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• W1991332036 abstract "Methylation and acetylation of position-specific lysine residues in the N-terminal tail of histones H3 and H4 play an important role in regulating chromatin structure and function. In the case of H3-Lys4, H3-Lys9, H3-Lys27, and H4-Lys20, the degree of methyla-tion was variable from the mono- to the di- or trimethylated state, each of which was presumed to be involved in the organization of chromatin and the activation or repression of genes. Here we inves-tigated the interplay between histone H4-Lys20 mono- and trim-ethylation and H4 acetylation at induced (β-major/β-minor glo-bin), repressed (c-myc), and silent (embryonic β-globin) genes during in vitro differentiation of mouse erythroleukemia cells. By using chromatin immunoprecipitation, we found that the β-majorand β-minor promoter and the β-globin coding regions as well as the promoter and the transcribed exon 2 regions of the highly expressed c-myc gene were hyperacetylated and monomethylated at H4-Lys20. Although activation of the β-globin gene resulted in an increase in hyperacetylated, monomethylated H4, down-regulation of the c-myc gene did not cause a decrease in hyperacetylated, monomethylated H4-Lys20, thus showing a stable pattern of histone modifications. Immunofluorescence microscopy studies revealed that monomethylated H4-Lys20 mainly overlaps with RNA pol II-stained euchromatic regions, thus indicating an association with transcriptionally engaged chromatin. Our chromatin immunopre-cipitation results demonstrated that in contrast to trimethylated H4-Lys20, which was found to inversely correlate with H4 hyper-acetylation, H4-Lys20 monomethylation is compatible with histone H4 hyperacetylation and correlates with the transcriptionally active or competent chromatin state. Methylation and acetylation of position-specific lysine residues in the N-terminal tail of histones H3 and H4 play an important role in regulating chromatin structure and function. In the case of H3-Lys4, H3-Lys9, H3-Lys27, and H4-Lys20, the degree of methyla-tion was variable from the mono- to the di- or trimethylated state, each of which was presumed to be involved in the organization of chromatin and the activation or repression of genes. Here we inves-tigated the interplay between histone H4-Lys20 mono- and trim-ethylation and H4 acetylation at induced (β-major/β-minor glo-bin), repressed (c-myc), and silent (embryonic β-globin) genes during in vitro differentiation of mouse erythroleukemia cells. By using chromatin immunoprecipitation, we found that the β-majorand β-minor promoter and the β-globin coding regions as well as the promoter and the transcribed exon 2 regions of the highly expressed c-myc gene were hyperacetylated and monomethylated at H4-Lys20. Although activation of the β-globin gene resulted in an increase in hyperacetylated, monomethylated H4, down-regulation of the c-myc gene did not cause a decrease in hyperacetylated, monomethylated H4-Lys20, thus showing a stable pattern of histone modifications. Immunofluorescence microscopy studies revealed that monomethylated H4-Lys20 mainly overlaps with RNA pol II-stained euchromatic regions, thus indicating an association with transcriptionally engaged chromatin. Our chromatin immunopre-cipitation results demonstrated that in contrast to trimethylated H4-Lys20, which was found to inversely correlate with H4 hyper-acetylation, H4-Lys20 monomethylation is compatible with histone H4 hyperacetylation and correlates with the transcriptionally active or competent chromatin state. It has been proposed that distinct post-translational histone modifications act sequentially or in combination to form a “histone code” within chromatin (1.Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6679) Google Scholar). Acetylation and methylation of specific histone lysine residues can serve as a mark of either euchromatin or silent heterochromatin. Although methylation of H3-Lys4, H3-Lys36, and H3-Lys79 has been linked to transcriptional activation and protection of euchromatin, H3-Lys9, H3-Lys27, and H4-Lys20 methylation is generally thought to be associated with gene repression and heterochromatin formation (2.Sims R.J. Nishioka K. Reinberg D. Trends Genet. 2003; 19: 629-639Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar, 3.Fischle W. Wang Y. Allis C.D. Nature. 2003; 425: 475-479Crossref PubMed Scopus (551) Google Scholar, 4.Lachner M. O'Sullivan R.J. Jenuwein T. J. Cell Sci. 2003; 116: 2117-2124Crossref PubMed Scopus (538) Google Scholar). In this regard it must be noted that histone lysine residues can be mono-, di-, or trimethylated (5.Schotta G. Lachner M. Sarma K. Ebert A. Sengupta R. Reuter G. Reinberg D. Jenuwein T. Genes Dev. 2004; 18: 1251-1262Crossref PubMed Scopus (861) Google Scholar), thus extending the coding potential of a methylatable lysine position. Previous studies, however, focused only on detection of H3 (for review see Refs. 3.Fischle W. Wang Y. Allis C.D. Nature. 2003; 425: 475-479Crossref PubMed Scopus (551) Google Scholar and 4.Lachner M. O'Sullivan R.J. Jenuwein T. J. Cell Sci. 2003; 116: 2117-2124Crossref PubMed Scopus (538) Google Scholar) or H4 (6.Fang J. Feng Q. Ketel C.S. Wang H. Cao R. Xia L. Erdjument-Bromage H. Tempst P. Simon J.A. Zhang Y. Curr. Biol. 2002; 12: 1086-1099Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 7.Rice J.C. Nishioka K. Sarma K. Steward R. Reinberg D. Allis C.D. Genes Dev. 2002; 16: 2225-2230Crossref PubMed Scopus (202) Google Scholar, 8.Nishioka 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 (467) Google Scholar) lysine methylation regardless of the methylation status. Recently, it was shown that a distinction between di- and trimethylation of various lysines of histone H3 is important for processes of transcriptional regulation or gene silencing (9.Santos-Rosa H. Schneider R. Bannister A.J. Sherrif J. Bernstein B.E. Emre N.C.T. Schreiber S.L. Mellor J. Kouzarides T. Nature. 2002; 419: 407-411Crossref PubMed Scopus (1608) Google Scholar, 10.Tamaru H. Zhang X. McMillen D. Singh P.B. Nakayama J.-I. Grewal S.I. Allis C.D. Chenh X. Selker E.U. Nat. Genet. 2003; 34: 75-79Crossref PubMed Scopus (311) Google Scholar, 11.Czermin B. Melfi R. McCabe D. Seitz V. Imhof A. Pirrotta V. Cell. 2002; 111: 185-196Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar). Moreover, studies that focused on the in vivo distribution of mono-, di-, and trimethylated H3-Lys9 and H3-Lys27 demonstrate that mono- and dimethylated H3-Lys9 and H3-Lys27 are specifically localized to silent domains within euchromatin, whereas trimethylated H3-Lys9 and monomethylated H3-Lys27 were enriched at pericentric heterochromatin (12.Rice J.C. Briggs S.D. Ueberheide B. Barber C.M. Shabanowitz J. Hunt D.F. Shinkai Y. Allis C.D. Mol. Cell. 2003; 12: 1591-1598Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar, 13.Peters A.H.F.M. Kubicek S. Mechtler K. O'Sullivan R.J. Derijck A.A.H.A. Perez-Burgos L. Kohlmaier A. Opravil S. Tachibana M. Shinkai Y. Martens J.H.A. Jenuwein T. Mol. Cell. 2003; 12: 1577-1589Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). In contrast to findings suggesting a role for H4-Lys20 methylation in regulating gene expression, a recently published study demonstrates that H4-Lys20 methylation, in particular trimethylation, plays no apparent role in gene regulation or heterochromatin function but is involved in DNA damage response in fission yeast (14.Sanders S.L. Portoso M. Mata J. Bähler J. Allshire R.C. Kouzarides T. Cell. 2004; 119: 603-614Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). The first studies on methylation of H4-Lys20 with antibodies to dimethylated H4-Lys20 reported that dimethylated H4-Lys20 acts in antagonizing H4-Lys16 acetylation (7.Rice J.C. Nishioka K. Sarma K. Steward R. Reinberg D. Allis C.D. Genes Dev. 2002; 16: 2225-2230Crossref PubMed Scopus (202) Google Scholar, 8.Nishioka 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 (467) Google Scholar) and does not correlate with gene activity (6.Fang J. Feng Q. Ketel C.S. Wang H. Cao R. Xia L. Erdjument-Bromage H. Tempst P. Simon J.A. Zhang Y. Curr. Biol. 2002; 12: 1086-1099Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Trimethylated H4-Lys20 was found to be a marker of constitutive heterochromatin in murine interphase and metaphase cells (15.Kourmouli 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 (217) Google Scholar) enriched at pericentric heterochromatin (16.Sarg B. Helliger W. Talasz H. Koutzamani E. Lindner H.H. J. Biol. Chem. 2004; 279: 53458-53464Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Furthermore, it was shown that trimethylated H3-Lys9 is required for induction of H4-Lys20 trimethylation and that trimethylation of histone H3-Lys9 and H4-Lys20 functions as a repressive mark in gene-silencing mechanisms (5.Schotta G. Lachner M. Sarma K. Ebert A. Sengupta R. Reuter G. Reinberg D. Jenuwein T. Genes Dev. 2004; 18: 1251-1262Crossref PubMed Scopus (861) Google Scholar). ChIP 2The abbreviations used are: ChIP, chromatin immunoprecipitation; RNApol II, RNA Polymerase II; RT-PCR, Reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PMSF, phenylmethylsulfonyl fluoride; MEL, mouse erythroleukemia; DAPI, 4-6-diamidino-2-phenylindole; Me2SO, dimethyl sulfoxide; HILIC, hydrophilic interaction liquid chromatography; ESI-MS, electrospray ion-mass spectrometry; Ab, antibody; FITC, fluorescein isothiocyanate; fwd, forward; rev, reverse. experiments demonstrated that the human histone deacetylase SirT1-induced deacetylation of H4-Lys16 is accompanied by H1 enrichment and the spreading of trimethylated H3-Lys9 and monomethylated H4-Lys20 at the promoter region of a repressed reporter system (17.Vaquero A. Scher M. Lee D. Erdjument-Bromage H. Tempst P. Reinberg D. Mol. Cell. 2004; 16: 93-105Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar). Histone H3-Lys27 trimethylation and H4-Lys20 monomethylation were shown to be associated with Xist expression in ES cells and seem to mark the initiation of X inactivation. However, both H3-Lys27 trimethylation and H4-Lys20 monomethylation are maintained in the absence of transcriptional repression (18.Kohlmaier A. Savarese F. Lachner M. Martens J. Jenuwein T. Wutz A. PLoS Biol. 2004; 2: 991-1003Crossref Scopus (303) Google Scholar). Further investigations indicating a role of methylated H4-Lys20 in gene activation were performed by Beisel et al. (19.Beisel C. Imhof A. Greene J. Kremmer E. Sauer F. Nature. 2002; 419: 857-862Crossref PubMed Scopus (243) Google Scholar), who found that the Drosophila epigenetic activator ASH-1, a histone methyltransferase, activates transcription by dimethylation of H3-Lys4, H3-Lys9, and H4-Lys20 at the promoter of target genes. Significant differences in subnuclear localization of the mono- and trimethyl versions of histone H4-Lys20 were recently observed during mouse embryogenesis (20.Biron V.L. McManus K.J. Hu N. Hendzel M.J. Underhill D.A. Dev. Biol. 2004; 276: 337-351Crossref PubMed Scopus (71) Google Scholar). H4 monomethyl Lys20 was shown to be elevated in proliferating cells; in contrast, histone H4 trimethyl Lys20 became enriched in differentiating cells during the mouse developmental process. Most recently, we investigated the changes in Lys20 methylation states of various acetylated H4 histones as well as the interplay between the various H4-Lys20 methylation states and acetylation during in vitro differentiation of mouse erythroleukemia cells (16.Sarg B. Helliger W. Talasz H. Koutzamani E. Lindner H.H. J. Biol. Chem. 2004; 279: 53458-53464Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). We found that trimethylated H4-Lys20 histones increase during the differentiation process of mouse erythroleukemia (MEL) cells and that these hypermethylated H4 histones completely preclude histone H4 tri- and tetraacetylation. With these observations in mind, we decided to investigate the methylation states of H4-Lys20 in correlation with acetylation at induced (β major/minor globin) or repressed (c-myc) genes or at silent genes like embryonic β-globin in the MEL cell system. The β-globin locus, containing the erythroid-specific and developmentally regulated β-globin genes, is a particularly informative system for investigating the structure/function of histone modification patterns. The present study indicates that monomethylated H4-Lys20 is not a principal feature of repressed gene regions. On the contrary, after induction of expression of the adult β-globin gene, we found increased monomethylated H4-Lys20 paralleled by hyperacetylation of H4 at the β-major and β-minor promoter and β-globin transcribed region. Increased levels of monomethyl H4-Lys20 and hyperacetylated H4 at the promoter (exon 1) and the transcribed exon 2 region were also found at the highly expressed c-myc gene. Most interestingly, monomethylation and hyperacetylation of the H4 histone also persist at low expression levels of c-myc, indicating that neither hypoacetylation nor decreased monomethylation of H4 is a prerequisite for c-myc gene down-regulation. To confirm the findings obtained with ChIP, we investigated the H4 acetylation/methylation pattern using hydrophilic interaction liquid chromatography. These experiments revealed that monomethylation of H4-Lys20 excludes neither acetylation of H4-Lys16 nor hyperacetylation of the respective H4 histone molecule. These experiments therefore support our ChIP results showing a negative correlation between the patterns of trimethylated H4-Lys20 and H4 hyperacetylation but a positive correlation between the patterns of monomethylated H4-Lys20 and H4 hyperacetylation at distinct gene regions of the c-myc and β-globin genes. Immunofluorescence microscopy studies showed that trimethyl H4-Lys20 is enriched mainly within DAPI-dense regions, which almost completely overlap with HP1β-stained heterochromatin largely excluded, however, from active chromatin (RNApol II) regions. In contrast, monomethyl H4-Lys20 mainly overlaps with RNApol II-stained euchromatic regions and is largely excluded from HP1β-stained heterochromatin, indicating an association with transcriptionally engaged chromatin. To summarize our results, we conclude that hyperacetylated and monomethylated H4-Lys20 may be important in maintaining the transcriptionally active or competent chromatin state. Cell Culture—MEL cells (line F4N) were grown in Dulbecco's minimum Eagle's medium containing 2× nonessential amino acids, 1× penicillin/streptomycin, and 10% fetal calf serum. Cells were cultured at initial cell density of 5 × 104/ml at 37 °C and 5% CO2. Differentiation was induced by the addition of 2% Me2SO for 96 h or 1.75 mm sodium butyrate for 72 h. The percentage of benzidine-positive cells was determined as described by Orkin et al. (21.Orkin S.H. Harosi F.J. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 98-102Crossref PubMed Scopus (442) Google Scholar). Reverse Transcription (RT)-PCR—Expression analysis by RT-PCR was performed with the Titanium One-step RT-PCR kit (Clontech) by using the following primer pairs: GAPDH fwd 5′-ACGGGAAGCCCATCACCATCTTCCA-3′ and rev 5′-ATCCACGACGGACACATTGGGGGTA-3′; myc fwd 5′-GGCTGGATTTCCTTTGGGCGTTGGA-3′ and rev 5′-TCTTTGCGCGCAGCCTGGTAGGA-3′; and β-globin fwd 5′-GGTTGTCTACCCTTGGACCCAGC-3′ and rev 5′-GGTACTTGTGAGCCAGGGCAGT-3′. The RT-PCR analysis of the two adult β-globin genes (β-minor and β-major) was performed with a primer pair that coamplifies β-minor and β-major globin cDNAs. To distinguish the two β-globin products, restriction enzyme digestion with PvuI was performed, which generates two β-minor globin products (234 and 107 bp) and one undigested β-major globin product (341 bp). PCR samples were separated on a 2% agarose gel, standardized by using control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal, and quantified. Formaldehyde Cross-linking and Sonication—1 × 108 cells were fixed with 0.4% formaldehyde in minimum Eagle's medium for 10 min at room temperature with gentle agitation to generate protein-DNA cross-links. To quench the reaction, glycine (125 mm final) was added. Cells were collected by centrifugation at 600 × g for 10 min at 4 °C and then washed two times in phosphate-buffered saline. After nuclei were prepared in cell lysis buffer at 4 °C (10 mm Tris/HCl, pH 8.0, 10 mm NaCl, 0.2% Triton X-100, 1 mm PMSF, and complete protease inhibitor mixture (Roche Applied Science) as indicated in the product description), nuclei were lysed by incubation in nuclei lysis buffer (50 mm Tris/HCl, pH 8.1, 10 mm EDTA, 1% SDS, 1 mm PMSF, and complete protease inhibitor mixture) for 10 min on ice. The lysate was sonicated at 4 °C with 10 pulses of 30 s each at 40% of maximum power with a Bandelin electronic UW 2070 (70 watts) sonicator with a 2-mm tip to generate DNA fragments from ∼200 to 1000 bp with a maximum at ∼500 bp. After centrifugation (10,000 × g for 10 min at 4 °C), the supernatant was used as soluble chromatin for chromatin immunoprecipitation assay. Chromatin Immunoprecipitation Assay—ChIPs were performed as described by Forsberg et al. (22.Forsberg E.C. Downs K.M. Christensen H.M. Im H. Nuzzi P.A. Bresnick E.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14494-14499Crossref PubMed Scopus (211) Google Scholar) with minor modifications. Briefly, the supernatant of soluble chromatin corresponding to ∼4 × 106 cells (∼3 A260 units of DNA) were used for chromatin immunoprecipitations. One volume of soluble chromatin was diluted with 9 volumes of ChIP dilution buffer (17 mm Tris/HCl, pH 8.1, 1.2 mm EDTA, 167 mm NaCl, 1.1% Triton X-100, 0.01% SDS, 1 mm PMSF, and complete protease inhibitor mixture) and precleared with 100 μl of protein A-agarose (50% slurry in ChIP dilution buffer) for 2 h at 4 °C with gentle rocking. After centrifugation at 400 × g for 3 min at 4 °C, an aliquot (∼150 μl) of precleared chromatin was removed and used in the subsequent PCR as input. The remainder of the chromatin (∼800 μl) was incubated overnight at 4 °C with gentle rotating with 10 μg of the respective antibody or with an isotype-matched unspecific antibody as a negative control. Immune complexes were collected by incubation with protein A-agarose (50% slurry in ChIP dilution buffer) for 2 h at 4 °C with gentle rotating. Protein A-agarose pellets were washed twice with 1 ml of low salt immune complex (IC) wash buffer (20 mm Tris/HCl, pH 8.1, 2 mm EDTA, 150 mm NaCl, 0.1% SDS, 1% Triton X-100), once with high salt IC wash buffer (20 mm Tris/HCl, pH 8.1, 2 mm EDTA, 500 mm NaCl, 0.1% SDS, 1% Triton X-100), once with LiCl wash buffer (10 mm Tris/HCl, pH 8.1, 1 mm EDTA, 0.25 m LiCl, 1% Nonidet P-40, 1% deoxycholate), and twice with TE buffer (10 mm Tris/HCl, pH 8.0, 1 mm EDTA). Immune complexes were eluted twice as the bound fraction with 250 μl of elution buffer (0.1 m NaHCO3, 1% SDS). NaCl (0.2 m final) was added to the bound fraction, and the input chromatin and cross-links were reversed by incubation at 65 °C overnight. Incubation with RNase A (0.05 mg/ml) at 37 °C for 30 min was followed by digestion with proteinase K (50 μg/ml) at 45 °C for 2 h. DNA was purified by two extractions with phenol/chloroform and precipitated with ethanol. Purified DNA was resuspended in 50 μl of water and quantified using picogreen fluorescence (Molecular Probes). PCR—The following primers were used for PCR: HS6 (product size 245 bp) fwd 5′-TCTTTAGTAAGGCATCCCACACA-3′, rev 5′-AGAGACCGAAGAAAAGGAGAATG-3′; HS3 (product size 254) fwd 5′-CTGATGAGGATTCAATGGGTTAG-3′, rev 5′-TCATCTATCTGAGCCAGCATGTA-3′; HS2 (product size 295 bp) fwd 5′-TCCTACACATTAACGAGCCTCTG-3′, rev 5′-TGATCACTCACAGCTGAAAACAT-3′; HS1 (product size 260 bp) fwd 5′-CATCCCTGGACAGATAAACAAGA-3′, rev 5′-CAAAATTCATAACACGGAAAAGC-3′; Ey-globin promoter (product size 245 bp) fwd 5′-AGAGAGTTTTTGTTGAAGGAGGA-3′, rev 5′-CACAGGAGTGTCAGAAGCAAGTA-3′; β-H1-globin promoter (product size 246 bp) fwd 5′-AGGTCCAGGGTGAAGAATAAAAG-3′, rev 5′-ATAGAAACCCTGGAAATTTCTGC-3′; β-major globin promoter (product size 179 bp) fwd 5′-GTACCCAGAGCTGAGACTCCTAA-3′, rev 5′-AGCAACTGATCCTACCTCACCTT-3′; β-minor globin promoter (product size 255 bp) fwd 5′-AGAATTAGCTGCGAGGATAAGA-3′, rev 5′-GCAAGTCAACACAACAGACTCA-3′; β-globin transcribed region 1 (product size 179 bp) fwd 5′-CCCAGCGGTACTTTGATAGC-3′, rev 5′-ATGCAGCTTGTCACAGTGGA-3′; β-globin transcribed region 2 (product size 246 bp) fwd 5′-CCCAGCGGTACTTTGATAGC-3′, rev 5′-AGAATAGCCAGGGGAAGGAA-3′; myc exon 1 (product size 264 bp) fwd 5′-GGGGAAGGGAGAAAGAGAGATT-3′, rev 5′-CTGGAATTACTACAGCGAGTCAGA-3′; and myc exon 2 transcribed region (product size 239 bp) fwd 5′-TTTCTATCACCAGCAACAGCAG-3′, rev 5′-CTCCTCCAAGTAACTCGGTCAT-3′. PCRs were performed in 1× buffer (16 mm (NH4)2SO4, 67 mm Tris/HCl, pH 8.8, 1.5 mm MgCl2, 0.01% Tween 20), 0.25 mm dNTPs, 2% Me2SO, 2 units of Bio Therm Taq-DNA polymerase, 0.25 μm of each primer set, 5 ng (10 ng) of DNA from bound fraction, control, or input. We used a touchdown PCR protocol starting at 95 °C for 3 min (10 min in the case of hot start DNA polymerase), followed by 5 cycles of 95 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min; then 5 cycles of 95 °C for 30 s, 53 °C for 1 min, and 72 °C for 1 min; and finally 30 cycles of 95 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min. PCR products were collected after 20, 25, 30, 35, and 40 cycles and separated on 2% agarose gels. Gels were scanned using the Typhoon gel and blot imager (Amersham Biosciences), and trace or volume intensity was measured using Quantity One software (Bio-Rad). Curves from input and bound were generated for each primer pair, and the enrichment or depletion of the bound fraction as compared with the input was calculated using the linear range of the two curves. Antibodies—The antibodies used were as follows: rabbit polyclonal to tetraacetylated H4 (amino acids 2-19) (Upstate 06-866), Western blotting 1:2500, ChIP 15 μl/4 × 106 cells; rabbit polyclonal to tetraacetylated H4 (amino acids 2-19) (Upstate 06-598), immunofluorescence 1:200; rabbit polyclonal to H4-acetyl-Lys16 (Abcam Ab1762), Western blotting 1:1000, ChIP 15 μl/4 × 106 cells; rabbit polyclonal to H4-mono-me-Lys20 (Abcam Ab9051), Western blotting 1:1000, immunofluorescence 1:200, ChIP 10 μg/4 × 106 cells; rabbit polyclonal to H4-di-me-Lys20 (Upstate 07-367), Western blotting 1:5000; rabbit polyclonal to H4-tri-me-Lys20 (Abcam Ab9053), Western blotting 1:1000, immunofluorescence 1:200, ChIP 10 μg/4 × 106 cells; rat monoclonal to HP1β (Abcam Ab10811), immunofluorescence 1:50; mouse monoclonal to RNA polymerase II (Abcam Ab5408), immunofluorescence 1:50. AffiniPure rabbit anti-rat IgG (H+L) (Jackson ImmunoResearch 312-005-003) was used as an isotype-matched antibody for the ChIP control studies. For Western blotting, goat anti-rabbit IgG peroxidase conjugate (Sigma A0545) 1:5000 was used as secondary antibody. The secondary antibodies for immunofluorescence were as follows: goat anti-rabbit IgG FITC conjugate (Sigma F9887) 1:160; goat anti-rabbit IgG (H+L) AffiniPure Fab fragment (Jackson ImmunoResearch 111-167-003) 1:100; goat anti-rat IgG Cy3 conjugate (Jackson ImmunoResearch 112-165-062) 1:160; and goat anti-mouse IgG Cy3 conjugate (Jackson ImmunoResearch 115-165-062) 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—Mouse erythroleukemia cells were grown in suspension culture, collected by centrifugation, and washed with phosphate-buffered saline. An amount of 105 cells was 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), blocked for 30 min with 2.5% bovine serum albumin/KCM, and sequentially incubated with the primary and secondary antibodies (in 1% bovine serum albumin/KCM). Cells were fixed for 5 min with 4% paraformaldehyde in KCM, stained with DAPI for 1 min, and mounted in ProLong antifade mounting medium (Molecular Probes). For double labeling with rabbit polyclonal to tetraacetylated H4 as first primary antibody and either rabbit polyclonal to H4-mono-me-Lys20 or rabbit polyclonal to H4-tri-me-Lys20 as second primary antibody, we used Cy3-conjugated Fab fragment goat anti-rabbit IgG as first secondary antibody to achieve effective blocking of the first primary antibody (tetraacetylated H4). Staining was visualized using a ×100 objective on a Zeiss Axioplan 2 microscope and SPOT camera (Diagnostic Instruments). Images were captured using MetaVue software (Universal Imaging Corp.), processed by deconvolution analysis using AutoDeblur Deconvolution software (AutoQuant Imaging, Inc.), and analyzed using Adobe Photoshop 8.0. Hydrophilic Interaction Liquid Chromatography—The histone fraction H4 (∼120 μg) was isolated by reversed phase-high performance liquid chromatography as described (23.Sarg B. Koutzamani E. Helliger W. Rundquist I. Lindner H.H. J. Biol. Chem. 2002; 277: 39195-39201Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) and 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 multistep gradient starting at solvent A/solvent B (100:0) (solvent A: 70% acetonitrile, 0.015 m triethanolamine/H3PO4, pH 3.0; solvent B: 65% acetonitrile, 0.015 m triethanolamine/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 modified histone H4 isoforms obtained by HILIC were identified by ESI-MS as described (23.Sarg B. Koutzamani E. Helliger W. Rundquist I. Lindner H.H. J. Biol. Chem. 2002; 277: 39195-39201Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Distribution of Monomethyl H4-Lys20 and Trimethyl H4-Lys20 during Interphase and at Metaphase in MEL Cells—To localize modified histones, we used immunofluorescence with monospecific antibodies. The distribution of mono- and trimethylated H4-Lys20 during MEL cell interphase was investigated by dual immunofluorescence staining (Fig. 1). We found that trimethylated H4-Lys20 is enriched mainly in DAPI-dense regions with almost complete overlapping with HP1β-stained heterochromatin (Fig. 1A), which is largely excluded from active chromatin (RNApol II) (Fig. 1B) regions. In contrast, monomethylated H4-Lys20 mainly overlaps with RNApol II-stained euchromatic regions (Fig. 1D) and not with DAPI-dense regions (Fig. 1C), thus indicating an association with transcriptionally engaged chromatin. A comparable distribution pattern of both monomethylated (data not shown) and trimethylated H4-Lys20 was also seen in differentiated MEL cells (16.Sarg B. Helliger W. Talasz H. Koutzamani E. Lindner H.H. J. Biol. Chem. 2004; 279: 53458-53464Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Mono- and Trimethyl H4-Lys20 Histones Are Enriched after Induction of MEL Cell Differentiation—We next asked whether the levels of modified histones change during the MEL cell differentiation process using Me2SO. Histones from induced and uninduced MEL cells were prepared and run on SDS-PAGE. After blotting to nitrocellulose, specific antibodies to mono-, di-, and trimethylated H4-Lys20, acetylated H4-Lys16, and hyperacetylated H4 were used for immunological Western blot analysis. Although the level of dimethylated H4-Lys20 did not change after treatment with Me2SO, the monomethylated H4-Lys20 level increased about 1.5-fold and that of trimethylated H4-Lys20 about 2-fold (Fig. 2). The level of acetylated H4-Lys16 and the level of hyperacetylated histone H4 were clearly diminished in Me2SO-treated MEL cells as compared with untreated controls (Fig. 2). These results prompted us to analyze the relationship between the distribution of various epigenetic marks and gene activation or repression during MEL cell differentiation. Because it is well known that during MEL cell differentiation the adult β-globin gene is largely expressed (24.Sawado T. Igarashi K. Groudine M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10226-10231Crossref PubMed Scopus (117) Google Scholar) and the c-myc gene is rapidly down-regulated (25.Lachman H.M. Skoultchi A.I. Nature. 1984; 310: 592-594Crossref PubMed Scopus (255) Google Scholar, 26.Kume T.S. Takada S. Obinata M. J. Mol. Biol. 1988; 202: 779-786Crossref PubMed Scopus (36) Google Scholar, 27.Kohlhuber F. Strobl L.J. Eick D. Oncogene. 1993; 8: 1099-1102PubMed Google Scholar), we used these gene loci to investigate histone H4 acetylation and methylation status at distinct sites. As determined by RT-PCR (Fig. 3), the increase in β-major globin transcript is about 60-fold and that in β-minor about 3-fold after 96 h of induction (Fig. 3, A and B). The myc transcripts decreased by about 10-15-fold after 96 h of induction, which is consistent with earlier reports (25.Lachman H.M. Skoultchi A.I. Nature. 1984; 310: 592-594Crossref PubMed Scopus (255) Google Scholar, 26.Kume T" @default.
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• W1991332036 title "Histone H4-Lysine 20 Monomethylation Is Increased in Promoter and Coding Regions of Active Genes and Correlates with Hyperacetylation" @default.
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