Release of Hypoacetylated and Trimethylated Histone H4 Is an Epigenetic Marker of Early Apoptosis

Nuclear events such as chromatin condensation, DNA cleavage at internucleosomal sites, and histone release from chromatin are recognized as hallmarks of apoptosis. However, there is no complete understanding of the molecular events underlying these changes. It is likely that epigenetic changes such as DNA methylation and histone modifications that are involved in chromatin dynamics and structure are also involved in the nuclear events described. In this report we have shown that apoptosis is associated with global DNA hypomethylation and histone deacetylation events in leukemia cells. Most importantly, we have observed a particular epigenetic signature for early apoptosis defined by a release of hypoacetylated and trimethylated histone H4 and internucleosomal fragmented DNA that is hypermethylated and originates from perinuclear heterochromatin. These findings provide one of the first links between apoptotic nuclear events and epigenetic markers. Nuclear events such as chromatin condensation, DNA cleavage at internucleosomal sites, and histone release from chromatin are recognized as hallmarks of apoptosis. However, there is no complete understanding of the molecular events underlying these changes. It is likely that epigenetic changes such as DNA methylation and histone modifications that are involved in chromatin dynamics and structure are also involved in the nuclear events described. In this report we have shown that apoptosis is associated with global DNA hypomethylation and histone deacetylation events in leukemia cells. Most importantly, we have observed a particular epigenetic signature for early apoptosis defined by a release of hypoacetylated and trimethylated histone H4 and internucleosomal fragmented DNA that is hypermethylated and originates from perinuclear heterochromatin. These findings provide one of the first links between apoptotic nuclear events and epigenetic markers. Apoptosis is a form of cell death essential for the morphogenesis, development, differentiation, and homeostasis of eukaryotic multicellular organisms. The activation of a genetically controlled cell death program leading to apoptosis results in characteristic biochemical and morphological features that take place both outside and inside the nucleus (1Lawen A. BioEssays. 2003; 25: 888-896Crossref PubMed Scopus (350) Google Scholar). The biochemical mechanisms responsible for key nuclear events, such as chromatin condensation, DNA fragmentation, and release of nuclear proteins, although commonly used as markers for apoptosis, are not fully understood (2Martelli A.M. Zweyer M. Ochs R.L. Tazzari P.L. Tabellini G. Narducci P. Bortul R. J. Cell. Biochem. 2001; 82: 634-646Crossref PubMed Scopus (132) Google Scholar). The regulated nature of apoptosis makes it likely that nuclear changes experienced by apoptotic cells are mediated by epigenetic markers. This epigenetic information is basically stored as DNA methylation and post-translational histone modifications. These two groups of modifications play an active role in organizing, compartmentalizing, and regulating genetic information encoded in DNA by defining nuclear architecture, and gene expression (3Khorasanizadeh S. Cell. 2004; 116: 259-272Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). With regard to gene regulation, the major functional consequence of DNA methylation is the repression of transcription (4Cedar H. Cell. 1988; 53: 3-4Abstract Full Text PDF PubMed Scopus (727) Google Scholar). In the case of histone modifications, the type of modification (acetylation, methylation, phosphorylation, etc.) and the specific amino acid residue that is modified determine the functional effect. Histone modifications also determine the nature of chromatin regions, such as heterochromatin. For example, the inactive X chromosome is characterized by trimethylation of Lys-27 of H3 and dimethylation of Lys-9 of H3 (5Plath K. Fang J. Mlynarczyk-Evans S.K. Cao R. Worringer K.A. Wang H. de la Cruz C.C. Otte A.P. Panning B. Zhang Y. Science. 2003; 300: 131-135Crossref PubMed Scopus (945) Google Scholar, 6Silva J. Mak W. Zvetkova I. Appanah R. Nesterova T.B. Webster Z. Peters A.H. Jenuwein T. Otte A.P. Brockdorff N. Dev. Cell. 2003; 4: 481-495Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 7Okamoto I. Otte A.P. Allis C.D. Reinberg D. Heard E. Science. 2004; 303: 644-649Crossref PubMed Scopus (615) Google Scholar), whereas Lys-9 trimethylation and Lys-27 monomethylation of H3 and Lys-20 trimethylation of H4 are characteristic of pericentric heterochromatin (8Peters A.H. Kubicek S. Mechtler K. O'Sullivan R.J. Derijck A.A. Perez-Burgos L. Kohlmaier A. Opravil S. Tachibana M. Shinkai Y. Martens J.H. Jenuwein T. Mol. 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Wesierska-Gadek J. Int. J. Cancer. 2003; 106: 486-495Crossref PubMed Scopus (80) Google Scholar), although some authors have interpreted this result to be a loss of hyperacetylated histones by degradation rather than the consequence of the active hypoacetylation of histones during apoptosis (19Hendzel M.J. Nishioka W.K. Raymond Y. Allis C.D. Bazett-Jones D.P. Th'ng J.P. J. Biol. Chem. 1998; 273: 24470-24478Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Thus, there is a need to clarify the role of these modifications in different nuclear events during apoptosis. Regarding apoptotic release of histones and other nuclear proteins, it has been proposed that this process is associated with chromatin condensation and DNA fragmentation (20Wu D. Ingram A. Lahti J.H. Mazza B. Grenet J. Kapoor A. Liu L. Kidd V.J. Tang D. J. Biol. Chem. 2002; 277: 12001-12008Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 21Scaffidi P. Misteli T. Bianchi M.E. Nature. 2002; 418: 191-195Crossref PubMed Scopus (3303) Google Scholar). Histone release may not merely be a simple by-product of chromatin condensation or DNA fragmentation and could be the result of specific chromatin modifications in particular nuclear compartments. In fact, during apoptosis a series of nuclear matrices and membrane proteins that are fundamental to the maintenance of internal nuclear structures are degraded by caspases (22Lazebnik Y.A. Cole S. Cooke C.A. Nelson W.G. Earnshaw W.C. J. Cell Biol. 1993; 23: 7-22Crossref Scopus (424) Google Scholar, 23Rao L. Perez D. White E. J. Cell Biol. 1996; 135: 1441-1455Crossref PubMed Scopus (511) Google Scholar, 24Weaver V.M. Carson C.E. Walker P.R. Chaly N. Lach B. Raymond Y. Brown D.L. Sikorska M. J. Cell Sci. 1996; 109: 45-56Crossref PubMed Google Scholar). For instance, degradation of nuclear lamins, which maintain nuclear structure by attaching chromatin to the nuclear membrane through heterochromatic structures (25Pyrpasopoulou A. Meier J. Maison C. Simos G. Georgatos S.D. EMBO J. 1996; 15: 7108-7119Crossref PubMed Scopus (139) Google Scholar, 26Makatsori D. Kourmouli N. Polioudaki H. Shultz L.D. McLean K. Theodoropoulos P.A. Singh P.B. Georgatos S.D. J. Biol. Chem. 2004; 279: 25567-25573Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), coincides with chromatin condensation and DNA fragmentation in apoptotic cells. Although the breakdown of internal nuclear structures is a prerequisite for chromatin condensation and DNA fragmentation (27Krystosek A. Cell Biol. 1999; 111: 265-276Google Scholar, 28Robertson J.D. Orrenius S. Zhivotovsky B. J. Struct. Biol. 2000; 129: 346-358Crossref PubMed Scopus (257) Google Scholar), it has been proposed that the maintenance of high order chromatin structures is essential for proper chromatin condensation (29Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (500) Google Scholar). In fact, drugs that modify chromatin structure are able to block apoptotic chromatin condensation (30Johnson C.A. Padget K. Austin C.A. Turner B.M. J. Biol. Chem. 2001; 276: 4539-4542Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In this report, we have focused our attention on the global characterization of histone modification and DNA methylation changes associated with the apoptotic process, in particular in connection with the release of nuclear material to the cytosol. Significant global deacetylation of histones H3 and H4 associated with its release during apoptosis has been observed. Most interestingly, we have identified a unique pattern of post-translational modifications that is characteristic of histones released early on in apoptosis. Released histone H4 is specifically hypoacetylated and trimethylated at Lys-20, whereas released histone H3 is hypoacetylated, demethylated, and dephosphorylated. These histones are released and cofractionate with internucleosomally fragmented DNA with a greater 5-methylcytosine content than the DNA that remains in the nucleus. This released DNA originates in the heterochromatic perinuclear region of the cell. These data, together with the observed pattern of modification of released histones, suggest that the released material results from the degradation of perinuclear heterochromatin and internal structures of the nucleus early on in apoptosis. Materials—Jurkat and HL60 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum. The following rabbit polyclonal antibodies were used: anti-histone H3 (Abcam) directed against a C-terminal peptide of H3, anti-dimethyl Lys-4 histone H3 (Upstate Biotechnologies, Inc.), anti-dimethyl Lys-9 histone H3 (Upstate Biotechnologies, Inc.), anti-phospho Ser-10 histone H3 (Upstate Biotechnologies, Inc.), anti-acetyl histone H3 (Upstate Biotechnologies, Inc.), anti-acetyl Lys-9 histone H3 (Abcam), anti-acetyl histone H4 (Upstate Biotechnologies, Inc.), anti-acetyl Lys-5 histone H4 (Abcam), anti-acetyl Lys-8 histone H4 (Abcam), anti-acetyl Lys-12 histone H4 (Upstate Biotechnologies, Inc.), and anti-acetyl Lys-16 histone H4 (Upstate Biotechnologies, Inc.). Camptothecin and etoposide (Sigma) were used as apoptosis inducers. Apoptosis was analyzed using the Vybrant® apoptosis assay kit, 4-YO-PRO®-1/propidium iodide (Molecular Probes/Invitrogen). This kit is based on the use of the green fluorescent YO-PRO®-1 dye that specifically stains apoptotic cells that remain impermeant to propidium iodide (a dead cell stain). Live cells are not stained with YO-PRO®-1. Cells are then sorted by flow cytofluorometry. Induction of Apoptosis—Jurkat and HL60 cells were exposed to 100 μm etoposide or 2 μg/ml of camptothecin and incubated for 3–8 h (30Johnson C.A. Padget K. Austin C.A. Turner B.M. J. Biol. Chem. 2001; 276: 4539-4542Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Apoptosis was monitored by flow cytometry. Isolation of Histones—Histones were extracted from cell pellets by acid extraction on 0.25 m HCl followed by acetone precipitation (31Van Holde K.E. Chromatin. Springer-Verlag, New York1988Google Scholar). To investigate the release of histone to the cytosolic fraction during apoptosis, we performed hypotonic lysis with 20 mm pH 8.0 buffer containing 150 mm NaCl and 1% Triton X-100 as described in Wu et al. (20Wu D. Ingram A. Lahti J.H. Mazza B. Grenet J. Kapoor A. Liu L. Kidd V.J. Tang D. J. Biol. Chem. 2002; 277: 12001-12008Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Two fractions are obtained in this case: a cell lysate, containing the cytosolic fraction, and a nuclear pellet. In this case, the supernatant corresponding to the cytosolic fraction was precipitated using 20% trichloroacetic acid on ice for 30 min, centrifuged at 4 °C for 10 min, and washed once with acetone. Histones were then obtained by acid extraction as described above. Quantification of Global Histone Acetylation by High-performance Capillary Electrophoresis—The degree of histone acetylation was quantified by a modification of a previously described method (32Lindner H. Helliger W. Dirschlmayer A. Jaquemar M. Puschendorf B. Biochem. J. 1992; 283: 467-471Crossref PubMed Scopus (64) Google Scholar, 33Lund G. Andersson L. Lauria M. Lindholm M. Fraga M.F. Villar-Garea A. Ballestar E. Esteller M. Zaina S. J. Biol. Chem. 2004; 279: 29147-29154Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Individual histones were fractionated by reversed-phase high-performance liquid chromatography (HPLC) 4The abbreviations used are: HPLC, high performance liquid chromatography; HPCE, high-performance capillary electrophoresis; MS, mass spectrometry; mC, methylcytosine. on a Delta-Pak C18 column (Waters) eluted with an acetonitrile gradient (20–60%) in 0.3% trifluoroacetic acid (34Ballestar E. Abad C. Franco L. J. Biol. Chem. 1983; 271: 18817-18824Abstract Full Text Full Text PDF Scopus (100) Google Scholar) using a Beckman HPLC gradient system. Purity of histones was measured by PAGE. The non-, mono-, di-, tri-, and tetraacetylated histone derivatives of H3 fraction were resolved by high-performance capillary electrophoresis (HPCE). A non-coated fused silica capillary (Beckman-Coulter) (60.2 cm × 75.0 mm, effective length 50.0 cm) was used in a CE system (P/ACETM MDQ; Beckman-Coulter) connected to a data processing station (32 Karat™ Software). The running buffer was 110 mm phosphate buffer, pH 2.0, containing 0.03% (w/v) hydroxylpropylmethyl-cellulose. Running conditions consisted of a temperature of 25 °C and an operating voltage of 12 kV. On-column absorbance was monitored at 214 nm. Before each run, the capillary system was conditioned by washing with 0.1 m NaOH for 3 min and 0.5 m H2SO4 for 2 min and equilibrated with the running buffer for 3 min. Samples were injected under pressure (0.3 psi) for 3 s. Samples were obtained in triplicate, and all samples were analyzed in duplicate. Error bars in graphs represent standard deviation. Western Blotting—Histones were separated on 15% SDS-PAGE gel and blotted onto a polyvinylidene difluoride membrane of 22-μm pore size (Immobilon PSQ; Millipore). The membrane was blocked in 5% milk PBS-T (phosphate-buffered saline with 0.1% Tween-20) and immunoprobed with antibodies raised against different peptides containing different histone modifications as described above. The secondary antibodies used were goat anti-rabbit conjugated to horseradish peroxidase (1:3000) (Amersham Biosciences) and sheep anti-mouse horseradish peroxidase (1:3000). Bands obtained in Western blot were scanned and analyzed by Quantity One software (Gel Doc 2000; Bio-Rad). Experiments were performed in triplicate. Semiquantitative significance of the differences was estimated by direct comparison of the obtained values. Mass Spectrometry Analysis of Histones—Histone H4 global acetylation and acetylation at the specific lysine 16 site were analyzed by mass spectrometry. We separated acid-extracted histones by SDS-PAGE, excised the Coomassie-stained bands corresponding to histone H4, subjected them to acetylation with D6-acetic anhydride, and finally digested with trypsin as previously described (35Bonaldi T. Regula J.T. Imhof A. Methods Enzymol. 2003; 377: 111-130Crossref Scopus (43) Google Scholar). Supernatants were collected, vacuum-dried, and redissolved in 0.5 ml of 0.1% trifluoroacetic acid. Matrix-assisted laser desorption ionization time-of-flight MS analysis of the samples was carried out in a mass spectrometer Autoflex (Bruker Daltonics) in a positive ion reflector mode. Samples were added to a matrix consisting of 0.5 ml of 5 mg/ml of 2,5-dihydroxybenzoic acid in water:acetonitrile (2:1) with 0.1% trifluoroacetic acid. The ion acceleration voltage was 20 kV. Each spectrum was internally calibrated with the masses of two trypsin autolysis products. MS/MS analyses were performed in a linear LTQ ion trap mass spectrometer (Thermo Finnigan) equipped with a nano-electrospray ionization source by using coated GlassTip PicoTip emitters (New Objective). Samples were desalted and concentrated with Zip Tips (Millipore, Bedford, MA) following the manufacturer's protocol. The spectrometer was operated according to the manufacturer's instructions with manual adjustment of the collision energies. Fragment spectra were interpreted manually. Global 5-Methylcytosine Quantification—The 5-methylcytosine (mC) content was quantified by HPCE as previously described (36Fraga M.F. Esteller M. BioTechniques. 2002; 33: 632-649Crossref PubMed Scopus (342) Google Scholar). In brief, DNA samples were speed-back preconcentrated to 0.1 mg/ml and enzymatically hydrolyzed in a final volume of 5 ml. Samples were then directly injected into a Beckman MDQ high-performance capillary electrophoresis apparatus, and mC content was determined as the percentage of mC of total cytosine: mC peak area × 100/(C peak area + mC peak area). Error bars in graphs represent standard deviation. Data are representative of three independent experiments. Competitive Hybridization of Apoptotic DNA Fractions in Metaphase Chromosomes—To study the distribution along the chromosomes of the DNA isolated from nuclear pellet and cell lysate fractions, we modified the competitive genomic hybridization strategy (37Cigudosa J.C. Rao P.H. Calasanz M.J. Odero M.D. Michaeli J. Jhanwar S.C. Chaganti R.S. Blood. 1998; 91: 3007-3010Crossref PubMed Google Scholar). In this hybridization, we compared the DNA isolated from each of the fractions (either the cell lysate or the nuclear pellet) with the DNA isolated from intact cells. The DNA isolated from each of the fractions was labeled with Spectrum Red dUTP by CGH nick-translation kit (Vysis, Inc., Downer Grov, IL), and the total genomic DNA was labeled with Spectrum Green. The metaphases were captured using a fluorescence microscope (Olympus BX60) equipped with a CCD camera (Photometrics Sensys camera) and then analyzed using the chromofluor image analysis system (Cytovision; Applied Imaging Ltd, Newcastle, UK). 13–25 chromosomes were analyzed for each hybridization. Staining with Fluorescent DNA Probes and Fluorescence Microscopy— Control cells were fixed in 3.7% formaldehyde for 30 min at room temperature or methanol (-20 °C) for 1 min and permeabilized with phosphate-buffered saline-0.5% Triton X-100 for 10 min at room temperature as previously described (38Espada J. Ballestar E. Fraga M.F. Villar-Garea A. Juarranz A. Stockert J.C. Robertson K.D. Fuks F. Esteller M. J. Biol. Chem. 2004; 279: 37175-37184Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). DNA from the cell lysate fraction labeled with Spectrum Red was used to hybridize fixed cells. Confocal optical sections were obtained using a Leica TCS SP confocal microscope (Leica Microsystems, Heidelberg GMbH) equipped with krypton and argon lasers, and images were processed using Adobe Photoshop 5.0 (Adobe Systems Inc., Mountain View, CA). Global Reduction in Histone Acetylation Occurs during Apoptosis— As a first screening approach to investigating a potential connection between epigenetic alterations and nuclear changes in apoptosis, we analyzed global changes in the modification status of histones during apoptosis by Western blot. A change in the pattern of histone modifications could be associated with morphological changes that occur in the nucleus in apoptosis. We treated Jurkat cells with etoposide and acid extracted their histones at different times. For each sample, the level of apoptosis was monitored by flow cytometry (Fig. 1A, bottom). Antibodies against the global hyperacetylated forms of histones H3 and H4 and others against the monoacetylated form at specific residues of histone H3 (Lys-9) and histone H4 (Lys-5, -8, -12, and -16) were used. Antibodies against other modifications, such as phospho-Ser-10, dimethyl-Lys-4 and dimethyl-Lys-9 of histone H3, were also included. We first observed a significant decrease in global levels of histone H4 acetylation in apoptotic samples (Fig. 1A). However, when looking at specific lysine residues of H4 only Lys-8 and Lys-16 exhibited a loss of acetylation during apoptosis. No significant variations of acetylation were observed at Lys-5 and Lys-12. These results are consistent with previous reports that also suggest that the acetylation patterns of the pairs Lys-8 and Lys-16, on one hand, and Lys-5 and Lys-12, on the other, are coupled (39Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 38: 269-272Crossref Scopus (505) Google Scholar). In contrast to H4, no significant changes in acetylation (either global or site-specific) were observed for histone H3. Also, phosphorylation of Ser-10 and methylation of Lys-4 and Lys-9 of H3 appeared to remain stable (Fig. 1A). Because the only changes observed were associated with acetylation status, an alternative quantitative approach was used to estimate changes in histone acetylation of H3 and H4. The method requires the fractionation of histones by reversed phase HPLC followed by HPCE separation (35Bonaldi T. Regula J.T. Imhof A. Methods Enzymol. 2003; 377: 111-130Crossref Scopus (43) Google Scholar, 36Fraga M.F. Esteller M. BioTechniques. 2002; 33: 632-649Crossref PubMed Scopus (342) Google Scholar, 37Cigudosa J.C. Rao P.H. Calasanz M.J. Odero M.D. Michaeli J. Jhanwar S.C. Chaganti R.S. Blood. 1998; 91: 3007-3010Crossref PubMed Google Scholar). This allows the resolution and quantification of all acetylated forms of histones H3 and H4. In this analysis, for histone H4 each of the non-, mono-, di-, tri-, and tetraacetylated histone derivatives appeared as a doublet of peaks (Fig. 1B, top panel). The first and second peaks of the doublet had been previously identified as di- and trimethyl-Lys-20 histone H4, respectively (40Fraga M.F. Ballestar E. Villar-Garea A. Boix-Chornet M. Espada J. Schotta G. Bonaldi T. Haydon C. Petrie K. Ropero S. Perez-Rosado A. Calvo E. Lopez J.A. Cano A. Piris M.A. Ahn N. Imhof A. Caldas C. Jenuwein T. Esteller M. Nat. Genet. 2005; 37: 391-400Crossref PubMed Scopus (1495) Google Scholar). Trimethylation of Lys-20 in H4 is a marker of constitutive heterochromatin (9Schotta G. Lachner M. Sarma K. Ebert A. Sengupta R. Reuter G. Reinberg D. Jenuwein T. Genes. Dev. 2004; 18: 1251-1262Crossref PubMed Scopus (843) Google Scholar, 41Nishioka K. Rice J.C. Sarma K. Erdjument-Bromage H. Werner J. Wang Y. Chuikov S. Valenzuela P. Tempst P. Steward R. Lis J.T. Allis C.D. Reinberg D. Mol. Cell. 2002; 9: 1201-1213Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 42Kourmouli N. Jeppesen P. Mahadevhaiah S. Burgoyne P. Wu R. Gilbert D.M. Bongiorni S. Prantera G. Fanti L. Pimpinelli S. Shi W. Fundele R. Singh P.B. J. Cell Sci. 2004; 117: 2491-2501Crossref PubMed Scopus (212) Google Scholar) and aging (43Sarg B. Koutzamani E. Helliger W. Rundquist I. Lindner H.H. J. Biol. Chem. 2002; 277: 39195-39201Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). In Jurkat cells, relative losses of about 8 and 4% in the acetylated species of H4 were observed concomitantly with equivalent increases in non-acetylated forms for the etoposide (70% apoptosis after 8 h) and camptothecin (56% apoptosis after 8 h) treatments, respectively. The results obtained for HL60 cells also showed relative losses of 10 and 5% of acetylated H4 for the etoposide (78% apoptosis after 8 h) and camptothecin (60% after 8 h) treatments, respectively (Fig. 1C, bottom panel). In the case of histone H3, treatment of HL60 cells with etoposide and camptothecin resulted in respective relative losses of 5 and 3% of acetylated forms (Fig. 1C, bottom panel). These decreases were concomitant with an increase in the non-acetylated form of H3. Identical results were obtained with Jurkat cells (data not shown). These small decreases had not been observed when using Western blot to detect changes in the acetylation status of H3, although the HPCE analysis indicated that this is a very reproducible result. On the other hand, it is possible that the acetyl-H3 antibody does not detect all acetylated forms (mono-, di-, tri- and tetra-) that are quantified by HPCE. At any rate, the decrease in acetylation of H3 was significantly smaller than the variations observed for H4. Histone H4 Released from Nuclei Early in Apoptosis Is Hypoacetylated and Trimethylated—Having analyzed the global changes in histone H3 and H4 modifications, we next compared the specific modifications exhibited by histones that are massively released from the nucleus during apoptosis (23Rao L. Perez D. White E. J. Cell Biol. 1996; 135: 1441-1455Crossref PubMed Scopus (511) Google Scholar) with those that are retained. Thus, we induced apoptosis with etoposide and camptothecin and treated the cells with a lysis buffer containing 1% Triton X-100 (see "Experimental Procedures"). Under these conditions, in which cells are lysed but the integrity of nucleus is preserved (20Wu D. Ingram A. Lahti J.H. Mazza B. Grenet J. Kapoor A. Liu L. Kidd V.J. Tang D. J. Biol. Chem. 2002; 277: 12001-12008Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 44Shelton E.R. Wassarman P.M. DePamphilis M.L. J. Biol. Chem. 1980; 225: 771-782Abstract Full Text PDF Google Scholar), it is possible to separate histones released to the cytosol in apoptosis from those remaining in the chromatin. Therefore, each sample yielded two fractions that we call cell lysate, corresponding to the cytosolic fraction and nuclear pellet (Fig. 2). Histones were virtually absent from the cell lysate of uninduced control samples (Fig. 3A). In apoptotic samples, we observed a time-dependent increase of histones in the cell lysates, which were in fact released from the nucleus during apoptosis. The amount of histones reached about a quarter of that remaining in the pelleted fraction 8 h after inducing apoptosis (Fig. 3A). The analysis of H4 by HPCE showed that histones retained in the nuclear pellet did not exhibit significant differences between control and apoptotic samples (Fig. 3B), unlike the results obtained when total histone H4 had been analyzed where there were 5–10% decreases in acetylated histone H4 (Fig. 1B). However, a comparison of cell lysates with their corresponding nuclear pellets showed striking differences in the modification pattern of histone H4. Histone H4 isolated from cell lysates was found to be consistently hypoacetylated compared with its counterpart in apoptotic nuclear pellets, with an up to 15% relative decrease in the acetylated form of histone H4 during early apoptosis (Fig. 3B). Interestingly, differences in H4 acetylation between released and retained histone H4 became smaller at longer incubation times. Thus, shortly after apoptosis was induced, released histone H4 was enriched in hypoacetylated forms and the proportion of acetylated forms increased as apoptosis advanced, and the released histones more closely resembled those retained in the nuclear pellet. However, the most striking finding about histone H4 modification profiles was that released and retained H4 fractions exhibited different patterns of methylation. Most specifically, the characteristic doublets corresponding to different methylation forms of histone H4 were absent from both fractions, and whereas the cell lysate exhibited only the trimethylated form of histone H4, the nuclear pellet only contained the dimethylated form for short times after apoptosis induction (Fig. 3C). This is evident from the comparison of the electropherograms of these two samples, where each of the single peaks that corresponds to the di- and trimethylated forms of H4 had been fractionated during the isolation of the released histones (Fig. 3C). These data indicated that 3 h after apoptosis induction, almost 100% of histone H4 was trimethylated in the cell lysate (Fig. 3, D and E). This specific release of trimethylated H4 was progressively lost as apoptosis advanced. Indeed, after 6 and 8 h of incubation the relative trimethylation of released histone H4 dropped to 50 and 25%, respectively,