Maheshri:Micrococcal

Micrococcal Nuclease Assay

Adapted from: Felix H. Lam, David J. Steger and Erin K. O'Shea. | "Mapping nucleosome positions in S. cerevisiae by quantitative PCR" Assoc. w/ "Chromatin decouples promoter threshold from dynamic range". Nature, 453, 246-250 (8 May 2008).

Introduction
This protocol describes a method of assaying nucleosome positions in S. cerevisiae by nuclease protection and quantitative PCR (Q-PCR). Briefly, yeast cells are spheroplasted, treated with micrococcal nuclease, and single-nucleosome length DNA is gel-isolated and quantitated by Q-PCR using overlapping primer pairs tiling the length of a region of interest. In the accompanying Nature paper, this technique is used to map the promoter nucleosome positions of several genes in the phosphate starvation response (PHO) pathway in order to determine the accessibility of Pho4 (transcription factor) binding sites. While this protocol has been used extensively with S. cerevisiae, it should, in principle, be readily adaptable for use in other yeast species.

Materials
Reagents

1 M sorbitol - At room temperature.

1 M sorbitol - Chilled to 4°C.

Tris-EDTA (TE) - 10 mM Tris, pH 7.5; 1 mM EDTA.

β-ME buffer (11 ml) - 20 mM EDTA; 0.7 M β-ME. Prepare fresh at room temperature before each experiment.

Lyticase buffer (11 ml) - 1 M sorbitol; 50 mM Tris, pH 7.8; 5 mM β-ME. Prepare fresh at room temperature before each experiment.

Buffer A (4 ml) - 1 M sorbitol; 10 mM Tris, pH 7.5; 50 mM NaCl; 5 mM MgCl2; 1 mM CaCl2; 1 mM β-ME. Prepare fresh on ice for each experiment.

Buffer B (1.25 ml) - Buffer A with 0.15% NP-40. Prepare fresh on ice for each experiment.

Lyticase (from Arthrobacter luteus; catalog# L2524, Sigma) - Dilute powder to make a 20,000 U/ml stock in ddH2O. Store at -20°C.

Micrococcal nuclease (MNase) (catalog# N5386, Sigma) - Dilute powder to make a 2 U/μl stock in 10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM CaCl2; 50% glycerol. Store at -20°C.

MNase stop buffer (0.5 ml) - 5% SDS; 250 mM EDTA. Store at room temperature. Heat to 37°C or higher to dissolve SDS before using.

Proteinase K (0.5 ml) - 20 μg/μl in ddH2O. Store at -20°C.

RNase A (1 ml) - 0.1 μg/μl in TE. Store at -20°C.

Phenol:Chloroform

4 M NaCl

Ethanol (EtOH), 100%

Ethanol (EtOH), 70%

QIAEX II Gel Extraction kit

10x Taq buffer - 100 mM Tris, pH 8.3; 500 mM KCl; 20 mM MgCl2

dNTP mix - 8 mM total dNTPs (i.e., 2 mM each d(A|C|G|T)TP)

SYBR Green I (catalog# S-7567, Invitrogen) - Dilute 10,000x concentrate to a 100x stock using DMSO. Store protected from light at -20°C.

Taq polymerase

Q-PCR primer sets - Design primer pairs to produce overlapping ~ 100 bp products tiling a particular region of the genome, centered every 30-50 bp, and with Tm's as close as possible to 60°C.

Yeast genomic DNA - Prepare from the same yeast strain where nucleosome positions will be assayed. Used for Q-PCR quantitation standards.

Equipment
Applied Biosystems 7300 (in Prather Lab)

Time Taken
2-3 days

Spheroplasting
1. Grow ~ 250 ml of yeast to OD600 of ~ 0.5 (mid-log phase) under your condition of interest.

2. Pellet cells at 3800 g, 5 min at room temperature.

3. Decant pellet and resuspend in 10 ml β-ME buffer. Incubate at 30°C for 30 min. Shake gently every 10 min to prevent cells from settling.

4. Transfer sample to 15 ml conical tube, and pellet at 2000 g, 5 min at room temperature.

5. Wash pellet: resuspend in 15 ml room temperature 1 M sorbitol. Pellet at 2000 g, 5 min at room temperature, and decant.

6. Resuspend pellet in 9.5 ml lyticase buffer. Add 0.5 ml lyticase for a final concentration of 1000 U/ml. Incubate at 30°C for 30 min* on roller drum, or invert tube gently every 10 min to prevent cells from settling.
 * Incubation time is strain specific! During a pilot run, determine an appropriate incubation time by monitoring spheroplasts lyse in water under a light microscope.

7. Pellet spheroplasts at 2000 g, 5 min at room temperature. Pipet off supernatant (spheroplasts are fragile and do not pack as tightly as yeast).

8. Wash spheroplasts: resuspend gently in 15 ml 4°C 1 M sorbitol. Pellet, and pipet off supernatant.

Micrococcal nuclease (MNase) titration
9. Assuming a 4 point MNase titration series, resuspend spheroplasts in 0.8 ml buffer A. On ice, aliquot 0.2 ml spheroplasts to 4 microfuges tubes labeled "2 U", "1 U", "0.5 U", and "0 U".

10. Prepare 3 two-fold serial dilutions of MNase enzyme in buffer B on ice. To tube labeled "2 U MNase", add 2.1 μl of MNase stock (2 U/μl) to 408 μl buffer B. To tube labeled "1 U MNase", add 205 μl of "2 U MNase" to 205 μl buffer B, etc.

11. On ice, add 200 μl of the "2 U MNase", "1 U MNase", and "0.5 U MNase" enzyme dilutions to the correspondingly labeled 200 μl aliquots of spheroplasts. Add 200 μl of buffer B to the "0 U" spheroplast aliquot.

12. Incubate at 37°C for 20 min.

13. Stop digestions by adding 40 μl of MNase stop buffer. Vortex well.

14. Add 12 μl of proteinase K to each sample. Vortex well, and incubate at 37°C for 1-5 hrs.

Purify mono-nucleosome length DNA
15. Add 20 μl of 4 M NaCl and 1 volume (450 μl) of phenol to all samples. Vortex thoroughly (==== 20 sec). Add 1 volume (450 μl) of chloroform to all samples. Vortex thoroughly (==== 20 sec).

16. Spin 13-16,000 g for 10 min at room temperature to separate phases.

17. Transfer aqueous phase to pre-spun Eppendorf Phase Lock tubes. Add 1 volume of 1:1 phenol:chloroform. Invert mixture several times to mix. Spin 13-16,000 g for 5 min at room temperature.

18. Add 1 volume (450 μl) of phenol:chloroform. Mix and spin 13-16,000 g for 5 min at room temperature.

19. Transfer aqueous phase to new microfuge tubes. Add 1 ml of 100% EtOH. Mix well and precipitate DNA at -20°C for at least 30 min.

20. Spin samples 13-16,000 g for 20 min at room temperature. Aspirate off supernatant.

21. Resuspend DNA pellets in 200 μl of RNase A. Incubate at 37°C for 1-2 hrs.

22. Add 10 μl of 4 M NaCl and 0.5 ml of 100% EtOH. Precipitate DNA at -20°C for at least 30 min.

23. Spin samples 13-16,000 g for 20 min at room temperature. Aspirate off supernatant.

24. Wash pellets once with 1 ml of 70% EtOH. Spin and aspirate off EtOH.

25. Thoroughly air dry DNA pellets, and resuspend in 50 μl TE.

26. Run all samples (3 μl each) on a 2% agarose gel. You should see ladders showing DNA fragment sizes corresponding to integral multiples of ~ 150 bp. Ideally, you have will at least one sample enriched in 150 bp / mono-nucleosome length fragments that does not show any sign of over-digestion. Since the extent of digestion is dependent on such factors such as the amount of cells harvested, efficiency of spheroplasting, effective activity of your MNase stock, etc., you may need to revise steps 9-12 and consider a higher-fold dilution series of MNase, more titration points, and/or varying the digestion time at 37°C.

27. Choose one or two samples with the greatest fraction of mono-nucleosome length fragments, and run 20-25 μl on a 1.5% agarose gel. Excise the ~ 150 bp band, and extract DNA using the QIAquick Gel Extraction Kit or equivalent method.

Tiling Q-PCR
28. Combine forward and reverse primer pairs (5 μM each) to make 10 μM working dilutions.

29. Prepare a 3 or 4 point ten-fold serial dilution of yeast genomic DNA for use as Q-PCR quantitation standards.

30. To prepare your Q-PCR template, start with a 1:10 dilution of the gel-purified mono-nucleosome length DNA. Depending on the resulting quantitated values, you may need to adjust the amount of template DNA used per reaction.

31. Prepare a working solution of 7.5x SYBR green by diluting the 100x stock with H2O. SYBR green is unstable in H2O and should be diluted fresh for each experiment.

32. Design the layout of your Q-PCR 96 well plate or strips, and determine the amounts necessary to assemble the following per-well Q-PCR reaction: 1 μl mono-nucleosome DNA, diluted 1:10; 5 μl 10x Taq buffer; 5 μl 2 mM dNTPs; 0.5 μl 7.5x SYBR green; 0.5 μl Taq; 5 μl 10 μM primer mix; 33 μl H2O. (ROX can be added to a final concentration of 300nM as a reference dye).

33. Use the following thermal cycling program: 1 cycle of {94°C for 5 min}; 40 cycles of {95°C for 30 sec, 57°C for 30 sec, 72°C for 1 min, 75°C for 10 sec, plate read} (on Prather lab AB 7300, read during 72°C extension step); 1 cycle of {72°C for 5 min}. Include a 70-95°C melting curve at the end of the Q-PCR program, reading all points or every 0.2°C.

34. Be sure all quantitated nucleosome DNA values fall within the interpolated linear range defined by the genomic DNA serial dilution; if not, adjust the amount of template DNA as appropriate. Check to make sure all reactions are yielding homogeneous PCR products (i.e., single peaks in the melting curve); if not, it may be necessary to design alternate primer pairs.

Troubleshooting
If you do not observe significant differences in nucleosome profiles under inducing and repressing conditions, it is possible that your promoters are becoming remodeled during the steps preceding MNase treatment. To test this possibility, you can try replicating your condition of interest in the β-ME and lyticase buffers, and the two 1 M sorbitol washes. For example, add 10 mM KH2PO4 if you are studying promoter chromatin structure under phosphate-repressing conditions and your yeast cultures were originally grown under these conditions.