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Journal of Visualized ECopyright 2011 Creative Commons Attribution-NonCommercial LicenseApril 2011|50|e2593|Page 1 of 4Video ArticleDetection of Post-translational Modifications on Native Intact Nucleosomes byELISABo Dai1,Farida Dahmani2,Joseph A.Cichocki3,Lindsey C.Swanson2,Theodore P.Rasmussen31Institute for Stem Cell Biology and Regenerative Medicine,Stanford University2Department of Molecular and Cell Biology,University of Connecticut3Department of Pharmaceutical Sciences,University of ConnecticutCorrespondence to:Theodore P.Rasmussen at theodore.rasmussenuconn.eduURL:http:/ Biology,Issue 50,Chromatin,Nucleosome,Epigenetics,ELISA,Histone,Modification,Methylation,AcetylationDate Published:4/26/2011Citation:Dai,B.,Dahmani,F.,Cichocki,J.A.,Swanson,L.C.,Rasmussen,T.P.Detection of Post-translational Modifications on Native IntactNucleosomes by ELISA.J.Vis.Exp.(50),e2593,doi:10.3791/2593(2011).AbstractThe 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 richin 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 thatis epigenetic in nature 2,3.We developed a method called nucleosome ELISA(NU-ELISA)to quantitatively determine global PTM signaturesof nucleosomes extracted from cells.NU-ELISA is more sensitive and quantitative than western blotting,and is useful to interrogate theepiproteomic state of specific cell types.This video journal article shows detailed procedures to perform NU-ELISA analysis.Video LinkThe video component of this article can be found at http:/ Cell CultureNU-ELISA can be performed on any mammalian cell type that can be grown in culture.We prefer to prepare moderate to large batches of cellsso that nucleosomes can be isolated in sufficient quantity to prepare several identically loaded ELISA plates,thus allowing assays with severaldifferent antibodies(Abs).The following culture scales provide ample material:For mouse embyronic stem cells,grow one or two 15 cm plates of cells without feeder cells.For fibroblasts,grow 5 to 10 15 cm plates toconfluence.2.Isolation of NucleiNote:All the steps are on ice with pre-chilled buffers,except as indicated.1.Trypsinize cells with 3 ml trypsin per plate,combine,and add 20 ml of ice-cold PBS/butyrate.Centrifuge at 1000 rpm for 5 min.2.Resuspend the cells in 10 ml PBS/butyrate and centrifuge at 1000 rpm for 5 min.3.Resuspend in 4 ml lysis buffer with protease inhibitors(Sigma-Aldrich P-8340).Dounce homogenize 20 strokes with type B pestle on ice.4.Centrifuge at 2000g for 10 min at 4C.(The supernatant contains cytoplasm,which is not needed for this protocol,but it can be saved forother uses if desired).5.Resuspend the pellet in 2 ml ice cold wash buffer C(with protease inhibitors).6.Layer the resuspended material over 5 ml 30%sucrose cushion,then centrifuge at 2400g for 5 min in a swinging bucket rotor.Nuclei willmigrate through cushion,and debris remains at the interface.7.Remove all liquid volume carefully and resuspend the nuclei in 250 l ice cold wash buffer C+protease inhibitors.3.Isolation of Nucleosomes by in situ Micrococcal Nuclease(MNase)DigestionWe use a procedure in which chromatin is digested in situ within nuclei by infusing them with MNase,followed by a hypotonic treatment to drivefree intact nucleosomes into the supernatant.Journal of Visualized ECopyright 2011 Creative Commons Attribution-NonCommercial LicenseApril 2011|50|e2593|Page 2 of 41.Add 3 l 0.1 M CaCl2 to the sample.Put in 37C heat block.Allow to assume 37C temperature.2.Add 2 units MNase(Micrococcal Nuclease,Sigma-Aldrich,dissolved at 2 units/10l in MNase buffer),then incubate for at 37C for 12 min,with frequent mixing using a pipet tip.3.Add 6 l of 0.5 M Na-EDTA,pH 8.0 to stop the reaction.Put on ice.4.Centrifuge at 2000g for 4 min,discard supernatant.Resuspend the pellet in 300 l 0.2 mM Na-EDTA.Store on ice for 1 h with occasionalgentle pipeting.(These hypotonic conditions liberate free nucleosomes from nuclei).5.Centrifuge at 3000 g for 4 min at 4C.Save the supernatant,which contains free nucleosomes,on ice.6.Resuspend the pellet again with 300 l 0.2 mM Na-EDTA.Store on ice for 1 h with occasional gentle pipeting.7.Centrifuge at 3000g for 4min at 4C.Retain the supernatant and combine with the first supernatant from step 5.The resultingmononucleosome preparations can be aliquoted and stored at-80C.Note:The amount of chromatin can be crudely quantitated by measuring absorbance at 260 nm of a 10 l sample added to 990 l of water.A260=10(after adjusting for the 1/100 dilution)corresponds to about 1mg/ml of chromatin.Also,during the above procedure,small samples maybe retained and later analyzed for their DNA content to monitor the quality and extent of MNase digestions,which should predominantly containDNA of 146 bp in length.Laddering is indicative of incomplete MNase digestion.Note:Fig.1 contains a diagrammed summary of nuclear isolation and MNase digestions steps.4.Nucleosome-ELISA(NU-ELISA)We detect PTMs on fractions containing native intact nuclesosomes that have been immobilized on 96 well ELISA microtiter plates.For eachsample,we make a series of 2-fold dilutions,and these are coated onto wells in triplicate.1.Coat MaxiSorp plates overnight at 4C with 50 l/well of nucleosomes diluted in coating buffer.Suggested serial twofold dilutions ofnucleosomes are prepared from top row to the bottom row by adding 0.1 g,0.05 g,0.025 g,0.0125 g,0.00625 g,0.00313 g,0.00156g,with coating buffer only(0 g chromatin)in the bottom row.(Note:plate covers are needed for this step.)2.In the morning,wash the plates 4 times with 200 l/well PBS/0.5%Tween-20 at room temperature(RT)for a combined total of 10 min with aplate washer.3.Block 1 hr at RT with 100 l/well PBS/0.05%Tween-20/5%BSA.4.Remove the blocking buffer by shaking the inverted plate briskly over a sink.Cover and store the plates at-20C,or go directly to the nextstep.5.Add 1 Abs in a volume of 50 l/well diluted 1:1000(or as needed)in PBS/0.05%Tween-20/5%BSA,incubate at RT for 1 hour on a rotator.6.Wash plates 4 times with 200 l/well PBS/0.5%Tween-20 at RT and for a combined total of 10 min in a plate washer.7.Add horse radish peroxidase(HRP)-conjugated 2 Abs,diluted 1:5000(or as needed)in PBS/0.05%Tween-20/5%BSA at RT for an hour ona rotator.8.Wash plates 4 times with 200 l/well PBS/0.5%Tween-20.Each wash is at RT and for a total of 10 minutes with a plate washer.9.Develop the plates by adding 50 l of TMB substrate to each well for 10 min at RT.Stop the reaction by adding 50 l/well of 2N H2SO4 andread the plates 450 nm.(Tip:Centrifuge at 1500 rpm for 2 min using a plate rotor to dissipate any air bubbles in the wells prior to readingabsorbances).10.Export the readings to spreadsheet files for quantitative and statistical analyses.ReagentsPBS/butyrate135 mM NaCl2.5 mM KCl8 mM Na2HPO41.5 mM KH2PO410 mM Na-butyrateLysis Buffer250 mM sucrose10 mM Tris-HCl,pH 7.410 mM Na-butyrate4 mM MgCl20.1%Triton X-100Wash buffer C250 mM sucrose10 mM Tris-HCl,pH 7.410 mM Na-butyrateJournal of Visualized ECopyright 2011 Creative Commons Attribution-NonCommercial LicenseApril 2011|50|e2593|Page 3 of 44 mM MgCl2Sucrose Cushion30%(w/v)sucrose in wash buffer CMicroccocal Nuclease Buffer5 mM NaPO4 Buffer,pH 7.00.025 mM CaCl2Coating buffer80ml Solution A+170ml Solution B+250ml dH2OSolution A:0.2 M Na2CO3Solution B:0.2 M NaHCO35.Representative ResultsUsing the NU-ELISA method,a series of several identical plates are prepared which can be loaded with chromatin prepared in the form ofmononucleosomes.We typically prepare series of identically-loaded plates so that each can be probed with different anti-PTM specific antibodies(Abs).We also always prepare a plate that can be probed with an Ab that detects histones without regard to their modification state totalchromatin loading control.This control is essential in order to later quantitatively compare PTM content of nucleosomes prepared from differentsamples.To quantitate the levels of PTMs within a given chromatin sample we correct the raw absorbance values using this approach:First,we subtract any background signal using values obtained from control wells containing no chromatin(background is usually negligible).Wethen standardize PTM-specific signals to NU-ELISA results obtained from an identically loaded plate that was assayed with an Ab that detectsnucleosomes independent of their modification state.(We have used polyclonal Abs specific for histones H2A or H2B for this purpose).Wethen determine means and variances for each dilution of chromatin,and use data obtained from the linear portion of the ELISA assay.Detailedexamples of NU-ELISA mathematical and statistical analyses have been reported previously4Journal of Visualized ECopyright 2011 Creative Commons Attribution-NonCommercial LicenseApril 2011|50|e2593|Page 4 of 4 Figure 1.Schematic diagam of NU-ELISA steps.A.Mammalian Cells are harvested;B.Nuclei are separated from cells by Douncehomogenization,C.Disrupted cells are loaded on top of a 30%w/v sucrose cushion,D.After centrifugation,nuclei are deposited in the pellet;E,F.Chromatin are digested in situ to predominantly mononucleomes by MNase;G,H,I.Soluble mononucleosomes are extracted by hypotonictreatment and repeated centrifugation of residual nuclear material.J.Mononucleosomes from different samples(labeled samp.1 and 2 in thiscase)are coated on microtiter wells in series of identically-loaded plates and interrogated with PTM-specific Abs and PTM-independent Abs todetermine total chromatin loading.K.Depiction of differential signal detection levels in two samples that differ in their PTM content,yet havesimilar overall chromatin content,as judged by anti-H2B immunoreactivity.DiscussionNU-ELISA provides a method to ascertain the global status of nucleosome PTMs present within a particular cell type.NU-ELISA studies haveshown that global nucleosome modification states differ in comparisons of divergent cell types 4.In addition,NU-ELISA PTM profiles changewhen cells are exposed to epigenetic modulatory agents such as trichostatin-A or when mouse embyronic stem cells are differentiated 4.Themethod has also been applied successfully to study the chromatin of human ES cells5.It is important to note the NU-ELISA,in its present form,can detect only the composite signature of PTMs present within the sum total of the cellular epigenome.This differs markedly from genome-widechromatin immunoprecipitation,which can determine the distribution of a specific PTM across specific genetic loci.The initial steps of the NU-ELISA procedures are adapted from previous methods to isolate mononucleosomes from mammalian nuclei6,7.Theseadapted methods provide an expedient way to obtain high-quality intact mononucleosomes from mammalian cells,and the resulting fractionsare comprehensive in their chromatin content,but contain a great deal of additional nuclear material.Since specific Abs are used,contaminatingnon-nucleosomal material is tolerated well in the NU-ELISA assay,unlike mass-spec approaches that require greater purity.However,NU-ELISA is more sensitive and quantitative than western blotting 4,thus providing good alternatives to westerns and mass spec for the detection ofnucleosomal PTMs.DisclosuresNo conflicts of interest declared.AcknowledgementsDevelopment of the NU-ELISA method was supported by NIH grant RO1AG023687.References1.Luger,K.,Mader,A.W.,Richmond,R.K.,Sargent,D.F.&Richmond,T.J.Crystal structure of the nucleosome core particle at 2.8 A resolution.Nature 389,251-60(1997).2.Jenuwein,T.&Allis,C.D.Translating the histone code.Science 293,1074-80(2001).3.Turner,B.M.Histone acetylation and an epigenetic code.Bioessays 22,836-45(2000).4.Dai,B.&Rasmussen,T.P.Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells.Stem Cells 25,2567-74(2007).5.Tanasijevic,B.et al.Progressive accumulation of epigen etic heterogeneity during human ES cell culture.Epigenetics 4,330-8(2009).6.Thomas,J.O.Isolation and fractionation of chromatin and linker histones.in Chromatin:a practical approach(ed.Gould,H.)12-14(OxfordUniversity Press,Oxford,1998).7.Thorne,A.W.,Cary,P.D.&Crane-Robinson,C.Extraction and separation of core histones and non-histone chromosomal proteins.inChromatin:a practical approach(ed.Gould,H.)38-39(Oxford University Press,Oxford,1998).