5:2973–2979
  4. Saka Y, Sutani T, Yamashita Y, Saitoh S, Takeuchi M, Nakaseko Y, Yanagida M (1994) Fission yeast cut3 and cut14, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis. EMBO J 13:4938–4952
  5. Haruki H, Nishikawa J, Laemmli UK (2008) The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol Cell 31:925–932
  6. Cheng TM, Heeger S, Chaleil RA, Matthews N, Stewart A, Wright J, Lim C, Bates PA, Uhlmann F (2015) A simple biophysical model emulates budding yeast chromosome condensation. Elife 4:e05565
  7. Charbin A, Bouchoux C, Uhlmann F (2014) Condensin aids sister chromatid decatenation by topoisomerase II. Nucleic Acids Res 42:340–348
  8. Ono T, Losada A, Hirano M, Myers MP, Neuwald AF, Hirano T (2003) Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 115:109–121
  9. Hirota T, Gerlich D, Koch B, Ellenberg J, Peters JM (2004) Distinct functions of condensin I and II in mitotic chromosome assembly. J Cell Sci 117:6435–6445
  10. Hudson DF, Vagnarelli P, Gassmann R, Earnshaw WC (2003) Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes. Dev Cell 5:323–336
  11. Houlard M, Godwin J, Metson J, Lee J, Hirano T, Nasmyth K (2015) Condensin confers the longitudinal rigidity of chromosomes. Nat Cell Biol 17:771–781
  12. Gibcus JH, Samejima K, Goloborodko A, Samejima I, Naumova N, Nuebler J, Kanemaki MT, Xie L, Paulson JR, Earnshaw WC, Mirny LA, Dekker J (2018) A pathway for mitotic chromosome formation. Science 359:6376. pii: eaao6135
  13. Maundrell K (1990) nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J Biol Chem 265:10857–10864
  14. Basi G, Schmid E, Maundrell K (1993) TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123:131–136
  15. Petrova B, Dehler S, Kruitwagen T, Heriche JK, Miura K, Haering CH (2013) Quantitative analysis of chromosome condensation in fission yeast. Mol Cell Biol 33:984–998
  16. Hermand D, Nurse P (2007) Cdc18 enforces long-term maintenance of the S phase checkpoint by anchoring the Rad3-Rad26 complex to chromatin. Mol Cell 26:553–563
  17. Sutani T, Sakata T, Nakato R, Masuda K, Ishibashi M, Yamashita D, Suzuki Y, Hirano T, Bando M, Shirahige K (2015) Condensin targets and reduces unwound DNA structures associated with transcription in mitotic chromosome condensation. Nat Commun 6:7815
  18. Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M (2009) An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods 6:917–922
  19. Kanke M, Nishimura K, Kanemaki M, Kakimoto T, Takahashi TS, Nakagawa T, Masukata H (2011) Auxin-inducible protein depletion system in fission yeast. BMC Cell Biol 12:8
  20. Kakui Y, Rabinowitz A, Barry DJ, Uhlmann F (2017) Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast. Nat Genet 49:1553–1557
Materials
Cell Culture:
1. Pombe Glutamate medium (PMG):
14.7 mM potassium hydrogen phthalate
15.5 mM Na2HPO4
3.75 g/L L-glutamic acid
Monosodium salt
2% (w/v) glucose
5.2 mM MgCl2
0.1 mM CaCl2
13.4 mM KCl
0.28 mM Na2SO4
4.2 μM pantothenic acid
81.2 μM nicotinic acid
55.5 μM inositol
40.8 nM biotin
8.09 μM boric acid
2.37 μM MnSO4
1.39 μM ZnSO4
0.74 μM FeCl2
0.247 μM molybdic acid
0.6 μM KI
0.16 μM CuSO4
4.76 μM citric acid
150 μg/mL Adenine, Leucine, Uracil, Lysine, Histidine are added where necessary.
2. 10 mg/mL thiamine solution: 10 mg/mL (w/v) thiamine in deionized water, filter-sterilized.
3. 0.5 M 3-indoleacetic acid (IAA): dissolved in methanol. Prepare this freshly.
4. Yeast strains used in this protocol are listed in Table 1. Two copies of Skp1-Tir1 fusion proteins are expressed in all cells for efficient condensin destabilization.


Note
Expression levels of the Skp1-Tir1 fusion proteins are crucial for efficient target protein degradation [19].

Reagents for Western Blotting:
1. 0.2 mL PCR tube.
2. 1.5 mL tubes.
3. Screw cap 2 mL tubes.
4. 15 mL tubes.
5. 50 mL tubes.
6. Acid-washed glass beads (425–600 μm).
7. Needles (23 G × 1″).
8. 20% trichloroacetic acid solution (TCA).
9. 1 M Tris. No need to adjust pH.
10. 1 M dithiothreitol (DTT): store at −20 °C.
11. SDS buffer:
100 mM Tris-HCl (pH 6.8)
4% (w/v) sodium dodecyl sulfate
0.2% (w/v) bromophenol blue
20% (v/v) glycerol
200 mM DTT
Note
Prepare SDS buffer without 200 mM DTT and keep at room temperature. Add 1/5 volume of 1 M DTT to the SDS buffer just before use.

12. Nitrocellulose membrane.
13. PBST:
137 mM NaCl
2.7 mM KCl
10 mM Na2HPO4
1.8 mM KH2PO4
1% (v/v) Tween 20
14. Primary antibodies.
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. DurationOvernight incubation at Temperature4 °C is recommended.

Anti-Tat1 antibody: Anti-Tat1 antibodies are comparatively strong. Incubation at TemperatureRoom temperature for Duration01:00:00 is recommended.

15. Secondary antibody: HRP conjugated anti-mouse antibody.
16. Enhanced chemiluminescent (ECL) detection reagents.


Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Depletion of the Condensin SMC2/Cut14 Subunit
Depletion of the Condensin SMC2/Cut14 Subunit
Culture cells in PGM at Temperature25 °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 DurationOvernight growth is recommended.



Overnight
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 Duration03:00:00 (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
Pipetting
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 Duration02:00:00 (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 Duration03:00:00 and Duration01:30:00 before the arrest endpoint, respectively [20].


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
Pipetting
Incubate for Duration03:00:00 at Temperature25 °C .
3h
Incubation
Collect cells.
Confirmation of Condensin Depletion by Western Blotting
Confirmation of Condensin Depletion by Western Blotting
38m
38m
Harvest 2.5 OD595 units of cells (5 × 107 cells) in 15 mL tubes.
Centrifuge at Centrifigation3000 rpm, 4°C, 00:05:00 .

Centrifigation
Discard the supernatant.
Pipetting
Suspend cells in Amount1 mL 20% TCA solution .

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

Pipetting
Pause
Centrifuge Centrifigation13000 rpm, 4°C, 00:01:00 .

Centrifigation
Discard supernatant.
Pipetting
Suspend cells in Amount1 mL 1 M Tris .

Pipetting
Centrifuge Centrifigation13000 rpm, 4°C, 00:01:00 .

Centrifigation
Discard supernatant. Remove all the liquid carefully.
Pipetting
Suspend cells in Amount100 µL SDS buffer .

Pipetting
Boil at Temperature95 °C for Duration00:02:00 ..

2m
Add Amount200 µL glass beads 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.

Boil at Temperature95 °C for Duration00:02:00 ..
2m
Break cells using a Multibead shocker (6.0 m/s for Duration00:00:40 , or until cells are broken).

40s
Boil at Temperature95 °C for Duration00:02:00 ..
2m
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.

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

Centrifuge 50 mL tubes (from previous step) at Centrifigation1000 rpm, 00:02:00 .

Centrifigation
Discard screw cap tubes, recover the 1.5 mL tubes that contain the protein extract.
Note
These 50 mL tubes can be reused.

Boil at Temperature95 °C for Duration00:02:00 ..
2m
Spin at Centrifigation10000 rpm, Room temperature, 00:02:00 to remove cell debris.
Centrifigation
Load Amount5 µL –Amount10 µL for analysis by SDS-PAGE.

Analyze
Transfer proteins to a nitrocellulose membrane.
Blocking: Incubate the membrane with 5% skim milk in PBST at TemperatureRoom temperature for Duration00:30:00 .

30m
Incubation
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. DurationOvernight incubation at Temperature4 °C is recommended.

Anti-Tat1 antibody: Anti-Tat1 antibodies are comparatively strong. Incubation at TemperatureRoom temperature for Duration01:00:00 is recommended.


Incubation
Wash the membrane with PBST at TemperatureRoom temperature for Duration00:05:00 . (1/4)

5m
Wash
Wash the membrane with PBST at TemperatureRoom temperature for Duration00:05:00 . (2/4)

5m
Wash
Wash the membrane with PBST at TemperatureRoom temperature for Duration00:05:00 . (3/4)

5m
Wash
Wash the membrane with PBST at TemperatureRoom temperature for Duration00:05:00 . (3/4)

5m
Wash
Incubate the membrane with Secondary antibody.
Incubation
Wash the membrane with PBST at TemperatureRoom temperature for Duration00:05:00 . (1/3)
5m
Wash
Wash the membrane with PBST at TemperatureRoom temperature for Duration00:05:00 . (2/3)
5m
Wash
Wash the membrane with PBST at TemperatureRoom temperature for Duration00:05:00 . (3/3)
5m
Wash
Detection of the protein. Follow the manufacturer’s instruction for using the ECL reagents.
Open image in new window.
Analyze