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• W1963936136 abstract "Histone methylation is unique among post-translational histone modifications by virtue of its stability. It is thought to be a relatively stable and heritable epigenetic mark for gene-specific regulation. In this study, we use quantitative in situ approaches to investigate the cell cycle dynamics of methylated isoforms of histone H3 lysine 9. Contrary to the expected stability of trimethylated lysines, our results for trimethylated lysine 9 (tMeK9) of H3 demonstrate that the genomic content of this methylation undergoes significant changes as cells progress through mitosis. Unexpectedly, there is a loss of tMeK9 that appears to reflect a robust demethylase activity that is active during the period between anaphase and cytokinesis. Subsequent investigations of mitoses in tMeK9-deficient cells revealed defects in chromosome congression and segregation that are distinct from the increased cohesion at centromeres previously reported in association with the loss of tMeK9. Collectively, these results identify a mitosis-specific trimethylation of Lys9 in pericentromeric heterochromatin that functions in the faithful segregation of chromosomes. Histone methylation is unique among post-translational histone modifications by virtue of its stability. It is thought to be a relatively stable and heritable epigenetic mark for gene-specific regulation. In this study, we use quantitative in situ approaches to investigate the cell cycle dynamics of methylated isoforms of histone H3 lysine 9. Contrary to the expected stability of trimethylated lysines, our results for trimethylated lysine 9 (tMeK9) of H3 demonstrate that the genomic content of this methylation undergoes significant changes as cells progress through mitosis. Unexpectedly, there is a loss of tMeK9 that appears to reflect a robust demethylase activity that is active during the period between anaphase and cytokinesis. Subsequent investigations of mitoses in tMeK9-deficient cells revealed defects in chromosome congression and segregation that are distinct from the increased cohesion at centromeres previously reported in association with the loss of tMeK9. Collectively, these results identify a mitosis-specific trimethylation of Lys9 in pericentromeric heterochromatin that functions in the faithful segregation of chromosomes. The core histones (H2A, H2B, H3, and H4) are small basic proteins that form the fundamental building block of chromatin structure, the nucleosome. They function to compact over 2 m of genomic DNA into a nucleus with a diameter of ∼10 μm. Collectively, the core histones are the targets of more than five different types of post-translational modifications. These include ADP-ribosylation, ubiquitination, phosphorylation, acetylation, and methylation and involve more than 40 different amino acid residues (1Zhang L. Eugeni E.E. Parthun M.R. Freitas M.A. Chromosoma. 2003; 112: 77-86Crossref PubMed Scopus (220) Google Scholar). The majority of these modifications occur within the N-terminal tails of the core histones. Most modifications function in the regulation of gene expression by modulating chromatin structure and access of regulatory proteins (reviewed in Refs. 2Goll M.G. Bestor T.H. Genes Dev. 2002; 16: 1739-1742Crossref PubMed Scopus (109) Google Scholar and 3Sims R.J. II I Nishioka K. Reinberg D. Trends Genet. 2003; 19: 629-639Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). The function of some post-translational histone modifications may be additionally explained by the existence of protein domains found in many key regulatory and transcription factors that specifically recognize and bind acetylated (bromodomain) or methylated (chromodomain) residues (reviewed in Refs. 4Brehm A. Tufteland K.R. Aasland R. Becker P.B. BioEssays. 2004; 26: 133-140Crossref PubMed Scopus (147) Google Scholar, 5Cavalli G. Paro R. Curr. Opin. Cell Biol. 1998; 10: 354-360Crossref PubMed Scopus (154) Google Scholar, 6Eissenberg J.C. Gene (Amst.). 2001; 275: 19-29Crossref PubMed Scopus (89) Google Scholar, 7Zeng L. Zhou M.M. FEBS Lett. 2002; 513: 124-128Crossref PubMed Scopus (554) Google Scholar). Histone methylation was originally identified in the mid-1960s (8Murray K. Biochemistry. 1964; 127: 10-15Crossref Scopus (329) Google Scholar) and was shown to result from histone methyltransferases that catalyze the transfer of methyl groups from S-adenosyl-l-methionine onto the ϵ-amino group of lysine, arginine, and histidine (9Paik W.K. Kim S. Biochem. Biophys. Res. Commun. 1967; 29: 14-20Crossref PubMed Scopus (117) Google Scholar). Histone methyltransferases have been found to be specific for either lysine or arginine residues (10Lachner M. Jenuwein T. Curr. Opin. Cell Biol. 2002; 14: 286-298Crossref PubMed Scopus (698) Google Scholar) and the number of methyl groups (e.g. mono-, di-, or trimethylations) they attach to the specific amino acid (9Paik W.K. Kim S. Biochem. Biophys. Res. Commun. 1967; 29: 14-20Crossref PubMed Scopus (117) Google Scholar) (see also Ref. 11Bannister A.J. Schneider R. Kouzarides T. Cell. 2002; 109: 801-806Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Interestingly, although all core histones contain arginine and lysine residues, only histones H3 and H4 are good histone methyltransferase substrates in vivo. Histone H3 harbors at least six distinct lysine residues that can be methylated (Lys4, Lys9, Lys23, Lys27, Lys36, and Lys79), whereas histone H4 contains at least three target sites (Lys12, Lys20, and Lys59) (1Zhang L. Eugeni E.E. Parthun M.R. Freitas M.A. Chromosoma. 2003; 112: 77-86Crossref PubMed Scopus (220) Google Scholar, 2Goll M.G. Bestor T.H. Genes Dev. 2002; 16: 1739-1742Crossref PubMed Scopus (109) Google Scholar, 12van Leeuwen F. Gafken P.R. Gottschling D.E. Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 13Ng H.H. Feng Q. Wang H. Erdjument-Bromage H. Tempst P. Zhang Y. Struhl K. Genes Dev. 2002; 16: 1518-1527Crossref PubMed Scopus (424) Google Scholar). Recent studies have revealed that histone methylation is an essential process required for the developmental regulation of the metazoan genome (3Sims R.J. II I Nishioka K. Reinberg D. Trends Genet. 2003; 19: 629-639Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar, 11Bannister A.J. Schneider R. Kouzarides T. Cell. 2002; 109: 801-806Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 14Rice J.C. Allis C.D. Curr. Opin. Cell Biol. 2001; 13: 263-273Crossref PubMed Scopus (565) Google Scholar). For example, methylated Lys9 (H3) is required for proper heterochromatin protein 1 binding and heterochromatin formation (15Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Nature. 2001; 410: 120-124Crossref PubMed Scopus (2184) Google Scholar, 16Fischle W. Wang Y. Jacobs S.A. Kim Y. Allis C.D. Khorasanizadeh S. Genes Dev. 2003; 17: 1870-1881Crossref PubMed Scopus (793) Google Scholar, 17Jacobs S.A. Taverna S.D. Zhang Y. Briggs S.D. Li J. Eissenberg J.C. Allis C.D. Khorasanizadeh S. EMBO J. 2001; 20: 5232-5241Crossref PubMed Scopus (330) Google Scholar, 18Lachner M. O'Carroll D. Rea S. Mechtler K. Jenuwein T. Nature. 2001; 410: 116-120Crossref PubMed Scopus (2177) Google Scholar, 19Nakayama J. Rice J.C. Strahl B.D. Allis C.D. Grewal S.I. Science. 2001; 292: 110-113Crossref PubMed Scopus (1379) Google Scholar). In contrast, methylated Lys4 (H3) preferentially associates with transcriptionally active chromatin (20Kiekhaefer C.M. Grass J.A. Johnson K.D. Boyer M.E. Bresnick E.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14309-14314Crossref PubMed Scopus (88) Google Scholar, 21Litt M.D. Simpson M. Gaszner M. Allis C.D. Felsenfeld G. Science. 2001; 293: 2453-2455Crossref PubMed Scopus (520) Google Scholar, 22Noma K. Allis C.D. Grewal S.I. Science. 2001; 293: 1150-1155Crossref PubMed Scopus (601) Google Scholar). Unfortunately, however, many of these previous studies fail to distinguish between mono-, di-, or trimethylated species. Nevertheless, in the few studies that have distinguished between various methylation levels, there do appear to be functional distinctions between them. For example, Santos-Rosa et al. (23Santos-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 (1603) Google Scholar) demonstrated that dimethylated Lys4 was enriched in active and inactive yeast genes, whereas trimethylated K4 (tMeK4) 5The abbreviations used are: tMeK4, trimethylated lysine 4; dMeK9, dimethylated lysine 9; mMeK9, monomethylated lysine 9; eGFP, enhanced green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; CPTS, copper phthalocyanine 3,4′,4″,4‴-tetrasulfonic acid tetrasodium salt; PI, propidium iodide; dn, double null; MEF, mouse embryo fibroblast; IMEF, immortalized mouse embryonic fibroblast; TSI, total signal intensity. was only enriched in active genes. Methylation is known to be much more stable than other posttranslational histone modifications. Metabolic studies using radiolabeled isotopic tracers incorporated into the acetyl-, phospho-, or methyl-groups covalently attached to histones revealed that acetylation and phosphorylation turnover is rapid and can occur within minutes but that methylation appears much more stable. In fact, several research groups found that the methylation turnover rate was not resolved from the turnover of the histones themselves (24Byvoet P. Shepherd G.R. Hardin J.M. Noland B.J. Arch. Biochem. Biophys. 1972; 148: 558-567Crossref PubMed Scopus (155) Google Scholar, 25Duerre J.A. Lee C.T. J. Neurochem. 1974; 23: 541-547Crossref PubMed Scopus (59) Google Scholar, 26Waterborg J.H. J. Biol. Chem. 1993; 268: 4918-4921Abstract Full Text PDF PubMed Google Scholar). However, more recent results have specifically demonstrated that arginine methylation can be subsequently modified by a deimination reaction to yield citrullination (27Cuthbert G.L. Daujat S. Snowden A.W. Erdjument-Bromage H. Hagiwara T. Yamada M. Schneider R. Gregory P.D. Tempst P. Bannister A.J. Kouzarides T. Cell. 2004; 118: 545-553Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 28Wang Y. Wysocka J. Sayegh J. Lee Y.H. Perlin J.R. Leonelli L. Sonbuchner L.S. McDonald C.H. Cook R.G. Dou Y. Roeder R.G. Clarke S. Stallcup M.R. Allis C.D. Coonrod S.A. Science. 2004; 306: 279-283Crossref PubMed Scopus (774) Google Scholar). Furthermore, it has recently been shown that LSD1, a nuclear homolog of amine oxidases, specifically demethylates dimethylated Lys4 (29Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (3177) Google Scholar). More recently, it was shown that this demethylase could remove methylation from mono- and dimethylated lysine 9 (29Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (3177) Google Scholar, 30Bannister A.J. Kouzarides T. Nature. 2005; 436: 1103-1106Crossref PubMed Scopus (391) Google Scholar, 31Metzger E. Wissmann M. Yin N. Muller J.M. Schneider R. Peters A.H. Gunther T. Buettner R. Schule R. Nature. 2005; 437: 436-439Crossref PubMed Scopus (1368) Google Scholar). However, to date, lysine 9 trimethylation is considered stable (30Bannister A.J. Kouzarides T. Nature. 2005; 436: 1103-1106Crossref PubMed Scopus (391) Google Scholar). The trimethylation of lysine 9 has previously been shown to be important in both the regulation of gene expression during interphase and chromosome segregation (32Peters A.H. O'Carroll D. Scherthan H. Mechtler K. Sauer S. Schofer C. Weipoltshammer K. Pagani M. Lachner M. Kohlmaier A. Opravil S. Doyle M. Sibilia M. Jenuwein T. Cell. 2001; 107: 323-337Abstract Full Text Full Text PDF PubMed Scopus (1363) Google Scholar). Interestingly, the responsible histone lysine methyltransferase, Suv39h1, has been shown to be preferentially recruited to pericentromeric heterochromatin during entry into mitosis (33Aagaard L. Schmid M. Warburton P. Jenuwein T. J. Cell Sci. 2000; 113: 817-829Crossref PubMed Google Scholar). In this study, we examined the spatial and temporal dynamics of lysine 9 methylation throughout the cell cycle. Remarkably, we find striking mitosis-specific changes in tMeK9, which rapidly increases as cells enter mitosis, attains maximal levels at metaphase, and rapidly decreases as cells exit mitosis. By early G1, the methylation state is returned to its steady-state interphase levels. These changes in lysine 9 trimethylation were even more dramatic in midgestation mouse embryos and nonimmortalized mouse primary cultures. Our results provide the first evidence of changes in histone methylation that preclude the faithful transmission of the methylated lysine residues present in metaphase chromosomes. Furthermore, analysis of mitotic events in cells lacking tMeK9 revealed overall increases in chromosome congression and segregation defects. These defects are distinct from the preponderance of “butterfly chromosomes” reported previously (32Peters A.H. O'Carroll D. Scherthan H. Mechtler K. Sauer S. Schofer C. Weipoltshammer K. Pagani M. Lachner M. Kohlmaier A. Opravil S. Doyle M. Sibilia M. Jenuwein T. Cell. 2001; 107: 323-337Abstract Full Text Full Text PDF PubMed Scopus (1363) Google Scholar) and were also observed in cells treated for 2 h with the methylation inhibitor adenosine dialdehyde. Cell Culture—HeLa (human epithelioid cervical carcinoma), IM (male Indian muntjac skin fibroblast), and 10T1/2 (mouse fibroblast) cells were cultured in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum, Ham's F-10 medium plus 20% fetal bovine serum, and α-minimal essential medium plus 10% fetal bovine serum, respectively, in a 37 °C incubator with 5% CO2. Immortalized embryonic fibroblast cell lines from Suv39h1 and Suv39h2 double null embryos were provided by Dr. Thomas Jenuwein and isolated according to established procedures (34Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (2004) Google Scholar). Immunofluorescent Labeling—Asynchronous cells were plated onto sterilized glass coverslips 1 day prior to immunostaining such that they were 50-80% confluent the following day. Cells were fixed, permeabilized, immunofluorescently labeled, and mounted as detailed elsewhere (35McManus K.J. Hendzel M.J. Mol. Cell Biol. 2003; 23: 7611-7627Crossref PubMed Scopus (67) Google Scholar). The following primary antibodies were used at the dilutions indicated: anti-mMeK9 (1:200; Abcam), anti-dMeK9 (1:200; Abcam), anti-tMeK9 (1:200; Abcam), anti-trimethylated Lys27 (1:500; Abcam), anti-centromeric antigen (1:1000; Dr. G. Chan), and anti-phosphohistone H3 (Ser10) (1:400; Upstate Biotechnology, Inc., Lake Placid, NY). Appropriate secondary antibodies (e.g. mouse or rabbit) conjugated to fluorophores (e.g. Alexa Fluor 488 or Cy-3) were used for visualization of primary antibodies and were purchased from Molecular Probes, Inc. (Eugene, OR) or Jackson ImmunoResearch Laboratories, Inc. and used at dilutions of 1:200. Generation of 10T1/2 Cells Stably Expressing H3.3-eGFP—10T1/2 cells stably transfected with eGFP histone H3.3 were kindly provided by Dr. John Th'ng (Northwestern Ontario Regional Cancer Center, Thunder Bay, Ontario, Canada). Adenosine Dialdehyde Treatment—Live cells were treated with 25 mm adenosine dialdehyde (Sigma), a known methyltransferase inhibitor, for either 1 or 2 h. Cells were fixed, permeabilized, and counterstained with DAPI, and phenotypic abnormalities were manually scored. Immunoblot Analysis—To confirm the availability and accessibility of all methylation epitopes and show their temporal regulation throughout the cell cycle, immunoblot analysis was conducted on protein extracts isolated from asynchronously growing cells and compared with extracts isolated from mitotically arrested cells. HeLa cells were mitotically arrested using 15 nm nocodazole (Sigma) for 12 h. Alternatively, cells were arrested at the G1/S-phase boundary by standard double thymidine block, washed extensively with PBS, and permitted to progress for 4 h prior to a 4-h incubation with either nocodazole (15 nm) or ALLN (40 μg/ml; Calbiochem). Approximately 10 × 106 cells were harvested as described under “Flow Cytometry,” with all centrifugation steps performed at 4 °C. Following the final PBS wash, cells were lysed in Nuclei Buffer containing 250 mm sucrose, 200 mm NaCl, 10 mm Tris-HCl (pH 8.0), 2 mm MgCl2, 1 mm CaCl2, 1% Triton X-100, and 1 mm phenylmethanesulfonyl fluoride. Nuclei were pelleted and resuspended in 0.4N H2SO4 and placed on ice for 30 min. Nuclear debris was cleared by centrifugation at 14,000 rpm for 10 min. Supernatants were collected and added to 60 μl of 1 m Tris (pH 8.0) and 40 μl of 10 n NaOH. The acid-extracted proteins from 2.0 × 105 asynchronously growing and mitotically arrested cells were resolved on a 15% SDS-polyacrylamide gel. Equivalent protein loading was confirmed by either Coomassie Blue staining of a parallel gel or by copper phthalocyanine 3,4′,4″,4‴-tetrasulfonic acid tetrasodium salt (CPTS) staining as described by Bickar and Reid (36Bickar D. Reid P.D. Anal. Biochem. 1992; 203: 109-115Crossref PubMed Scopus (51) Google Scholar). Proteins were transferred to polyvinylidene difluoride membranes, blocked with 5% nonfat milk, and incubated with the appropriate antibody overnight at 4 °C. Immunoblots were washed three times with TBS containing 1% Tween 20 prior to a 1-h incubation with anti-rabbit horseradish peroxidase (1:10,000; Jackson ImmunoResearch Laboratories). Chemiluminescence was performed as described by the manufacturer (ECL+; Amersham Biosciences). Flow Cytometry—Asynchronous and subconfluent cells were harvested using 0.53 mm EDTA. Cells were pelleted by centrifugation at 1,500 rpm for 5 min and resuspended in PBS. Cells were pelleted and resuspended two additional times prior to aliquoting 2 × 106 cells per tube. Cells were pelleted and fixed in 1 ml of 70% ice-cold ethanol. Fixed cells were maintained at 4 °C for up to 1 week prior to analysis. Cell aliquots were immunostained separately with 100 μl of a 1:200 dilution of one of the anti-methylation antibodies (listed above) for 30 min. Cells were then washed twice with PBS and incubated with anti-rabbit Alexa Fluor 488 (1:200; Molecular Probes) for 30 min. Cells were washed as above, and cell cycle stages were revealed by staining DNA with 60 μm propidium iodide (PI) for 30 min at 37 °C. Cells were pelleted, washed once with PBS, and resuspended in 500 ml of PBS prior to flow cytometric analysis using a FACSort (BD Biosciences). Appropriate controls were utilized and included unstained cells, PI-stained only, anti-rabbit Alexa Fluor 488 only, and anti-rabbit Alexa Fluor 488 with PI. Figures were compiled in Adobe Photoshop version 6.0, whereas statistical data were exported and analyzed as detailed below. Embryo Immunofluorescence—Embryonic day 9.5 embryos were collected from CD1 mice (Charles River Laboratories), washed in PBS, and fixed in 4% paraformaldehyde at 4 °C for 18 h. Embryos were cryoprotected in 30% sucrose-PBS, mounted in O.C.T. (Tissue-Tek), and sectioned at 14 μm using a Leica CM 1900 cryostat. Sections were immediately fixed in 4% paraformaldehyde for 7 min, washed twice for 10 min in PBS, permeabilized for 30 min in PBS plus 0.1% Triton X-100, blocked in PBS plus 0.1% Triton X-100 with 5% heat inactivated sheep serum for 30 min, and incubated overnight at 4 °C with an appropriate primary antibody. Sections were washed three times for 10 min each in PBS plus 0.1% Triton X-100, blocked for 30 min in PBS plus 0.1% Triton X-100 with 5% heat-inactivated sheep serum, incubated with secondary antibody (Alexa594-goat anti-rabbit; 1:300; Molecular Probes) for 2 h at room temperature, washed three times for 5 min each in PBS plus 0.1% Triton X-100, and mounted with 90% glycerol/PBS/DAPI (1 μg/ml). Images were collected as detailed above. Suv39h1/2 Double Null Immortalized Mouse Embryonic Fibroblasts—Suv39h1/2 double null (dn) immortalized mouse embryonic fibroblasts (IMEFs) (D5) and IMEF controls (W8) were generously provided by Dr. T. Jenuwein (Research Institute of Molecular Pathology, Vienna, Austria). Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and supplemented with 1% nonessential amino acids (Invitrogen) and 0.1 mm β-mercaptoethanol (Sigma). Aberrant mitoses were investigated using an Axioskop 2 plus digital microscope (Carl Zeiss, Inc.) equipped with a plan-neofluar, oil immersion × 40 (numerical aperture = 0.75) lens. Asynchronous D5 and W8 cells were seeded onto coverslips, paraformaldehyde-fixed, and counterstained with DAPI prior to examination. Based on standard chromosome morphological criteria, mitotic cells were classified as being in metaphase, anaphase, telophase, or cytokinesis/early G1. Cells were manually scored as normal or aberrant phenotypes, and the data were tabulated. Aneuploidy was investigated by flow cytometry and was performed as above. Histone Methylation in Asynchronous Cell Cultures—To explore the possibility of cell cycle-dependent changes in histone H3 lysine 9 methylation states, we examined asynchronous HeLa, IM, and mouse 10T1/2 cells by indirect immunofluorescence using a panel of Lys9 methylation level-specific antibodies (i.e. mono-, di- and trimethylated Lys9). The antibodies were deemed to be highly specific for the specified epitope based on indirect immunofluorescence peptide competition assays (supplemental Figs. 1 and 2A). Asynchronous cells provide representatives of all cell cycle stages on a single slide. A subjective observation of fluorescence intensities can easily be coupled to morphological markers of cell cycle position. This provides a rapid method to survey methylations for potential cell cycle-dependent changes in abundance and has been previously used to identify G2- and M-phase-specific changes in histone H3 phosphorylation at serine 10 (37Hendzel M.J. Wei Y. Mancini M.A. Van Hooser A. Ranalli T. Brinkley B.R. Bazett-Jones D.P. Allis C.D. Chromosoma. 1997; 106: 348-360Crossref PubMed Scopus (1516) Google Scholar). Fig. 1 illustrates the variation in the intensity of immunofluorescent staining of cells stained with antibodies directed against mono-, di-, and trimethyl modifications at Lys9 (mMeK9, dMeK9, and tMeK9) of histone H3. There appear to be significant changes in methylation when comparing mitotic and interphase HeLa and 10T1/2 cells labeled with anti-mMeK9, -dMeK9, and -tMeK9 (Figs. 1 and 2). Whereas an apparent increase in methylation will occur solely because of the change in DNA density, the relative change in trimethylation is so dramatic that it is difficult to represent the interphase cell staining without saturating the images of the mitotic cells.FIGURE 2High resolution images of various lysine methylation epitopes in mouse cells. Representative digital images of 10T1/2 cells immunofluorescently labeled with antibodies directed against mMeK9, dMeK9, and tMeK9. For reference purposes, at least one mitotic cell (indicated by an arrow) has been included for signal intensity comparisons with the interphase cells. The DAPI channel has been included, and pericentromeric heterochromatin is visible as DAPI intense staining regions. Scale bar, 3 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Phosphorylation of Serine 10 Does Not Increase tMeK9 Detection by Indirect Immunofluorescence—Surprisingly, the temporal progression pattern of tMeK9 closely resembles that of PhosS10, its neighboring residue. Because there is a possibility that the reactivity of the anti-tMeK9 antibody may be influenced by the presence or absence of PhosS10, we sought to establish whether the anti-tMeK9 antibody would still recognize its cognate epitope in the absence of PhosS10. Accordingly, we developed a calf intestinal alkaline phosphatase assay that is performed in situ. Live 10T1/2 cells were permeabilized and treated with or without calf intestinal alkaline phosphatase, paraformaldehyde-fixed, and immunofluorescently labeled with anti-tMeK9. This procedure displaced metaphase cells from the coverslip; however, prophase cells remained adhered to the coverslips and exhibited robust anti-tMeK9 immunostaining in both the presence and absence of PhosS10 (supplemental Fig. 2B). Furthermore, these prophase anti-tMeK9 signal intensities were visually more intense than those of interphase cells on the same coverslips (data not shown). These data establish that the phosphorylation status of Ser10 is not responsible for the observed increase in antibody staining of trimethylated lysine 9. Histone H3.3 Does Not Replace H3 Trimethylated at Lysine 9 during Mitotic Exit—During interphase, tMeK9 has been shown to be displaced from chromatin through the incorporation of histone H3.3 via the replication-independent nucleosome assembly pathway (38Janicki S.M. Tsukamoto T. Salghetti S.E. Tansey W.P. Sachidanandam R. Prasanth K.V. Ried T. Shav-Tal Y. Bertrand E. Singer R.H. Spector D.L. Cell. 2004; 116: 683-698Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). Although this process is likely to be restricted to interphase cells, one recent report demonstrated a small amount of histone H2B exchange during mitosis (39Thiriet C. Hayes J.J. Genes Dev. 2005; 19: 677-682Crossref PubMed Scopus (114) Google Scholar). Thus, it is possible that the incorporation of histone H3.3 occurs specifically during exit from mitosis. To test this hypothesis, we generated 10T1/2 cells stably transfected with eGFP-histone H3.3 and examined their association with pericentromeric heterochromatin during the earliest stages of G1, when the interphase nuclei are still decondensing. At this point, the incorporation of histone H3.3 is expected to be maximal if the loss of tMeK9 is dependent upon H3.3 incorporation. Fig. 3 shows deconvolved images of two daughter nuclei in early G1. DNA is shown in red, and eGFP-H3.3 is shown in green. Note that at this stage of early G1, these cells still partially maintain their metaphase chromosome organization. The arrows indicate regions of pericentromeric heterochromatin, which are easily recognized by their distinctive morphology in mouse fibroblast cell lines. There is no significant incorporation of histone H3.3 into pericentromeric heterochromatin. Epitope Accessibility Is Not Responsible for Changes in Trimethylated Lys9 Abundance during Metaphase—Perhaps the most obvious explanation for the indirect immunofluorescence results detailed above is the mitotic dissociation of proteins (e.g. heterochromatin protein 1), which may bind to, and mask, a subset of the epitopes during interphase or mitosis. This hypothesis can be easily examined by immunoblotting protein preparations isolated at specific cell cycle stages, where any such proteins will be displaced during SDS-PAGE. To establish that changes in epitope accessibility are not responsible for the methylation dynamics observed, immunoblotting was performed on acid-extracted proteins isolated from asynchronous or mitotically enriched populations. Approximately 55.2 ± 0.8% of asynchronous HeLa cells were in G0/G1, 16.9 ± 0.8% in S-phase, and 26.7 ± 1.2% in G2/M as determined by flow cytometry. Following overnight treatment with nocodazole, a 3.4-fold increase in the proportion of cells in G2/M was observed (91.1 ± 1.6%), whereas those in G0/G1 (3.6 ± 1.3%) and S-phase (5.4 ± 0.6%) were decreased markedly. Microscopy was used to confirm that the cells had accumulated in prometaphase (data not shown). Equivalent protein amounts from the two populations were resolved by SDS-PAGE and immunostained to assess cell cycle-associated differences in the methylated Lys9 derivatives (Fig. 4). Similar results were obtained with mitotically enriched populations generated by a double thymidine block followed by either nocodazole or ALLN treatment (data not shown). These results confirm that there are significant increases in the abundance of mMeK9 and tMeK9 during mitosis. By this approach, the change in the methylation status of dMeK9 appeared to be minimal. Global Histone Methylation Dynamics throughout the Cell Cycle—To better characterize the cell cycle-associated methylation dynamics in asynchronous HeLa cells, we examined each of the three Lys9 methylation epitopes by flow cytometry (Fig. 5). If Lys9 methylation is indeed stable, we would expect to observe only a 2-fold increase in total methylation over the course of a cell cycle with the principle methylation intensity decrease coinciding with the 4 to 2 N reduction of the genome during cytokinesis. These relationships are more apparent when expressed as mean intensities for the different stages of the cell cycle (G0/G1, S-phase, and G2/M) after normalizing for DNA content (supplemental Table 1). When expressed this way, the mMeK9 shows a doubling of the labeling intensity when progressing from the 2 n (PI intensity = 200) to 4 n (PI intensity = 400). The dMeK9 remains relatively constant across the cell cycle. In contrast, the tMeK9 isoforms of histone H3 reach peak intensities in G2/M (PI intensity = 400) that are greater than expected based on the change in DNA content. Similar results were obtained for 10T1/2 cells (data not shown). Because the G2/M peak contains primarily G2 cells, any change that is confined to cells in prophase through metaphase would not be reflected in the prominent G2/M cluster. For example, when histone H3 is phosphorylated at serine 10 o" @default.
• W1963936136 created "2016-06-24" @default.
• W1963936136 creator A5011336975 @default.
• W1963936136 creator A5037953672 @default.
• W1963936136 creator A5042375136 @default.
• W1963936136 creator A5045343897 @default.
• W1963936136 creator A5073472372 @default.
• W1963936136 date "2006-03-01" @default.
• W1963936136 modified "2023-10-03" @default.
• W1963936136 title "Dynamic Changes in Histone H3 Lysine 9 Methylations" @default.
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