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• W2016500746 abstract "Posttranslational modification of histones is a common means of regulating chromatin structure and thus diverse nuclear processes. Using a hydrophilic interaction liquid chromatographic separation method in combination with mass spectrometric analysis, the present study investigated the alterations in histone H4 methylation/acetylation status and the interplay between H4 methylation and acetylation during in vitro differentiation of mouse erythroleukemia cells and how these modifications affect the chromatin structure. Independently of the type of inducer used (dimethyl sulfoxide, hexamethylenebisacetamide, butyrate, and trichostatin A), we observed a strong increase in non- and monoacetylated H4 lysine 20 (H4-Lys20) trimethylation. An increase in H4-Lys20 trimethylation, however, to a clearly lesser extent, was also found when cells accumulated in the stationary phase. Since we show that trimethylated H4-Lys20 is localized to heterochromatin, the increase in H4-Lys20 trimethylation observed indicates an accumulation of chromatin-dense and transcriptionally silent regions during differentiation and during the accumulation of control cells in the stationary phase, respectively. When using the deacetylase inhibitors butyrate or trichostatin A, we found that H4 hyperacetylation prevents H4-Lys20 trimethylation, but not mono- or dimethylation, and that the nonacetylated unmethylated H4-Lys20 is therefore the most suitable substrate for H4-Lys20 trimethylase. Summarizing, histone H4-Lys20 hypotrimethylation correlates with H4 hyperacetylation and H4-Lys20 hypertrimethylation correlates with H4 hypoacetylation. The results provide a model for how transcriptionally active euchromatin might be converted to the compacted, transcriptionally silent heterochromatin. Posttranslational modification of histones is a common means of regulating chromatin structure and thus diverse nuclear processes. Using a hydrophilic interaction liquid chromatographic separation method in combination with mass spectrometric analysis, the present study investigated the alterations in histone H4 methylation/acetylation status and the interplay between H4 methylation and acetylation during in vitro differentiation of mouse erythroleukemia cells and how these modifications affect the chromatin structure. Independently of the type of inducer used (dimethyl sulfoxide, hexamethylenebisacetamide, butyrate, and trichostatin A), we observed a strong increase in non- and monoacetylated H4 lysine 20 (H4-Lys20) trimethylation. An increase in H4-Lys20 trimethylation, however, to a clearly lesser extent, was also found when cells accumulated in the stationary phase. Since we show that trimethylated H4-Lys20 is localized to heterochromatin, the increase in H4-Lys20 trimethylation observed indicates an accumulation of chromatin-dense and transcriptionally silent regions during differentiation and during the accumulation of control cells in the stationary phase, respectively. When using the deacetylase inhibitors butyrate or trichostatin A, we found that H4 hyperacetylation prevents H4-Lys20 trimethylation, but not mono- or dimethylation, and that the nonacetylated unmethylated H4-Lys20 is therefore the most suitable substrate for H4-Lys20 trimethylase. Summarizing, histone H4-Lys20 hypotrimethylation correlates with H4 hyperacetylation and H4-Lys20 hypertrimethylation correlates with H4 hypoacetylation. The results provide a model for how transcriptionally active euchromatin might be converted to the compacted, transcriptionally silent heterochromatin. In eukaryotes, histone proteins associate with DNA to form nucleosomes that are folded into higher order chromatin structures. Differences in higher order chromatin structures, which are important prerequisites for numerous biological processes including cellular proliferation, differentiation, development, gene expression, genome stability, and cancer, are thought to be realized by a variety of posttranslational modifications of histone N termini, particularly of histones H3 and H4. Besides acetylation, histones are subjected to phosphorylation, methylation, ubiquitination, ADP-ribosylation, and deamidation (1van Holde K.E. Rich A. Chromatin. Springer, New York1988: 111-148Google Scholar, 2Lindner H. Sarg B. Hoertnagl B. Helliger W. J. Biol. Chem. 1998; 273: 13324-13330Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Distinct combinations of covalent histone modifications including lysine acetylation, lysine and arginine methylation, and serine phosphorylation form the basis of the histone code hypothesis (3Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6530) Google Scholar, 4Turner B.M. BioEssays. 2000; 22: 836-845Crossref PubMed Scopus (959) Google Scholar, 5Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7570) Google Scholar). This hypothesis proposes that a pre-existing modification affects subsequent modifications on histone tails and that these modifications generate unique surfaces for the binding of various proteins or protein complexes responsible for higher order chromatin organization and gene activation and inactivation. Some of the histone-modifying enzymes (e.g. lysine methyltransferases) are, when deregulated, considered to be involved in carcinogenesis (6Schneider R. Bannister A.J. Kouzarides T. Trends Biochem. Sci. 2002; 27: 396-402Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Histone H4 is typically acetylated at lysines 5, 8, 12, and 16, methylated at arginine 3 and lysine 20, and phosphorylated at serine 1 (3Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6530) Google Scholar, 7Wang H. Huang Z.Q. Xia L. Feng Q. Erdjument-Bromage H. Strahl B.D. Briggs S.D. Allis C.D. Wong J. Tempst P. Zhang Y. Science. 2001; 293: 853-857Crossref PubMed Scopus (617) Google Scholar, 8Strahl B.D. Briggs S.D. Brame C.J. Caldwell J.A. Koh S.S. Ma H. Cook R.G. Shabanowitz J. Hunt D.F. Stallcup M.R. Allis C.D. Curr. Biol. 2001; 11: 996-1000Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). Unlike the dynamic process of histone acetylation and phosphorylation, histone methylation is regarded as a relatively static long-term signal with a low turnover of the methyl group. Whereas arginine can be either mono- or dimethylated (the latter in symmetric or asymmetric form), lysine methylation can occur as a mono-, di-, or trimethylated derivative. In contrast to histone H3 methylation, H4-Lys20 was long considered to be maximally dimethylated in mammals (9Annunziato A.T. Eason M.B. Perry C.A. Biochemistry. 1995; 34: 2916-2924Crossref PubMed Scopus (66) Google Scholar). Most recently, however, Sarg et al. (10Sarg B. Koutzamani E. Helliger W. Rundquist I. Lindner H.H. J. Biol. Chem. 2002; 277: 39195-39201Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) conducted a mass spectrometric analysis with a newly developed hydrophilic interaction chromatographic method enabling the simultaneous separation of methylated and acetylated forms and, for the first time, found in vivo evidence that H4-Lys20 is also trimethylated in mammalian tissue. Moreover, in rat liver and kidney the proportion of trimethylated histone H4 increases during aging. In Raji and K562 cells the trimethylated form was also detected, primarily when the cells were accumulated in the stationary phase (10Sarg B. Koutzamani E. Helliger W. Rundquist I. Lindner H.H. J. Biol. Chem. 2002; 277: 39195-39201Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Most recently, two novel SET domain histone methyltransferases, Suv4-20h1 and Suv4-20h2, acting as nucleosomal H4-Lys20 trimethylating enzymes, have been identified (11Schotta G. Lachner M. Sarma K. Ebert A. Sengupta R. Reuter G. Reinberg D. Jenuwein T. Genes Dev. 2004; 18: 1251-1262Crossref PubMed Scopus (842) Google Scholar). It was also shown that histone H3-Lys9 trimethylation is required for the induction of H4-Lys20 trimethylation (H4-tri-meLys20) and that trimethylation of histone H3-Lys9 and histone H4-Lys20 functions as a repressive mark in gene-silencing mechanisms (11Schotta G. Lachner M. Sarma K. Ebert A. Sengupta R. Reuter G. Reinberg D. Jenuwein T. Genes Dev. 2004; 18: 1251-1262Crossref PubMed Scopus (842) Google Scholar, 12Kourmouli 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 (211) Google Scholar). Studies investigating possible links between histone methylation and acetylation have revealed that methylation of Arg3 of H4 by the histone H4-specific protein methyltransferase PRMT 1 facilitates acetylation by p300 on histone H4-Lys8 and -Lys12 and that this methylation at position 3 plays an important role in transcriptional regulation (7Wang H. Huang Z.Q. Xia L. Feng Q. Erdjument-Bromage H. Strahl B.D. Briggs S.D. Allis C.D. Wong J. Tempst P. Zhang Y. Science. 2001; 293: 853-857Crossref PubMed Scopus (617) Google Scholar, 8Strahl B.D. Briggs S.D. Brame C.J. Caldwell J.A. Koh S.S. Ma H. Cook R.G. Shabanowitz J. Hunt D.F. Stallcup M.R. Allis C.D. Curr. Biol. 2001; 11: 996-1000Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). Acetylation on any of the four H4 lysines on the N-terminal tail (Lys5, Lys8, Lys12, Lys16), however, inhibits the methylation of Arg3 by PRMT 1 (7Wang H. Huang Z.Q. Xia L. Feng Q. Erdjument-Bromage H. Strahl B.D. Briggs S.D. Allis C.D. Wong J. Tempst P. Zhang Y. Science. 2001; 293: 853-857Crossref PubMed Scopus (617) Google Scholar, 8Strahl B.D. Briggs S.D. Brame C.J. Caldwell J.A. Koh S.S. Ma H. Cook R.G. Shabanowitz J. Hunt D.F. Stallcup M.R. Allis C.D. Curr. Biol. 2001; 11: 996-1000Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). These results demonstrating the interplay between histone H4 methylation and acetylation support the histone code hypothesis. Recent findings suggest that histone H4-Lys20 methylation inhibits H4-Lys16 acetylation and vice versa, leading to the hypothesis that methylation of H4-Lys20 maintains silent chromatin, in part by precluding neighboring acetylation on the H4 tail (13Nishioka 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, 14Rice J.C. Nishioka K. Sarma K. Steward R. Reinberg D. Allis C.D. Genes Dev. 2002; 16: 2225-2230Crossref PubMed Scopus (201) Google Scholar). These studies, however, did not discriminate between the individual methylation states (3Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6530) Google Scholar, 4Turner B.M. BioEssays. 2000; 22: 836-845Crossref PubMed Scopus (959) Google Scholar, 5Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7570) Google Scholar). Of particular interest are recent findings regarding the role of various states of histone H3/H4 methylation in the regulation of transcription. In contrast to the dimethylated state of histone H3-Lys4, which occurs at both inactive and active euchromatic genes, H3-Lys4 trimethylation is present exclusively at active genes (15Santos-Rosa H. Schneider R. Bannister A.J. Sherriff J. Bernstein B.E. Emre N.C. Schreiber S.L. Mellor J. Kouzarides T. Nature. 2002; 419: 407-411Crossref PubMed Scopus (1587) Google Scholar), whereas H3-Lys9 trimethylation and also H4-Lys20 trimethylation are present in repressive chromatin domains (11Schotta G. Lachner M. Sarma K. Ebert A. Sengupta R. Reuter G. Reinberg D. Jenuwein T. Genes Dev. 2004; 18: 1251-1262Crossref PubMed Scopus (842) Google Scholar, 15Santos-Rosa H. Schneider R. Bannister A.J. Sherriff J. Bernstein B.E. Emre N.C. Schreiber S.L. Mellor J. Kouzarides T. Nature. 2002; 419: 407-411Crossref PubMed Scopus (1587) Google Scholar). It is obvious, therefore, that not only methylation in itself but the precise methylation state and the position of the lysine determine gene activity/repression. In the present study we aimed to investigate the changes in Lys20 methylation states of non-, mono-, di-, tri-, and tetraacetylated H4 histones as well as the interplay between the various H4-Lys20 methylation states and acetylation during in vitro differentiation of mouse erythroleukemia cells induced by Me2SO 1The abbreviations used are: Me2SO, dimethyl sulfoxide; DAPI, 4-6-diamidino-2-phenylindole; ESI-MS, electrospray ionization mass spectrometry; HDAC, histone deacetylase; HILIC, hydrophilic interaction liquid chromatography; HMBA, hexamethylenebisacetamide; HP1, heterochromatin protein-1; MEL, mouse erythroleukemia; RP-HPLC, reversed-phase high performance liquid chromatography; TEA, triethylamine; TSA, trichostatin A.1The abbreviations used are: Me2SO, dimethyl sulfoxide; DAPI, 4-6-diamidino-2-phenylindole; ESI-MS, electrospray ionization mass spectrometry; HDAC, histone deacetylase; HILIC, hydrophilic interaction liquid chromatography; HMBA, hexamethylenebisacetamide; HP1, heterochromatin protein-1; MEL, mouse erythroleukemia; RP-HPLC, reversed-phase high performance liquid chromatography; TEA, triethylamine; TSA, trichostatin A./HMBA and the deacetylase inhibitors sodium butyrate/TSA, respectively. For the analysis we used a hydrophilic interaction liquid chromatographic method recently developed in our laboratory (2Lindner H. Sarg B. Hoertnagl B. Helliger W. J. Biol. Chem. 1998; 273: 13324-13330Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 16Sarg B. Helliger W. Hoertnagl B. Puschendorf B. Lindner H. Arch. Biochem. Biophys. 1999; 372: 333-339Crossref PubMed Scopus (25) Google Scholar, 17Lindner H. Sarg B. Meraner C. Helliger W. J. Chromatogr. A. 1996; 743: 137-144Crossref PubMed Scopus (99) Google Scholar, 18Lindner H. Sarg B. Helliger W. J. Chromatogr. A. 1997; 782: 55-62Crossref PubMed Scopus (87) Google Scholar, 19Lindner H. Helliger W. Methods Mol. Biol. 2004; 251: 75-88PubMed Google Scholar) in combination with a mass spectrometric analysis enabling the simultaneous separation of non-, mono-, di-, and trimethylated Lys20 of non-, mono-, di-, tri-, and tetraacetylated H4 histones. In all cases and regardless of the nature of the inducer we found a strong increase in the trimethylated Lys20 of non- and monoacetylated histone H4 (ac0 H4-tri-meLys20 and ac1 H4-tri-meLys20, respectively) during differentiation. By using an antibody that specifically recognizes trimethylated Lys20 of histone H4, it was possible to demonstrate that H4-tri-meLys20 localizes to heterochromatin. The observed increase in non- and monoacetylated H4-tri-meLys20 thus results in an increase in heterochromatic regions, thereby indicating chromatin compaction and repression of gene activity in the course of erythroid differentiation. Treatment of mouse erythroleukemia cells with sodium butyrate or TSA resulted in H4 histone hyperacetylation. With regard to the individual acetylated forms in detail, we found diacetylated H4-Lys20 to be trimethylated in traces, whereas tri- and tetraacetylated H4-Lys20 were not trimethylated at all. We therefore suggest that in contrast to non- and monoacetylated H4 histones, which correlate with H4-tri-meLys20, histone H4 hyperacetylation precludes Lys20 trimethylation, thus providing further support for the histone code hypothesis. Independently of the degree of acetylation, however, we found histone H4-di-meLys20 and, to a far lesser extent, histone H4-mono-meLys20. Materials—Sodium perchlorate (NaClO4), triethylamine (TEA), acetonitrile, and trifluoroacetic acid were purchased from Fluka (Buchs, Switzerland). All other chemicals were purchased from Merck (Darmstadt, Germany) if not otherwise indicated. Cell Culture—Mouse erythroleukemia (MEL) cells (line F4N) were grown in Dulbecco's minimum Eagle's medium (Biochrom, Berlin, Germany) containing 2× nonessential amino acids, 1× penicillin-streptomycin, and 10% fetal calf serum. Cells were cultured at initial cell densities of 5.104-7.104/ml at 37 °C and 5% CO2. Differentiation was induced by the addition of 2% Me2SO (for 144 h), 5 mm HMBA (144 h), 4 ng/ml TSA (144 h), or 1.75 mm sodium butyrate (72 h). The untreated control cells were harvested after 72 h in the log phase or after 144 h in stationary phase. The percentage of benzidine-positive cells was determined as described by Orkin et al. (20Orkin S.H. Harosi F.I. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 98-102Crossref PubMed Scopus (442) Google Scholar). Preparations of Histones—The histones were extracted from MEL cells with sulfuric acid (0.2 m) according to the procedure of Helliger et al. (21Helliger W. Lindner H. Grubl-Knosp O. Puschendorf B. Biochem. J. 1992; 288: 747-751Crossref PubMed Scopus (32) Google Scholar). High Performance Liquid Chromatography—The equipment used consisted of a 127 Solvent Module and a Model 166 UV-visible region detector (Beckman Instruments). The effluent was monitored at 210 nm, and the peaks were recorded using Beckman System Gold software. Solvent compositions are expressed as v/v throughout this text. Reversed-phase HPLC—The separation of core histones was performed on a Nucleosil C4 column (250 × 8-mm inner diameter, 5-μm particle pore size, 30-nm pore size, end-capped; Seibersdorf). Samples (∼500 μg) were injected onto the column. The histone sample was chromatographed as described previously (10Sarg B. Koutzamani E. Helliger W. Rundquist I. Lindner H.H. J. Biol. Chem. 2002; 277: 39195-39201Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 22Lindner H. Helliger W. Puschendorf B. J. Chromatogr. 1988; 450: 309-316Crossref PubMed Scopus (19) Google Scholar). Hydrophilic Interaction Liquid Chromatography—The histone fraction H4 (∼100 μg) isolated by RP-HPLC was further separated on a SynChropak CM300 column (250 × 4.6-mm inner diameter, 6.5-μm particle size, 30-nm pore size; Agilent Technologies, Vienna, Austria) at 30 °C and at a constant flow of 1.0 ml/min using a multi-step gradient starting at solvent A-solvent B (100:0) (solvent A: 70% acetonitrile, 0.015 m TEA/H3PO4, pH = 3.0; solvent B: 65% acetonitrile, 0.015 m TEA/H3PO4, pH = 3.0, and 0.68 m NaClO4). The concentration of solvent B was increased from 0 to 10% B in 2 min and from 10 to 40% in 30 min and was then maintained at 40% for 10 min. The isolated protein fractions were desalted using RP-HPLC. Histone fractions obtained in this way were collected and, after the addition of 20 μl of 2-mercaptoethanol (0.2 m), were lyophilized and stored at -20 °C. Endoproteinase Glu-C Digestion—Histone H4 (∼ 10 μg) obtained by HILIC fractionation was digested with Staphylococcus aureus V8 protease (Roche Applied Science; 1:20 w/w) in 50 μl of 25 mm NH4HCO3 buffer (pH 4.0) for1hat room temperature. The digest was subjected to RP-HPLC as described previously (10Sarg B. Koutzamani E. Helliger W. Rundquist I. Lindner H.H. J. Biol. Chem. 2002; 277: 39195-39201Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Endoproteinase Lys-C Digestion—N-terminal peptides obtained by Glu-C digestion were further cleaved with endoproteinase Lys-C (Roche Applied Science; 1:5 w/w) in 15 μl of 25 mm Tris-HCl buffer (pH 8.7) for 2 h at 37 °C. The digest was subjected to RP-HPLC-ESI-MS. Mass Spectrometric Analysis—Histone H4 peptide fractions obtained by endoproteinase Lys-C cleavage were injected onto a PepMap C18 column (150 × 1-mm inner diameter, 3-μm particle size; ICT, Vienna, Austria). The column eluate was coupled directly to a Finnigan LCQ ion trap instrument (San Jose, CA) equipped with an electrospray source (RP-HPLC-ESI-MS). Samples of ∼1 μg were chromatographed within 55 min at a constant flow of 35 μl/min with a two-step acetonitrile gradient starting at solvent A - solvent B (90:10) (solvent A: water containing 0.1% trifluoroacetic acid; solvent B: 85% acetonitrile and 0.093% trifluoroacetic acid). The concentration of solvent B was increased linearly from 10 to 40% over a 45-min period and from 40 to 100% over 20 min. Antibodies—The antibodies used were: rabbit polyclonal to H4-tri-meLys20 (Abcam), Western blotting 1:1000, immunofluorescence 1:200; rat monoclonal to HP1β (Abcam), immunofluorescence 1:50; mouse monoclonal to RNA polymerase II (Abcam), immunofluorescence 1:50. For Western blotting, goat anti-rabbit IgG peroxidase conjugate (Sigma) 1:5000 was used as secondary antibody. The secondary antibodies for immunofluorescence were: goat anti-rabbit IgG fluorescein isothiocyanate conjugate (Sigma), 1:160; goat anti-rat IgG Cy3 conjugate (Jackson ImmunoResearch), 1:160; goat anti-mouse IgG Cy3 conjugate (Jackson ImmunoResearch), 1:160. Western Blotting—Histones were resolved in SDS-loading buffer, fractionated by SDS-PAGE, and transferred to a nitrocellulose membrane (Hybond ECL, Amersham Biosciences). The membrane was probed with antibodies using standard techniques and detected by enhanced chemiluminescence (ECL, Amersham Biosciences). Immunofluorescence—MEL cells were grown in suspension culture, collected by centrifugation, and washed with phosphate-buffered saline. 105 cells were cytospun onto slides. Cells were permeabilized using Triton X-100 (1% for 2 min and 0.1% for 10 min) in KCM buffer (120 mm KCl, 20 mm NaCl, 0.5 mm EDTA, 10 mm Tris/HCl, pH 7.5) and incubated sequentially with the primary and secondary antibodies. Cells were fixed for 5 min with 4% paraformaldehyde in KCM, stained with DAPI for 1 min, and mounted in mounting medium (Dako). Staining was visualized using an ×100 objective on a Zeiss Axioplan 2 microscope and a SPOT camera (Diagnostic Instruments). Images were captured using MetaVue software (Universal Imaging Corp.) and analyzed with Corel Photo-Paint 10. HILIC Separation of Modified Forms of Histone H4 of Uninduced MEL Cells and Cells Cultured in the Presence of Me2SO, HMBA, Sodium Butyrate, and TSA—Untreated MEL cells and cells treated with Me2SO, HMBA, sodium butyrate, and TSA were grown in culture. After 72 and 144 h and before harvesting the cells, the number of benzidine-positive cells indicating hemoglobin accumulation was determined. In the case of butyrate-treated cells, however, the study was limited to the first 72 h, because butyrate is metabolized by the cells, and after a certain time its concentration is reduced to a level below that required to stimulate differentiation (23Friend C. Zajac-Kaye M. Holland J.G. Pogo B.G. Cancer Res. 1987; 47: 378-382PubMed Google Scholar). Whereas throughout the entire period study untreated cultures were found to contain a very low level (less than 2%) of spontaneously differentiated cells, cultures containing Me2SO, HMBA, or butyrate produced ∼80–95% benzidine-positive cells and those containing TSA nearly 70%. The sulfuric acid-extracted core histones were fractionated using an RP-HPLC procedure described previously (10Sarg B. Koutzamani E. Helliger W. Rundquist I. Lindner H.H. J. Biol. Chem. 2002; 277: 39195-39201Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 22Lindner H. Helliger W. Puschendorf B. J. Chromatogr. 1988; 450: 309-316Crossref PubMed Scopus (19) Google Scholar), and the histone H4 fractions obtained were subjected to HILIC, a chromatographic technique developed in our laboratory for separating modified core and H1 histones (2Lindner H. Sarg B. Hoertnagl B. Helliger W. J. Biol. Chem. 1998; 273: 13324-13330Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 16Sarg B. Helliger W. Hoertnagl B. Puschendorf B. Lindner H. Arch. Biochem. Biophys. 1999; 372: 333-339Crossref PubMed Scopus (25) Google Scholar, 17Lindner H. Sarg B. Meraner C. Helliger W. J. Chromatogr. A. 1996; 743: 137-144Crossref PubMed Scopus (99) Google Scholar, 18Lindner H. Sarg B. Helliger W. J. Chromatogr. A. 1997; 782: 55-62Crossref PubMed Scopus (87) Google Scholar, 19Lindner H. Helliger W. Methods Mol. Biol. 2004; 251: 75-88PubMed Google Scholar). A number of major and minor peaks could be received, which were identified by ESI-MS as described previously (10Sarg B. Koutzamani E. Helliger W. Rundquist I. Lindner H.H. J. Biol. Chem. 2002; 277: 39195-39201Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Fig. 1 shows the pattern of histone H4 acetylation and methylation of untreated (control) MEL cells at 72 h (Fig. 1A) and 144 h (Fig. 1B) after seeding, respectively, and of cells grown for 72 h in the presence of butyrate (Fig. 1C) and 144 h in the presence of Me2SO (Fig. D). The histone H4 acetylation/methylation pattern of MEL cells grown for 144 h in the presence of TSA and HMBA resembled that obtained by treating with butyrate and Me2SO, respectively (data not shown). In control cells and cells treated with Me2SO and HMBA, histone H4 was hypoacetylated, containing primarily non acetylated (ac0) and monoacetylated (ac1) forms with Lys16 as the most frequently acetylated lysine residue. In response to Me2SO treatment, a clear increase in trimethylated unacetylated and trimethylated monoacetylated H4 species was observed (Fig. 1, D versus B). Treatment of cells with butyrate and TSA, known HDAC inhibitors, caused hyperacetylation of H4, in which up to four acetyl groups are bound to the N-terminal tail. The results revealed that H4-Lys20 is methylated in all of the acetylated isoforms, as well as in the nonacetylated isoform, apart from a nonmethylated H4 present in varying amounts. In general, the dimethylated form was found to be the main methylation product followed, in smaller amounts, by the mono- and trimethylated forms. To facilitate an unambiguous assignment, the double peak of triacetylated H4 histones (designated in Fig. 1C with an asterisk) had to be investigated in more detail. Characterization of Hyperacetylated H4 Fractions from Butyrate-treated Cells Obtained by HILIC—To precisely determine the modification status of hyperacetylated histone H4 shown in Fig. 1C, the HILIC fractions were isolated, desalted by RP-HPLC, and treated with endoproteinase Glu-C as described previously (10Sarg B. Koutzamani E. Helliger W. Rundquist I. Lindner H.H. J. Biol. Chem. 2002; 277: 39195-39201Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The fragmentation yielded an N-terminal peptide (residues 1–52 and 1–53) that was further cleaved with endoproteinase Lys-C. Endoproteinase Lys-C is a serine protease, and its activity is inhibited at lysine residues that have been modified by an acetyl or methyl group (10Sarg B. Koutzamani E. Helliger W. Rundquist I. Lindner H.H. J. Biol. Chem. 2002; 277: 39195-39201Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The digests were analyzed by RP-HPLC-ESI-MS using a 150 × 1.0-mm inner diameter microbore column. The diacetylated (ac2) form was found to be acetylated at Lys16 and Lys12, and as can be seen from the separation pattern in Fig. 1C, was mainly dimethylated followed by the monomethylated derivative and, present in trace amounts only, the trimethylated one. In the case of the triacetylated H4 protein (marked with an asterisk), which is separated into two HILIC subfractions, several peptide fragments were detected (Fig. 2). ESI-MS analysis revealed a triacetylated H4-di-meLys20 form with acetyl groups at either Lys8-Lys12-Lys16 or Lys5-Lys12-Lys16 and, in very small amounts, at Lys5-Lys8-Lys16. This finding that the various lysines are partially occupied in the triacetylated form agrees well with observations made by other investigators (24Turner B.M. O'Neill L.P. Allan I.M. FEBS Lett. 1989; 253: 141-145Crossref PubMed Scopus (102) Google Scholar, 25Clarke D.J. O'Neill L.P. Turner B.M. Biochem. J. 1993; 294: 557-561Crossref PubMed Scopus (57) Google Scholar). Interestingly, a peptide corresponding to the tetraacetylated (Lys5-Lys8-Lys12-Lys16) nonmethylated form, which could not be separated by the HILIC system, was found in the ac3 double peak. A triacetylated trimethylated H4 histone, however, was undetectable. Concerning the tetraacetylated H4 protein, two HILIC subfractions containing one or two methyl groups were identified. Similar to triacetylated H4, a tetraacetylated trimethylated H4 was also not seen. At any rate, however, the H4-di-meLys20 form predominates in the hyperacetylated histone H4 fractions. The Levels of H4-tri-meLys20 Are Raised in Hypoacetylated H4 Fractions during MEL Cell Differentiation—We recently showed for the first time that histone H4 from mammalian tissue is not only mono- and dimethylated but also trimethylated at Lys20 and that the trimethylated form accumulates with age and in cells in the stationary phase (10Sarg B. Koutzamani E. Helliger W. Rundquist I. Lindner H.H. J. Biol. Chem. 2002; 277: 39195-39201Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Novel studies on methylated histones have revealed that H4-tri-meLys20 is a repressive mark in gene silencing and is important for heterochromatin assembly (11Schotta G. Lachner M. Sarma K. Ebert A. Sengupta R. Reuter G. Reinberg D. Jenuwein T. Genes Dev. 2004; 18: 1251-1262Crossref PubMed Scopus (842) Google Scholar, 12Kourmouli 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 (211) Google Scholar). We were thus interested in investigating the occurrence of trimethylated H4 in differentiated cells. To obtain quantitative data, we conducted a HILIC analysis of histone H4 from MEL cells treated with various inducers and quantified the results. As depicted in Fig. 3A, an increase of about 70% in trimethylated H4 (ac0 + ac1) forms appears in non-growing cells. Upon induction a remarkable further increase to the 4–5-fold level occurs in differentiated cel" @default.
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• W2016500746 title "Histone H4 Hyperacetylation Precludes Histone H4 Lysine 20 Trimethylation" @default.
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