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

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.


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.
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 auxininducible 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. 14. Primary antibodies (see Notes 3 and 4).
15. Break cells using a Multibead shocker (6.0 m/s for 40 s, or until cells are broken).
17. Puncture the bottom of the screw cap tubes using a 23 G needle (see Note 10).
21. Discard screw cap tubes, recover the 1.5 mL tubes that contain the protein extract (see Note 11).
23. Spin at 10,000 rpm at room temperature for 2 min to remove cell debris.  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 OD 595 ¼ 0.05 (approximately 1 Â 10 6 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.