Enrichment of Methylated DNA by Methyl-CpG Immunoprecipitation

Abstract

Normal DNA methylation is an epigenetic modification required for proper development. Aberrant DNA methylation, in contrast, is frequently observed in many different malignancies including leukemias and lymphomas. Global DNA methylation profiling addresses the methylated sequences (methylome) of patient genomes to identify disease-specific methylation patterns. Workload in methylome analyses can be considerably reduced by methylome enrichment using proteins or antibodies with high affinity to methylated DNA. Methyl-CpG Immunoprecipitation (MCIp) employs an immobilized recombinant human methyl-CpG binding domain protein 2, MBD2, which binds methylated CpGs in double-stranded DNA. Elution with increasing salt concentrations allows the fractionated enrichment of different degrees of methylation.

Appendices

Production of MBD2-Fc protein

Materials

2.1 Cell Culture

• 1. Drosophila melanogaster S2 cells, stably transfected with pMTBip/MBD-Fc and pCoHygro (11).

• 2. Cell culture flasks 75 cm² (Greiner Bio-One, Frickenhausen, Germany).

• 3. 2 L cell culture roller bottles (Corning, Lowell, MA, USA).

• 4. Insect-Xpress medium, protein-free with L-glutamine (Lonza, Cologne, Germany).

• 5. Penicillin/Streptomycin (e.g., Pen/Strep, Invitrogen).

• 6. Hygromycin B (50 mg/ml, Clontech, Saint-Germain-en-Laye, France).

• 7. Microscope and Neubauer counting chamber.

• 8. 100 mM CuSO₄, sterile filtered; working concentration is 0.5 mM.

• 9. Cooling incubator (e.g., Thermo Fisher Scientific, Karlsruhe, Germany) with roller equipment (e.g., Cellroll, Integra Biosciences, Fernwald, Germany).

• 10. Centrifuge tubes (e.g., 200 ml one way conical tubes, BD Biosciences, Heidelberg, Germany).

• 11. Benchtop cooling centrifuge (e.g., 4 K15, Sigma, Osterode, Germany) with swinging bucket rotor and buckets including adapters for 250 ml conical centrifuge bottles.

2.2 Protein Isolation

• 1. Dialysis tubing (e.g., Spectra/Por MWCO 6–8,000, Serva, Heidelberg, Germany) and suitable tubing clamps.

• 2. rProtein A-sepharose fast flow (e.g., GE Healthcare, München, Germany).

• 3. High-speed centrifuge with fixed angle rotor for 250 ml centrifuge bottles (e.g., Beckman J-25 with rotor JA 14, Beckmann Coulter, Krefeld, Germany) and suitable 250 ml reusable polypropylene centrifuge bottles.

• 4. Chromatography column (e.g., XK 16, GE Healthcare).

• 5. Peristaltic pump (e.g., RP-1, Rainin, Emeryville, CA, USA).

• 6. TBS, pH 7.4.

• 7. TBS, pH 7.4, 500 mM NaCl.

• 8. TBS, pH 7.4, 20% ethanol.

• 9. 3 M KCl.

• 10. 0.1 M citric acid, pH 3.

• 11. 1.5 M Tris–HCl, pH 8.8.

• 12. UV spectrophotometer (e.g., BioPhotometer, Eppendorf) and UV-compatible cuvettes.

• 13. Dialysis cassette (e.g., Slide-A-Lyzer, 0.5–3 ml capacity, 3,500 MWCO, Thermo Fisher Scientific).

• 14. Protease inhibitor cocktail (e.g., Roche, Mannheim, Germany).

• 15. 100% glycerol.

Methods

An overview of cell numbers required for the different steps of protein production is given in Table 2 (2.4). A short summary of the protein production procedure is provided in Table 3 (2.4).

Table 2 Culturing Drosophila S2 cells

Table 3 MBD-Fc protein production

3.1 Cell Culture of Drosophila S2 Cells

• 1. For initial expansion in a cell culture flask, inoculate Xpress medium containing hygromycin and Pen/Strep from a frozen stock of S2 cells at a titer of 5–10  ×  10⁵ cells/ml and incubate at 21°C (see Note 1).

• 2. Split the culture if the cell number reaches 5  ×  10⁶ cells/ml. Do not dilute below 5  ×  10⁵ cells/ml.

3.2 Induction of MBD2-Fc Protein Expression

• 1. Seed two cultures of 300 ml Xpress medium containing 2  ×  10⁶ cells/ml, Pen/Strep and Hygromycin at 21°C in 2 L roller bottles until the cell number reaches approximately 1  ×  10⁷ cells/ml which usually takes 3 days (see Note 2). Set the roller to a speed of 6 rounds/min.

• 2. At about 1  ×  10⁷ cells/ml, distribute the 2  ×  300 ml cultures to four conical one way centrifuge bottles and centrifuge using a swinging bucket rotor at 15°C for 8 min, 300  ×  g.

• 3. To induce the MBD2-Fc gene, discard the supernatant and resuspend the cells in Xpress medium containing Pen/Strep and 0.5 mM CuSO₄ but no hygromycin (see Note 3).

• 4. Retransfer the four suspensions to the two roller bottles and continue incubation at 21°C for 4 days (see Note 4) in which the cell number usually reaches 2.5–3  ×  10⁷ cells/ml.

• 5. Distribute the 2  ×  300 ml cell cultures to four conical centrifuge bottles and centrifuge using a swinging bucket rotor at 15°C for 8 min, 300  ×  g.

• 6. Transfer the supernatants without cellular carryover to four 250 ml centrifuge bottles and centrifuge in a fixed angle rotor at 4°C for 60 min, 15,000  ×  g (see Note 5).

3.3 MBD-Fc Protein Isolation by Affinity Chromatography on rProtein A-Sepharose Fast Flow

• 1. Dialyze the cleared supernatants from the previous step at 4°C against 5 L TBS, pH 7.4, with 3–4 buffer changes (see Note 6).

• 2. Pack the chromatography column with 5 ml rProtein A-sepharose fast flow and equilibrate the sepharose matrix with 80 ml cold TBS (see Note 7).

• 3. Load the dialyzed culture supernatant from Subheading 2.3 item 1 (Appendix) onto the matrix.

• 4. Wash the matrix with 80 ml TBS containing 500 mM NaCl, then with 80 ml TBS (see Note 8).

• 5. Elute the MBD-Fc protein with 0.1 M citric acid, pH 3 in 20 fractions of 1 ml each into Eppendorf tubes containing 50 μl 1.5 M Tris–HCl, pH 8.8 (see Note 9).

• 6. Identify the fractions with the highest protein content by measuring absorbance at 280 nm using a UV spectrophotometer. Keep fractions with absorbance higher than 2 (see Note 10).

• 7. Combine these fractions and transfer the mixture to a dialysis cassette. Dialyze against 1 L cold (4°C) TBS with two buffer changes.

• 8. Transfer dialysate to a 50 ml tube and add 10% NaN₃ to a final concentration of 0.02%, 4 μl protease inhibitor cocktail per ml dialysate and 100% glycerol to a final concentration of 20%. Homogenize carefully but thoroughly and store in 0.5–1 ml aliquots at −20°C.

See Note 11.

See Note 12.

See Note 13.

3.4 Overview of Cell Culturing and Protein Production

See Tables 2 and 3.

Notes

• 1. The initial expansion medium should contain 20–30% conditioned medium, i.e., supernatant of a previous culture.

• 2. Starting with a titer of more than 1  ×  10⁷ cells/ml may hamper optimal protein production though may be tolerable. At this step, a weekly production cycle starts which is continued 3 days later with induction of protein expression.

• 3. CuSO₄ induces the expression of the MBD-Fc gene via the metallothionein promoter.

• 4. During these 4 days of incubation, the MBD-Fc protein is expressed and excreted into the medium via an excretion signal engineered in the expression vector.

• 5. Cells can be recycled: Resuspend the cells in one of the conical centrifuge bottles with 50 ml Xpress medium containing Pen/Strep and Hygromycin and start a next round of production (refer to subheading 2.2 item 1 (Appendix)). The weekly protein production cycle may be continued until a considerable decrease in the amount of produced protein becomes obvious.

• 6. Use a funnel to facilitate transfer to dialysis tubing. Dialyze in cold room using a 10 L plastic bucket placed on a magnetic stirrer. Add 2.5 g sodium azid (NaN₃) to 5 L TBS to prevent fungal growth.

• 7. Set the speed of the peristaltic pump such that 80 ml buffer or dialysate passes the sepharose matrix within about 60 min.

• 8. Presence of 500 mM NaCl at this step proved very efficient to remove possible Drosophila DNA contamination derived from the cell culture.

• 9. 1.5 M Tris–HCl, pH 8.8 neutralizes the acidic elution buffer and, hence, stabilizes the eluted protein.

• 10. Fractions with absorbance higher than 2 usually start with fractions 7–9 and end with fractions 11–13.

• 11. The protein concentration may be determined by any established protein quantitation method, e.g., the Pierce Protein Assay. MBD-Fc protein yield from 600 ml S2-cell culture usually ranges between 5 and 15 mg. Protein purity may be tested by SDS PAGE. Protein size is approximately 40 kDa.

• 12. Avoid repeated freezing and thawing since this may harm protein stability.

• 13. To restore the rProtein A-sepharose column matrix, wash with 80 ml 3 M KCl, then with 80 ml TBS and finally with TBS containing 20% ethanol. Keep the column with sepharose matrix at 4°C until next use. The column matrix may be reused four to five more times. It should be replaced if the yield drops considerably or the amount of contaminating protein as observed by SDS-PAGE considerably increases.