Chromatin Fractionation Assay in Fission Yeast

Materials and Reagents

1. Yeast strain (Schizosaccharomyces pombe)
   
2. Flask (IWAKI PUMPS, catalog number: 4980FK500 )
   
3. Conical tube (BD Biosciences, Falcon^®, catalog number: 2070 )
   
4. 1.5 ml microcentrifuge tube (Eppendorf, catalog number: 022364111 )
   
5. Lysing enzymes from Trichoderma harzianum (Sigma-Aldrih, catalog number: L1412 )
   
6. Zymolyase 100T (Seikagaku Corporation, catalog number: 120493-1 )
   
7. Complete Mini (Roche Diagnostics, catalog number: 11836153001 )
   
8. Anti-GFP antibody (Roche Diagnostics, catalog number: 11814460001 )
   
9. Anti-histone H3 antibody (Millipore, Upstate Biotechnology, catalog number: 05-499 )
   
10. Anti-alpha-tubulin antibody (generously provided by Dr. A. Baines)
    Note: Commercially available antibodies against alpha-tubulin (such as Abcam, catalog number: ab6161 ) can be used.
    
11. Goat HRP conjugated-anti-mouse antibody (Life Technologies, Biosource, catalog number: A10551 )
    
12. YES liquid medium (see Recipes)
    
13. STOP buffer (see Recipes)
    
14. PEMS buffer (see Recipes)
    
15. 2x HBS buffer (see Recipes)
    
16. Lysis buffer (see Recipes)
    
17. 2x Laemmli protein sample buffer (see Recipes)
    

Equipment

1. Air- (Tykyo Rikakikai, Eyela, model: FMC-1000 ) or water bath- (Taitec, model: MM-10 ) incubator shaker
   
2. Centrifuges equipped with 50 ml tubes (Tomy Digital Biology, model: AX-501 ) and 1.5 ml microtubes (Tomy Digital Biology, model: MX-107 )
   
3. Heat block (TAITEC, model: DTU-1BN) or water bath (Taitec, model: EXN-B )
   
4. Light microscope (Nikon Corporation, model: Eclipse E200 )
   
5. Electrophoresis apparatus (Bio-Rad Laboratories, catalog numbers: 165-8002JA and 164-5052 )
   
6. Transfer unit (Bio-Rad Laboratories, catalog numbers: 170-3930JA and 170-3935JA )
   

Procedure

1. Culture fission yeast cells in 200 ml of YES medium in a 500 ml flask at 26 °C.
   Note: Stop culturing during the mid-log phase (OD₅₉₅ = ~1.0).
   
2. Prepare approximately 2.5 x 10⁸ cells by dilution of inoculum to 25 ml at OD₅₉₅ = 0.5.
   
3. Harvest the cells by centrifugation at 400 x g for 5 min at 4 °C.
   
4. Resuspend the cell pellet in 1 ml of ice-cold STOP buffer and transfer the cell suspension to a 1.5 ml microtube.
   
5. Centrifuge the cell suspension at 400 x g for 5 min at 4 °C and pour out the supernatant (STOP buffer) by aspiration.
   
6. Place the microtube containing the cell pellet on ice for 5 min.
   
7. Resuspend the cell pellet in 1 ml of PEMS buffer dissolving 1 mg/ml lysing enzymes and 1 mg/ml Zymolyase 100T.
   
8. Incubate the cell suspension for 20 min at 37 °C for making spheroplasts which readily burst for enabling to fractionate chromatin-bound and -unbound proteins at the later step.
   Note: To test whether these lysing enzymes work adequately, take a small volume of the cell suspension, add the equal volume of 10% SDS, and then monitor yeast lysis progression by light microscopy. If lysing is successful, almost all cells (spheroplasts) are expected to burst in the presence of SDS.
   
9. Centrifuge the spheroplast suspension at 400 x g for 5 min at 4 °C. Pour out the supernatant (PEMS buffer) carefully by aspiration.
   
10. Wash the spheroplast pellet twice by resuspending in 1 ml of 1.2 M sorbitol to avoid bursting of spheroplasts, followed by centrifugation at 400 x g for 5 min at 4 °C.
    
11. Lyse the spheroplasts by resuspending in 0.5 ml of ice-cold lysis buffer, followed by incubation on ice for 5 min.
    
12. Take 1/10 aliquot of the resulting lysate and save it as the whole cell extract (optional).
    Note: To check the protein expression of our interest, we recommend an immunoblot of the whole cell extract with the other samples.
    
13. Centrifuge the rest of the lysate at 22,000 x g for 15 min at 4 °C.
    
14. Save the supernatant as the chromatin-unbound fraction.
    
15. Wash the pellets twice by resuspending in 1 ml of lysis buffer, followed by centrifugation at 22,000 x g for 5 min at 4 °C.
    
16. Resuspend the pellet in 0.45 ml of lysis buffer and save it as the chromatin-bound fraction.
    
17. Add an equal volume of 2x Laemmli protein sample buffer to all of the saved fractions.
    
18. Apply an equal volume of these fractions to the 8% SDS-PAGE gel and immunoblot using antibodies against histone H3 (1/5,000 dilution), alpha-tubulin (1/5,000 dilution) and specific protein(s).
    Note: We detected the chromatin association of Pcf1-GFP protein by using the anti-GFP antibody (1/1,000 dilution). The secondary antibody was diluted at 1/10,000.
    

Representative data

Figure 1. The cdc10-M17 mutant cells were grown in YES medium at 26 °C, synchronized to G1 phase by incubation for 4 h at 36 °C, and returned to 26 °C. Released cells into S phase were harvested at indicated time(s) and subjected to chromatin fractionation assay to monitor the chromatin association of Pcf1-GFP.

Notes

1. The proper performance of the assay can be easily confirmed by immunoblots using anti-histone H3 and anti-alpha-tubulin antibodies, since histone H3 and alpha-tubulin are fractionated to the chromatin-bound and -unbound fractions, respectively. In some cases as shown in Figure 1, alpha-tubulin was fractionated into the chromatin-bound fraction. This contamination was probably due to low efficiency of cell lysis or insufficient washout of the chromatin-unbound proteins. Therefore, we recommend the reader(s) to check the former possibility by monitoring yeast lysis progression in the presence of SDS and adjusting cell number, concentration of lysing enzymes, and/or reaction time, if necessary. For the latter possibility, additional wash of the pellets should be done to remove the chromatin-unbound proteins completely.

Recipes

1. YES liquid medium
   5 g/L Bacto yeast extract
   30 g/L glucose
   225 mg/L adenine, histidine, leucine, uracil and lysine hydrochloride
   
2. STOP buffer
   150 mM NaCl
   50 mM NaF
   10 mM EDTA
   1 mM NaN₃
   pH 8.0
   
3. PEMS buffer
   100 mM PIPES
   50 mM EDTA
   10 mM MgSO₄
   1.2 M sorbitol
   pH 6.9
   
4. 2x HBS buffer
   50 mM MOPS
   120 mM beta-glycerophosphate
   30 mM MgCl₂
   30 mM EGTA
   30 mM p-nitrophenylphosphate
   0.2 mM Na₃VO₄
   pH 7.2
   
5. Lysis buffer
   2x HBS buffer 5 ml
   10 % Triton X-100 0.5 ml
   2 M sorbitol 2 ml
   100 mM dithiothreitol (DTT) 0.1 ml
   0.1 mM phenylmethanesulfonyl fluoride (PMSF) 0.1 ml
   Complete mini 1 tablet
   Fill up to 10 ml with distilled water
   
6. 2x Laemmli protein sample buffer
   0.1 M Tris-HCl
   4 % sodium dodecyl sulfate (SDS)
   20 % glycerol
   0.2 % bromophenolblue (BPB)
   20 % 2-mercaptoethanol (2-ME)
   pH 6.8
   

Acknowledgments

This protocol was described in our previous report (Kunoh and Habu, 2014). We express our sincere appreciation to Dr. Mahito Sadaie (Graduate School of Biostudies, Kyoto University) for his technical advice on this protocol. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan to T.H. and JST-CREST to Professor Jun Takada, Okayama University.

References

1. Kunoh, T. and Habu, T. (2014). Pcf1, a large subunit of CAF-1, required for maintenance of checkpoint kinase Cds1 activity. Springerplus 3: 30.
   
2. Ogawa, Y., Takahashi, T. and Masukata, H. (1999). Association of fission yeast Orp1 and Mcm6 proteins with chromosomal replication origins. Mol Cell Biol 19(10): 7228-7236.
   
3. Sadaie, M., Kawaguchi, R., Ohtani, Y., Arisaka, F., Tanaka, K., Shirahige, K. and Nakayama, J.. (2008). Balance between distinct HP1 family proteins controls heterochromatin assembly in fission yeast. Mol Cell Biol  28(23): 6973-6988.