We thank Diana Adjaoud for technical assistance. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (2015-2020), Genome Quebec (2016-2019) and Genome Canada (2016-2019) to E.B.A. E.B.A. is a senior scholar of the Fonds de la Recherche du Qu&#233;bec-Sant&#233; (FRQ-S). L.M and N.S.K. have a PhD scholarship from the FRQ-S. H.B had a PhD scholarship from the Ministry of Higher Education and from Scientific Research of Tunisia and the Cole Foundation.

1. GSH-agarose Affinity Purification of the GST-RING1B(1-159)-BMI1(1-109) E3 Ubiquitin Ligase Complex
<ol>
	<li>Use the pGEX6p2rbs-GST-RING1B (1-159 aa) - BMI1 (1 -109aa) bacteria expression construct to transform BL21 (DE3) bacteria (see Table of Materials)23. This construct allows the expression of murine RING1B domain 1-159 fused to BMI1 domain 1-109 with GST tag in pGEX-6P-2 backbone.</li>
	<li>Perform an overnight starter culture by inoculating RIL-bacteria expressing GST-RING1B (1-159 aa) - BMI1(1 -109 aa) in 20 mL of LB broth medium in presence of 100 &#181;g/mL ampicillin and 50 &#181;g/mL chloramphenicol. Incubate by shaking at 37 &#176;C overnight. On the next day, add the starter culture to a 1 L flask containing 500 mL of fresh LB broth medium (1/26 dilution) with ampicillin and incubate the culture in the shaker at 37 &#176;C for 2 to 4 h.</li>
	<li>During the incubation time, measure the OD at 600 nm with a spectrophotometer. When bacteria culture reaches 0.6 OD units at 600 nm, take a 1 mL aliquot in an 1,5 mL tube as the non-induced sample. Add 400 &#181;M of Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) to the 1 L culture to induce protein expression. Incubate bacteria at 25 &#176;C for 6 h to overnight.
	<ol>
		<li>Centrifuge the 1 mL aliquots at 14,000 x g for 30 s, discard the supernatant, resuspend the pellet in 200 &#956;L of Laemmli sample buffer, and keep for SDS-PAGE and Coomassie blue analysis of protein induction.</li>
	</ol>
	</li>
	<li>Transfer the induced bacteria culture from the flask into a clean centrifugation bottle and spin down at 3,500 x g for 15 min at 4 &#176;C. Discard the supernatant and resuspend the pellet with 40 mL of cold PBS and spin down at 3,500 x g for 15 min at 4 &#176;C.</li>
	<li>Discard the supernatant and freeze the pellet at -80 &#176;C or proceed to the next step. If the proteins are induced at the expected molecular weight, proceed to the next step.</li>
	<li>Resuspend the bacteria pellet in 25 mL of ice-cold lysis buffer A (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% NP40, 1 mM PMSF, 0.5 mM DTT, and 1/100 anti-protease cocktail containing AEBSF, Aprotinin, Bestatin, E-64, Leupeptin, and Pepstatin A). Make sure that all the bacteria pellet is resuspended. PMSF and anti-protease cocktail must be freshly added to the lysis buffer, only at the time of bacteria lysis. 
	CAUTION: Always keep the sample on ice. 
	NOTE: Lysozyme at 100 &#181;g/mL can be added to enhance bacteria lysis.</li>
	<li>Incubate the bacteria lysate on ice for 15 min. Use a probe sonicator and sonicate at 70% output amplitude for 30 s (4&#8211;5x), and then centrifuge at 21,000 x g for 20 min at 4 &#176;C. 
	CAUTION: During sonication, keep always the samples on ice.</li>
	<li>During the time of centrifugation, take 500 &#181;L packed GSH-agarose beads (50% slurry) in a 15 mL tube and add 10 mL of buffer A without PMSF and anti-proteases. Mix briefly and spin at 2,500 g for 3 min at 4 &#176;C, repeat this wash step two more times and keep the beads on ice.</li>
	<li>Following bacterial lysate centrifugation, collect the supernatant and transfer it into a 50 mL conical tube. Take an aliquot of 100 &#956;L as a total lysate and add 100 &#956;L of 2x Laemmli sample buffer for SDS-PAGE and Coomassie blue analysis.</li>
	<li>Filter the lysate through 0.45 &#956;m pore syringe filter and mix it with the GSH beads. Incubate at 4 &#176;C with shaking for 5 h. Spin down the beads at 2,500 x g for 3 min, remove, and save the supernatant as the flow through. Wash the proteins-GSH bound beads 6 times (5 mL each) with buffer A containing anti-protease cocktail and PMSF.</li>
	<li>After the last wash, transfer the beads along with 10 mL of buffer into an empty chromatography column, add 1.5 mL buffer A containing 25 mM glutathione, and collect the elution by gravity into 1.5 mL microtubes. Repeat the elution procedure 4 times. 
	NOTE: Glutathione stock solution is prepared at 200 mM concentration in 50 mM Tris-HCl, pH 7.5</li>
	<li>Take an aliquot of 10 &#181;L from each one of the 4 elutions and add 10 &#181;L of 2x Laemmli sample buffer. These samples will be used for SDS-PAGE and Coomassie blue analysis to determine the presence and the purity of the purified proteins. 
	NOTE: Following the last elution, keep the beads in buffer at 4 &#176;C for recycling and future use.</li>
	<li>Choose the eluted fractions showing reasonable amounts of purified proteins for further analysis. Make small aliquots of the preparation. Store the purified proteins in -80&#176;C. Usually, protein concentration will range from 0.5 &#181;g to 2 &#181;g per &#181;L and samples can be stored in this state.</li>
	<li>OPTIONAL: Concentrate the sample or change buffer using a 10K centrifugal filter unit</li>
</ol>
2. Purification of the BAP1/DEUBAD (ASXL2) Deubiquitinase Complex
<ol>
	<li>Use pET30a+-His-BAP138 and pDEST-MBP-DEUBAD (ASXL2)35 bacterial expression constructs to transform BL21 (DE3) RIL bacteria for the production of His-BAP1 and MBP-DEUBAD respectively.</li>
	<li>Perform two separate overnight starter cultures by incubating His-BAP1 and MBP-DEUBAD (ASXL2) expressing bacteria in 20 mL of LB broth of ampicillin medium containing 50 &#181;g/mL chloramphenicol and the corresponding antibiotic for each plasmid, 100 &#181;g/mL of kanamycin or 100 &#181;g/mL of ampicillin respectively. Place on shaker at 37&#176;C.</li>
	<li>Perform steps 1.3&#173;&#8211;1.6.</li>
	<li>Resuspend the bacteria pellets in 25 mL of ice-cold lysis buffer B (50 mM Tris pH 7.3, 500 mM NaCl, 3 mM &#946;-Mercaptoethanol, 0.2% Triton X-100, 1 mM PMSF and 1/100 anti-protease cocktail). Mix equal volumes of the His-BAP1 with the MBP-DEUBAD lysates and incubate on ice for 15 min. Sonicate and then centrifuge the lysate as indicated in protocol 1 (step 9).
	<ol>
		<li>During the centrifugation, prepare 500 &#181;L packed Ni-NTA agarose beads (50% slurry) by washing them three times with buffer B without PMSF and anti-protease cocktail.</li>
	</ol>
	</li>
	<li>Following bacterial lysate centrifugation, collect the supernatant and transfer it into a 50 mL conical tube. Take an aliquot of 100 &#956;L as a total lysate and add 100 &#956;L of 2x Laemmli sample buffer&#160;for SDS-PAGE and Coomassie blue analysis.</li>
	<li>Filter the lysate through 0.45 &#956;m pore syringe filter and mix it with the Ni-NTA beads. Incubate at 4&#176;C with shaking for 5 h. Spin down the beads at 2,500 x g for 3 min, remove, and save the supernatant as the flow through.
	<ol>
		<li>Wash the beads 6 times (5 mL each) with buffer B containing 20 mM 1,3-Diaza-2,4-cyclopentadiene (Imidazole). 
		NOTE: Make a stock solution of 2 M imidazole at pH 7.3.</li>
	</ol>
	</li>
	<li>After the last wash, transfer the beads with 10 mL of buffer into an empty chromatography column, add 1 mL buffer B containing 200 mM Imidazole and collect the elution by gravity into 1.5 mL microtubes containing 10 &#181;L DTT (500 mM) and 2 &#181;L EDTA (500 mM, pH: 8). Repeat the elution procedure 4 times. Take 10 &#181;L of each elution fraction and add 10 &#181;L of 2x Laemmli sample buffer for SDS-PAGE and Coomassie blue analysis. Pool the desired eluted fractions. 
	NOTE: Keep the beads in buffer at 4 &#176;C for recycling and future use.</li>
	<li>Dilute the eluted complex 3 times with buffer C (50 mM Tris pH 7.3, 300 mM NaCl, 1% Triton X-100, 5 mM DTT, 1 mM EDTA, 1 mM PMSF, and 1/100 protease inhibitor cocktail) prior to incubation with the amylose agarose beads.
	<ol>
		<li>Prepare amylose agarose beads (500 &#181;L packed) by washing them 2 times with the buffer C without adding PMSF and anti-protease cocktail. Add the beads to the diluted elution and incubate overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Centrifuge at 2,500 x g for 3 min and keep the flow through. Wash the proteins-bounds beads with 5 mL of buffer C (5&#8211;6x).</li>
	<li>Keep the purified His-BAP1/MBP-DEUBAD complex on the amylose agarose beads in buffer D (50 mM HEPES pH 8.0, 50 mM NaCl, 10% Glycerol, and 1 mM DTT) and store at -80 &#176;C. Take 20 &#181;L of the beads fraction (50% beads solution) and add 20 &#181;L of 2x Laemmli sample buffer for SDS-PAGE and Coomassie blue analysis.</li>
</ol>
3. Purification of the Nucleosomes from HEK293T Cells
<ol>
	<li>Culture HEK293T cells in complete Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 5% newborn calf serum (NBS), 2 mM L-glutamine, and 1% penicillin/streptomycin.</li>
	<li>Plate around 12 x 106 HEK293T cells in 20 mL of complete media per 15 cm culture dish one day before the transfection (10 dishes were used). Before transfection, change the cell&#8217;s medium&#160;to 12 mL of serum-free medium&#160;and transfect the cells with 21 &#181;g of pCDNA-Flag-H2A using 63 &#181;l of polyethylenimine (PEI) at 1 mg/mL36. Change to complete medium 12 h later.</li>
	<li>Three days post-transfection, wash the cells with 15 mL ice-cold PBS (twice) and harvest them in 3 mL of ice-cold PBS using a cell scraper. Centrifuge at 2,100 x g for 8 min at 4 &#176;C. Discard the PBS and proceed to the lysis step or freeze the cell pellet at -80 &#176;C.</li>
	<li>Resuspend the cell pellet in about 10 volumes of buffer E (50 mM Tris-HCl pH 7.3, 420 mM NaCl, 1% NP-40, 1 mM MgCl2, 1 mM PMSF, 1/100 protease inhibitor cocktail, and 20 mM of N-methylmaleimide (NEM)) and incubate on ice for 20 min. Adding N-methylmaleimide (NEM) is critical to inhibit DUBs that co-purify with nucleosomes.</li>
	<li>After centrifugation at 3,000 x g for 5 min, discard the supernatant and resuspend the chromatin pellet in 10 volumes of buffer E. Mix by inversion and centrifuge at 3,000 x g for 5 min.
	<ol>
		<li>Repeat the wash step another time using buffer E and two times using buffer F &#34;MNase Buffer&#34; (20 mM Tris-HCl pH 7.5, 100 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 0.3 M sucrose, 0.1% NP-40, 1 mM PMSF, and 1/100 protease inhibitor cocktail). 
		CAUTION: Pellet will float during washes and centrifugation.</li>
	</ol>
	</li>
	<li>Resuspend the chromatin in 5 mL buffer F and treat the pellet with Micrococcal nuclease (MNase) (3 U/mL for 10 min at room temperature). 
	NOTE: The reaction can be aided by mixing using a Dounce Homogenizer.</li>
	<li>After incubation, take a 40 &#956;L aliquot of the mixture for analysis of nucleosomal DNA fragments. Mix this aliquot with 40 &#956;L of Phenol/chloroform and 20 &#956;L of 6x DNA loading buffer, vortex, spin at 18,000 x g for 2 min, and load the DNA on a 2% agarose gel.
	<ol>
		<li>When the DNA is predominantly a 147 bp fragment corresponding to mononucleosomes, stop the reaction by adding 5 mM EDTA at final concentration.</li>
	</ol>
	</li>
	<li>Following centrifugation at 21,000 x g for 20 min at 4&#176;C, incubate the soluble chromatin fraction with anti-Flag resin overnight at 4 &#176;C. Wash the beads with 6x Buffer G (50 mM Tris-HCl, pH 7.3, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 1 mM PMSF, 1 mM DTT, 1/100 protease inhibitors cocktail).</li>
	<li>Transfer the beads with 5 mL buffer G into an empty chromatography column. Elute the beads-bound nucleosomes with 200 &#956;g/mL of Flag elution peptide. Use an elution buffer composed of buffer G containing 200 &#956;g/mL Flag elution peptides (see Table of Materials) and 1/5 (vol:vol) 1 M Tris, pH 8.0. Elute the nucleosomes 3x with 260 &#956;L Flag elution Buffer (2 h each elution).</li>
	<li>Test the 3 elutions by taking an aliquot for phenol-chloroform extraction and load the DNA in a 2% agarose gel. Take 10 &#181;L of each elution fraction and add 10 &#181;L of Laemmli sample buffer 2x for SDS-PAGE and Coomassie blue analysis and load each elution for Western Blot detection of ubiquitinated H2A. Store the elution at -80 &#176;C. In general, purification from human cells yields less amount of proteins than that from bacteria, with concentrations ranging from 0.1 to 0.5 &#956;g/&#956;L.</li>
</ol>
4. Nucleosome Ubiquitination Assay Using BMI1/RING1B E3 Ubiquitin Ligase Complex
<ol>
	<li>Centrifuge the nucleosomes through 10K pore 0.5 mL centrifugal filters to change the substrate from the elution buffer to the reaction buffer H (25 mM Tris, pH 7.5; 10 mM MgCl2, and 5 mM ATP). Alternatively, commercially available nucleosomes can be used for the assay. 
	NOTE: Changing substrate suspension solution to reaction buffer ensures reproducibility and prevents potential E3 ligase inhibition by compounds present in the Flag elution mixture.</li>
	<li>Incubate 1 &#956;g of the nucleosomes in 40 &#956;L total volume of buffer H. Add Ub Activating Enzyme (UBE1) (250 ng), Ub (50 ng/&#956;L) and E2 Ub-conjugating enzymes (672 ng), and 1 &#956;g of BMI1/RING1B E3 ubiquitin ligase complex. Incubate the reaction for 3 h at 37 &#176;C with occasional shaking. 
	NOTE: Several controls can be conducted in parallel including omitting, E1, E2, E3, ATP and ubiquitin. This ensures the specificity for the ubiquitination reaction.</li>
	<li>Stop the reaction by adding 40 &#956;L of 2x Laemmli sample buffer and analyze histone H2A K119 ubiquitination by SDS-PAGE and western blotting, according to standard procedures. Use either anti-H2A or anti-H2A K119ub antibodies (see Table of Materials).</li>
</ol>
5. In Vitro Nucleosomal H2A DUB Assay Using BAP1/DEUBAD
<ol>
	<li>Use the purified nucleosomes for&#160;the in vitro deubiquitination assay. Resuspend 100&#8211;500 ng of nucleosomes in 40 &#956;L buffer I (50 mM Tris-HCl, pH 7.3, 1 mM MgCl2, 50 mM NaCl, and 1 mM DTT). Add 1 &#956;g of BAP1 bacteria-purified His-BAP1 and MBP-DEUBAD. Carry out the deubiquitination reaction for 3 h at 37 &#176;C with occasional shaking.</li>
	<li>Stop the in vitro reaction by adding 40 &#956;L of 2x Laemmli buffer and analyze by immunoblotting.</li>
</ol>

<ol>
	<li>Ye, Y., Rape, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+ubiquitin+chains:+E2+enzymes+at+work.">Building ubiquitin chains: E2 enzymes at work.</a> Nature Reviews Molecular Cell Biology. 10, 755-764 (2009).</li><li>Komander, D., Clague, M. J., Urbe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+the+chains:+structure+and+function+of+the+deubiquitinases.">Breaking the chains: structure and function of the deubiquitinases.</a> Nature Reviews Molecular Cell Biology. 10, 550-563 (2009).</li><li>Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unravelling+of+the+ubiquitin+system.">The unravelling of the ubiquitin system.</a> Nature Reviews Molecular Cell Biology. 16, 322-324 (2015).</li><li>Nakayama, K. I., Nakayama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+ligases:+cell-cycle+control+and+cancer.">Ubiquitin ligases: cell-cycle control and cancer.</a> Nature Reviews Cancer. 6, 369-381 (2006).</li><li>Senft, D., Qi, J., Ronai, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+ligases+in+oncogenic+transformation+and+cancer+therapy.">Ubiquitin ligases in oncogenic transformation and cancer therapy.</a> Nature Reviews Cancer. 18, 69-88 (2018).</li><li>Zheng, N., Shabek, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+Ligases:+Structure,+Function,+and+Regulation.">Ubiquitin Ligases: Structure, Function, and Regulation.</a> Annual Review of Biochemistry. 86, 129-157 (2017).</li><li>Iwai, K., Fujita, H., Sasaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+ubiquitin+chains.+NF-kappaB+signalling,+cell+death+and+beyond.">Linear ubiquitin chains. NF-kappaB signalling, cell death and beyond.</a> Nature Reviews Molecular Cell Biology. 15, 503-508 (2014).</li><li>Kulathu, Y., Komander, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atypical+ubiquitylation+-+the+unexplored+world+of+polyubiquitin+beyond+Lys48+and+Lys63+linkages.">Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages.</a> Nature Reviews Molecular Cell Biology. 13, 508-523 (2012).</li><li>Yau, R., Rape, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+increasing+complexity+of+the+ubiquitin+code.">The increasing complexity of the ubiquitin code.</a> Nature Cell Biology. 18, 579-586 (2016).</li><li>Mevissen, T. E. T., Komander, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Deubiquitinase+Specificity+and+Regulation.">Mechanisms of Deubiquitinase Specificity and Regulation.</a> Annual Review of Biochemistry. 86, 159-192 (2017).</li><li>Bedford, L., Lowe, J., Dick, L. R., Mayer, R. J., Brownell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-like+protein+conjugation+and+the+ubiquitin-proteasome+system+as+drug+targets.">Ubiquitin-like protein conjugation and the ubiquitin-proteasome system as drug targets.</a> Nature Reviews Drug Discovery. 10, 29-46 (2011).</li><li>Minton, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammasomes:+Ubiquitin+lines+up+for+inflammasome+activity.">Inflammasomes: Ubiquitin lines up for inflammasome activity.</a> Nature Reviews Immunology. 14, 580-581 (2014).</li><li>Popovic, D., Vucic, D., Dikic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+in+disease+pathogenesis+and+treatment.">Ubiquitination in disease pathogenesis and treatment.</a> Nature Medicine. 20, 1242-1253 (2014).</li><li>Upadhyay, A., Amanullah, A., Chhangani, D., Mishra, R., Mishra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+multifaceted+E3+ubiquitin+ligases+barricade+extreme+defense:+Potential+therapeutic+targets+for+neurodegeneration+and+ageing.">Selective multifaceted E3 ubiquitin ligases barricade extreme defense: Potential therapeutic targets for neurodegeneration and ageing.</a> Ageing Research Reviews. 24, 138-159 (2015).</li><li>Hammond-Martel, I., Yu, H., Affar el, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+ubiquitin+signaling+in+transcription+regulation.">Roles of ubiquitin signaling in transcription regulation.</a> Cellular Signalling. 24, 410-421 (2012).</li><li>Schwertman, P., Bekker-Jensen, S., Mailand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+DNA+double-strand+break+repair+by+ubiquitin+and+ubiquitin-like+modifiers.">Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers.</a> Nature Reviews Molecular Cell Biology. 17, 379-394 (2016).</li><li>Uckelmann, M., Sixma, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+ubiquitination+in+the+DNA+damage+response.">Histone ubiquitination in the DNA damage response.</a> DNA Repair. 56, 92-101 (2017).</li><li>Robzyk, K., Recht, J., Osley, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rad6-dependent+ubiquitination+of+histone+H2B+in+yeast.">Rad6-dependent ubiquitination of histone H2B in yeast.</a> Science. 287, 501-504 (2000).</li><li>Hwang, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+RING+finger+protein+required+for+histone+H2B+monoubiquitination+and+cell+size+control.">A conserved RING finger protein required for histone H2B monoubiquitination and cell size control.</a> Molecular Cell. 11, 261-266 (2003).</li><li>Kim, J., Hake, S. B., Roeder, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+homolog+of+yeast+BRE1+functions+as+a+transcriptional+coactivator+through+direct+activator+interactions.">The human homolog of yeast BRE1 functions as a transcriptional coactivator through direct activator interactions.</a> Molecular Cell. 20, 759-770 (2005).</li><li>Wood, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=an+E3+ubiquitin+ligase+required+for+recruitment+and+substrate+selection+of+Rad6+at+a+promoter.">an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter.</a> Molecular Cell. 11, 267-274 (2003).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+histone+H2A+ubiquitination+in+Polycomb+silencing.">Role of histone H2A ubiquitination in Polycomb silencing.</a> Nature. 431, 873-878 (2004).</li><li>Buchwald, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+E3-ligase+activity+of+the+Ring-Ring+complex+of+polycomb+proteins+Bmi1+and+Ring1B.">Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1B.</a> The EMBO Journal. 25, 2465-2474 (2006).</li><li>Scheuermann, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H2A+deubiquitinase+activity+of+the+Polycomb+repressive+complex+PR-DUB.">Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB.</a> Nature. 465, 243-247 (2010).</li><li>Jensen, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAP1:+a+novel+ubiquitin+hydrolase+which+binds+to+the+BRCA1+RING+finger+and+enhances+BRCA1-mediated+cell+growth+suppression.">BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.</a> Oncogene. 16, 1097-1112 (1998).</li><li>Harbour, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+mutation+of+BAP1+in+metastasizing+uveal+melanomas.">Frequent mutation of BAP1 in metastasizing uveal melanomas.</a> Science. 330, 1410-1413 (2010).</li><li>Abdel-Rahman, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GermLine+BAP1+mutation+predisposes+to+uveal+melanoma,+lung+adenocarcinoma,+meningioma,+and+other+cancers.">GermLine BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers.</a> Journal of Medical Genetics. , (2011).</li><li>Bott, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+deubiquitinase+BAP1+is+commonly+inactivated+by+somatic+mutations+and+3p21.1+losses+in+malignant+pleural+mesothelioma.">The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma.</a> Nature Genetics. 43, 668-672 (2011).</li><li>Goldstein, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GermLine+BAP1+mutations+and+tumor+susceptibility.">GermLine BAP1 mutations and tumor susceptibility.</a> Nature Genetics. 43, 925-926 (2011).</li><li>Testa, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GermLine+BAP1+mutations+predispose+to+malignant+mesothelioma.">GermLine BAP1 mutations predispose to malignant mesothelioma.</a> Nature Genetics. 43, 1022-1025 (2011).</li><li>Wiesner, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GermLine+mutations+in+BAP1+predispose+to+melanocytic+tumors.">GermLine mutations in BAP1 predispose to melanocytic tumors.</a> Nature Genetics. 43, 1018-1021 (2011).</li><li>Dey, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+tumor+suppressor+BAP1+causes+myeloid+transformation.">Loss of the tumor suppressor BAP1 causes myeloid transformation.</a> Science. 337, 1541-1546 (2012).</li><li>Pena-Llopis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAP1+loss+defines+a+new+class+of+renal+cell+carcinoma.">BAP1 loss defines a new class of renal cell carcinoma.</a> Nature Genetics. 44, 751-759 (2012).</li><li>Bononi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAP1+regulates+IP3R3-mediated+Ca2++flux+to+mitochondria+suppressing+cell+transformation.">BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation.</a> Nature. 546, 549-553 (2017).</li><li>Daou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoubiquitination+of+ASXLs+controls+the+deubiquitinase+activity+of+the+tumor+suppressor+BAP1.">Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1.</a> Nature Communications. 9, 4385 (2018).</li><li>Daou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BAP1/ASXL2+Histone+H2A+Deubiquitinase+Complex+Regulates+Cell+Proliferation+and+Is+Disrupted+in+Cancer.">The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and Is Disrupted in Cancer.</a> The Journal of Biological Chemistry. 290, 28643-28663 (2015).</li><li>Mashtalir, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autodeubiquitination+protects+the+tumor+suppressor+BAP1+from+cytoplasmic+sequestration+mediated+by+the+atypical+ubiquitin+ligase+UBE2O.">Autodeubiquitination protects the tumor suppressor BAP1 from cytoplasmic sequestration mediated by the atypical ubiquitin ligase UBE2O.</a> Molecular Cell. 54, 392-406 (2014).</li><li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+carboxyl+hydrolase+BAP1+forms+a+ternary+complex+with+YY1+and+HCF-1+and+is+a+critical+regulator+of+gene+expression.">The ubiquitin carboxyl hydrolase BAP1 forms a ternary complex with YY1 and HCF-1 and is a critical regulator of gene expression.</a> Molecular and Cellular Biology. 30, 5071-5085 (2010).</li><li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+suppressor+and+deubiquitinase+BAP1+promotes+DNA+double-strand+break+repair.">Tumor suppressor and deubiquitinase BAP1 promotes DNA double-strand break repair.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 285-290 (2014).</li><li>Dai, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAP1+inhibits+the+ER+stress+gene+regulatory+network+and+modulates+metabolic+stress+response.">BAP1 inhibits the ER stress gene regulatory network and modulates metabolic stress response.</a> Proceedings of the National Academy of Sciences of the United States of America. 114, 3192-3197 (2017).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAP1+links+metabolic+regulation+of+ferroptosis+to+tumour+suppression.">BAP1 links metabolic regulation of ferroptosis to tumour suppression.</a> Nature Cell Biology. 20, 1181-1192 (2018).</li><li>Kolluri, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+functional+BAP1+augments+sensitivity+to+TRAIL+in+cancer+cells.">Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells.</a> eLife. 7, (2018).</li><li>Machida, Y. J., Machida, Y., Vashisht, A. A., Wohlschlegel, J. A., Dutta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+BAP1+regulates+cell+growth+via+interaction+with+HCF-1.">The deubiquitinating enzyme BAP1 regulates cell growth via interaction with HCF-1.</a> The Journal of Biological Chemistry. , (2009).</li><li>Sahtoe, D. D., van Dijk, W. J., Ekkebus, R., Ovaa, H., Sixma, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAP1/ASXL1+recruitment+and+activation+for+H2A+deubiquitination.">BAP1/ASXL1 recruitment and activation for H2A deubiquitination.</a> Nature Communications. 7, 10292 (2016).</li></ol>

The authors declare no competing financial interests.

There are several advantages of establishing robust in vitro ubiquitination and deubiquitination assays for proteins of interest. These assays can be used to: (i) establish optimal conditions and define minimal requirement for these reactions, (ii) determine enzymatic kinetic and biochemical constants, (iii) define the roles of cofactors or inhibitors that can impact these reactions, (iv) identify interaction interfaces, (v) test the impact of artificial or disease-associated mutations and (vi) establish assay conditions...

Ubiquitination is one of the most conserved post-translational modifications and is critical for a wide variety of organisms including yeast, plants and vertebrates. Ubiquitination consists of the covalent attachment of ubiquitin, a highly conserved 76 amino acid polypeptide, to target proteins and occurs in three sequential steps involving three enzymes, i.e., E1-activating, E2-conjugating and E3 ligase1,2,3. This post-translational modification plays central roles in a wide spectrum of biological processes. Indeed, the E3 ligases, which provide the specificity of the reaction, constitute a large superfamily of enzymes and are the most abundant enzymes of the ubiquitin system4,5,6. The downstream effects of protein ubiquitination depend on nature of the modification: monoubiquitination, multi-monoubiquitination, and linear or branched polyubiquitination. Monoubiquitination is rarely associated with proteasomal degradation, but instead this modification is involved in mediating various signaling events. Polyubiquitination involves the N-terminal or the lysine residues in ubiquitin molecule itself, and the destiny of a polyubiquitinated protein depends on which residue is involved in ubiquitin chain extension. It has long been known that polyubiquitination mediated by lysine 48 of ubiquitin induces proteasomal degradation. On the contrary, polyubiquitination via lysine 63 of ubiquitin is often associated with protein activation7,8,9. Similar to other important post-translational modifications, ubiquitination is reversible and ubiquitin removal from proteins is ensured by specific proteases termed deubiquitinases (DUBs), which have emerged as important regulators of cellular processes2,10. Importantly, many DUBs are highly specialized, and regulate, through deubiquitination, specific substrates, indicating that a fine balance between ubiquitination and deubiquitination is critical for protein function. E3s and DUBs, along with the proteasome degradation machinery and accessory factors, form the Ubiquitin Proteasome System (UPS, with &#62;1200 genes) which regulates major signaling pathways, several of which are associated with cell growth and proliferation, cell fate determination, differentiation, cell migration, and cell death. Importantly, deregulation of several signaling cascades involving ubiquitination promotes tumorigenesis and neurodegeneration diseases5,11,12,13,14.
Ubiquitination plays pervasive roles in chromatin biology and DNA-dependent processes15,16,17. For instance, monoubiquitination of histone H2A on lysine 119 (hereafter H2A K119ub) is a critical post-translational modification involved in transcriptional repression and DNA repair18,19,20,21,22. H2A K119ub is catalyzed by the Polycomb Repressive Complex 1 (PRC1), which plays a key role in the maintenance of epigenetic information and is highly conserved from Drosophila to human. Canonical PRC1 is constituted notably by the RING1B and BMI1, which are the core E3 ubiquitin ligase complex responsible for the above-mentioned ubiquitination event22,23. In Drosophila, H2A monoubiquitination (H2A K118ub which corresponds to H2A K119ub in mammalians) is reversed by the DUB Calypso, which interacts with Additional Sex Comb (ASX) forming the Polycomb-repressive DUB (PR-DUB) complex24. The mammalian ortholog of calypso, BAP1, is a tumor suppressor deleted or inactivated in various human malignancies25,26,27,28,29,30,31,32,33. BAP1 regulates DNA-dependent processes in the nucleus and Calcium-signaling-mediated apoptosis at the endoplasmic reticulum33,34,35,36,37,38,39,40,41,42. BAP1 assembles multi-subunit protein complexes containing transcription regulators notably ASXL1, ASXL2 and ASXL3 (ASXLs), three orthologues of ASX38,43. ASXLs use the DEUBiquitinase ADaptor (DEUBAD) domain, also termed ASXM domain, to stimulate BAP1 DUB activity35,36,44. Hence, ASXLs play important roles in coordinating BAP1 DUB activity at chromatin and more broadly its tumor suppressor function.
Several methods exist to study ubiquitination and deubiquitination processes. Notably, biochemical assays using proteins purified from bacteria remain very powerful in demonstrating direct ubiquitination of, or removal of ubiquitin from, specific substrates. These experiments can be conducted to investigate a range of parameters such as the determining the requirement of minimal complexes, determining reactions kinetics, defining structure/function relationships, and understanding the impact of pathological gene mutations. Here, we provide protocols to conduct ubiquitination and deubiquitination reactions on chromatin substrates with purified components. As a model system, in vitro ubiquitination and deubiquitination of nucleosomal H2A protein is presented. Bacteria-purified proteins assembled in minimal complexes of RING1B/BMI1 and BAP1/DEUBAD are used for ubiquitination or deubiquitination of nucleosomal H2A, respectively.

<table><tbody><tr><td>Amylose agarose beads</td><td>New England Biolabs</td><td>#E8021</td><td></td></tr><tr><td>Amicon Ultra 0.5 mL centrifugal filters 10K</td><td>Sigma-Aldrich</td><td>#UFC501096</td><td></td></tr><tr><td>Anti-H2AK119ub (H2Aub)</td><td>Cell Signaling Technology</td><td>#8240</td><td></td></tr><tr><td>Anti-Flag-agarose beads</td><td>Sigma-Aldrich</td><td>#A4596</td><td></td></tr><tr><td>Anti-protease cocktail</td><td>Sigma-Aldrich</td><td>#P8340</td><td></td></tr><tr><td>BL21 (DE3) CodonPlus-RIL bacteria</td><td>Agilent technologies</td><td>#230240</td><td></td></tr><tr><td>DMEM</td><td>Wisent</td><td>#319-005-CL</td><td></td></tr><tr><td>Empty chromatography column</td><td>Biorad</td><td>#731-1550</td><td></td></tr><tr><td>Flag peptide</td><td>Sigma-Aldrich</td><td>#F3290</td><td></td></tr><tr><td>GSH-agarose beads</td><td>Sigma-Aldrich</td><td>#G4510</td><td></td></tr><tr><td>HEK293T</td><td>ATCC</td><td>#CRL-3216</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>#I5513</td><td></td></tr><tr><td>Micrococcal nuclease (MNase)</td><td>Sigma-Aldrich</td><td>#N3755</td><td></td></tr><tr><td>Ni-NTA agarose beads</td><td>ThermoFisher Scientific</td><td>#88221</td><td></td></tr><tr><td>N-methylmaleimide (NEM)</td><td>Bioshop</td><td>#ETM222</td><td></td></tr><tr><td>Pore syringe filter 0.45 &amp;mu;m</td><td>Sarstedt</td><td>#83.1826</td><td></td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polysciences Inc</td><td>#23966-1</td><td></td></tr><tr><td>pGEX6p2rbs-GST-RING1B(1-159)-Bmi1(1-109)</td><td>Addgene</td><td>#63139</td><td></td></tr><tr><td>Ub Activating Enzyme (UBE1)</td><td>Boston Biochem</td><td>#E-305</td><td></td></tr><tr><td>UBCH5C (UBE2D3)</td><td>Boston Biochem</td><td>#E2-627</td><td></td></tr></tbody></table>

in vitro ubiquitination and deubiquitination assays of nucleosomal histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

GST-BMI1 and RING1B proteins are well produced in bacteria and can be readily extracted in the soluble fraction. Figure 1A shows a Coomassie blue staining for a typical purification of the GST-BMI1-RING1B complex. The GST-BMI1 and RING1B bands migrate at the expected molecular weight, ~45 kDa and ~13 kDa respectively. Notably the E3 ligase complex is highly homogenous with very low levels of bacteria proteins contaminants and/or degradation products. Moreover...

Watch this Scientific Journal Video about In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones at JoVE.com

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

in-vitro-ubiquitination-deubiquitination-assays-nucleosomal

Research

JoVE Journal

Biochemistry

10.6K Views.  University of Montréal. Gezien de complexiteit van de alomtegenwoordigheid en deubiquitatiereacties in de cellen is het belangrijk om in vitro tests vast te stellen met gedefinieerde gezuiverde componenten. Dit helpt om de mechanismen van alomtegenwoordigheid en deubiquitatie te begrijpen en ook om te bepalen hoe mutaties geassocieerd met genen die de enzymen die verantwoordelijk zijn voor deze reactie coderen, menselijke ziekte kunnen veroorzaken. We kunnen de domeinen en motieven in deze componenten identificeren ...

Video: In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Biology

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Assessing DUB Variants in Neurodevelopmental Disorders Using a Cleavage Assay

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. Discrete disorders caused by single genes provide models to investigate mechanisms driving atypical neurodevelopment. Variants of genes encoding deubiquitylating enzyme (DUB) family proteins are associated with several NDDs, but there is a need to determine the pathogenic mechanisms of disorders driven by these gene variants. The impact of gene variants on DUB activity can be experimentally determined using a substrate-independent in vitro ubiquitin cleavage assay. This assay does not require knowledge of downstream substrates to directly measure catalytic activity. Here, the protocol for determining the impact of gene variants on enzymatic activity is modeled using the DUB Ubiquitin Specific Protease 27, X-linked (USP27X), which is mutated in X-linked intellectual disability 105 (XLID105). This experimental pipeline can be used to clarify the mechanisms underlying neurodevelopmental disorders driven by variants in DUB genes.

This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Samenvatting

Meer video's verkennen