Sumoylation and desumoylation assays for a chromatin-remodelling complex in vivo and in vitro
Related Journal & Article Information
Manuscript: NCB-B08340D
Journal: Nature Cell Biology
Introduction
Small ubiquitin-like modifier (SUMO) is a small molecule, but has a variety of regulatory functions in cells [1-3]. SUMO modification is involved in transcriptional regulation, subcellular localization, and protein-protein interactions. SUMO conjugation requires sequential E1-dependent activation, E2-dependent conjugation, and E3-dependent ligation steps. Similar to protein phosphorylation, ubiquitination, acetylation, and methylation, SUMO conjugation and deconjugation are a dynamic processes. SUMOylating enzymes add SUMO to protein substrates, whereas deSUMOylating enzymes remove SUMO from SUMO-conjugated proteins. Many transcription factors and coregulators are SUMOylated and SUMOylation of these factors are mainly involved in transcriptiona repression mechanism [4, 5].
SUMOylation and deSUMOylation assays are available to investigate the dynamics of SUMOylation/deSUMOylation process, and provide exciting ways to study transcriptional regulatory mechanisms. This protocol details both in vivo and in vitro SUMOylation/deSUMOylationg assays that provide combined molecular approaches to study cellular functions of chromatin remodelling complexes.
Materials
Reagents
SUMO1, reptin, SAE1/2 (E1), Ubc9 (E2), SENP1, SENP1C603A expression plasmids, PolyFect transfection reagent (Qiagen), complete protease inhibitor cocktail (Roche), Ni-NTA agarose beads (Qiagen), TNT T7 quick-coupled reticulocyte lysate kit (Promega), and protein A/G-sepharose beads (Sigma).
Equipment
Procedure
In vivo SUMOylation assay
1. HeLa cells grown in 35 mm dish were transfected with 0.3 mg of reptin, 0.6 mg of HisMax-SUMO1, and 0.6 mg of Ubc9 plasmids using PolyFect transfection reagents.
2. After 36 hrs, the cells were lysed in 500 ml of lysis buffer (150 mM NaCl, 25 mM Tris-HCl at pH 7.8, 0.1 % Nonidet P40, and 1 mM EDTA) supplemented with complete protease inhibitor cocktail and 0.2 % SDS.
3. The crude extracts were sonicated briefly for 3 sec (setting 3, Branson Sonifier 450), and were clarified by centrifugation at 13,000 rpm for 30 min.
4. The supernatants were incubated in 15 ml of Ni-NTA agarose (50 % slurry) beads for 2 hrs, and the slurry was washed with 1 ml of lysis buffer four times.
5. Proteins were separated by SDS-PAGE and the existence of SUMOylated reptin and non-SUMOylated reptin was confirmed by immunoblotting with anti-reptin IgG or anti-SUMO antibody.
In vitro SUMOylation assay
1. SUMO1, GST-SAE1/2, and His-Ubc9 proteins were purified for the in vitro SUMOylation assay.
2. The assay was performed in a 10 ml of reaction volumes containing 2 mg of purified SUMO protein, 0.3 mg of purified SAE1/2 protein, 0.5 mg of purified Ubc9 protein, 0.2 ml of 100 mM ATP, 1X protease inhibitor cocktail, and an ATP regenerating system (50 mM Tris-HCl at pH 7.6, 5 mM MgCl2, 2 mM ATP, 10 mM creatine phosphate, 3.5 U/ml creatin kinase, and 0.6 U/ml inorganic pyrophosphatase) with 1 ml of 35S-methionine-labelled in vitro-translated reptin protein.
3. Reaction products were incubated at 37 °C for 3 hrs, and analysed by autoradiography.
In vivo deSUMOylation assay
1. HeLa cells grown in 24-well plate were transfected with 0.05 mg of reptin, 0.15 mg of SUMO1, 0.15 mg of Ubc9, and 0.3 mg of SENP1 or SENP1C603A plasmids using PolyFect transfection reagents.
2. After 36 hrs, 100 ml of sampling buffer (25 mM Tris-HCl, pH. 6.8, 2 % b-mercaptoethanol, 0.8 % (w/v) SDS, 0.1 % bromophenol blue, and 10 % (v/v) glycerol) was added to each well for the purpose of cell lysis.
3. Each 20 ml of cell lysate was loaded for SDS-PAGE followed by immunoblotting.
In vitro deSUMOylation assay
1. Each 10 mg of myc-SENP1 or SENP1 C603A was transfected to 293 cells grown in 100 mm plate using calcium phosphate transfection reagents.
2. After 48 hrs, cells were lysed with 500 ml of cell lysis buffer (150 mM NaCl, 1 mM EDTA, 25 mM Tris-HCl, pH 7.8, 10 % Glycerol, 0.1 % Nonidet P-40, and protease inhibitor cocktal).
3. The lysates were incubated with anti-myc antibody for 2 hr, and 50 ml of 50 % protein A-Sepharose bead slurry was mixed with the lysates for an additional 1 hr.
4. Immunoprecipitated materials were washed three times with cell lysis buffer to eliminate non-specific binding, followed by incubation with 5 ml of in vitro SUMOylated reptin at 37 °C for 4 hrs.
5. Reaction products were incubated at 37 °C for 3 hrs, and analysed by autoradiography.
Isolation of SUMOylated and non-SUMOylated reptin chromatin remodelling proteins
1. To isolate SUMOylated reptin protein, HeLa cells were transfected with Flag-reptin, Flag-Ubc9, and HisMax-SUMO1 plasmids.
2. After 48 hrs, cells were harvested and lysed with ice-cold phosphate-buffered saline (PBS) containing 0.2 % Nonidet P-40 and 1 mM DTT.
3. The lysates were sonicated for 3 secs (setting 3, Branson sofier 450), and the supernatant was taken after centrifugation. The supernatant was incubated with Ni-NTA agarose resin, and the resins were washed with PBS containing 0.2 % Nonidet P-40.
4. The bound materials were eluted with 300 mM imidazole. The eluted SUMOylated reptin were diluted with PBS containing 0.2 % Nonidet P-40 and 1 mM DTT and then incubated with anti-Flag M2 antibody-immobilized agarose beads.
5. The resulting immunoprecipitate was washed with PBS containing 0.2 % Nonidet P-40, followed by three washes with buffer containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.2 % Nonidet P-40, and 10 % glycerol.
6. The bound materials were eluted with 3X Flag peptide (200 mg/ml) and used as SUMOylated Flag-tagged reptin proteins.
7. To prepare unmodified non-SUMOylated reptin protein, HeLa cells were transfected only with Flag-reptin plasmid, and the lysate was prepared as described above. The cell lysates were sonicated for 3 secs (setting 3, Branson sonifier 450). The resulting supernatant was incubated with anti-Flag M2 antibody-immobilized agarose beads and Flag-tagged non-SUMOylated reptin was eluted as described above.
Troubleshooting
Critical Steps
Anticipated Results
References
1. Kerscher, O., Felberbaum, R. & Hochstrasses, M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 22, 159-180 (2006).
2. Hay, R. T. SUMO: a history of modification. Mol. Cell 18, 1-12 (2005).
3. Baek, S. H. & Rosenfeld, M. G. Nuclear receptor coreglators: their modification codes and regulatory mechanism by translocation. Biochem. Biophys. Res. Commun. 319, 7007-714 (2004).
4. Gill, G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms? Genes Dev. 18, 2046-2059 (2004).
5. Baek, S. H. A novel link between SUMO modification and cancer metastasis. Cell Cycle 5, 1492-1495 (2006).
Acknowledgements
We thank Jason S. Lee (Seoul National University) for critical reading of the manuscript. This work was supported by Korea Research Foundation Grant, the Molecular and Cellular Biodiscovery Research Program and SRC program from MOST/KOSEF, Brain Korea 21 Research Program, and the National R&D program for cancer control from Ministry of Health & Welfare (S.H.B.), and Ubiquitome Research Program and SRC program from MOST/KOSEF (K.I.K.).
Keywords
SUMO, reptin, chromatin-remodelling complex, sumoylation, desumoylation