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Histone H3 Lysine 9 Methylation Occurs Rapidly at the Onset of Random X Chromosome Inactivation

2002; Elsevier BV; Volume: 12; Issue: 3 Linguagem: Inglês

10.1016/s0960-9822(02)00660-7

1879-0445

Jacqueline E. Mermoud, Bilyana Popova, Antoine H.F.M. Peters, Thomas Jenuwein, Neil Brockdorff,

Genomics and Chromatin Dynamics

In female mammals, a single X chromosome is stably and heritably silenced early in embryogenesis [1Lyon M.F. Gene action in the X chromosome of the mouse (Mus musculus L.).Nature. 1961; 190: 372-373Crossref PubMed Scopus (2452) Google Scholar]. The inactive X is characterized by asynchronous DNA replication [2Priest J.H. Heady J.E. Priest R.E. Delayed onset of replication of human X chromosomes.J. Cell Biol. 1967; 35: 483-487Crossref PubMed Scopus (44) Google Scholar, 3Takagi N. Differentiation of the X chromosome in early female mouse embryos.Exp. Cell Res. 1974; 86: 127-135Crossref PubMed Scopus (83) Google Scholar] and epigenetic chromatin modifications, including DNA methylation [4Mohandas T. Sparkes R.S. Shapiro L.J. Reactivation of an inactive X chromosome: Evidence for X-inactivation by DNA methylation.Science. 1981; 211: 393-396Crossref PubMed Scopus (467) Google Scholar], histone H3/H4 hypoacetylation [5Jeppesen P. Turner B.M. The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene-expression.Cell. 1993; 74: 281-289Abstract Full Text PDF PubMed Scopus (579) Google Scholar, 6Belyaev N.D. Keohane A.M. Turner B.M. Differential underacetylation of histones H2A, H3 and H4 on the inactive X chromosome in human female cells.Hum. Genet. 1996; 97: 573-578Crossref PubMed Scopus (97) Google Scholar, 7Boggs B.A. Connors B. Sobel R.E. Chinault A.C. Allis C.D. Reduced levels of histone H3 acetylation on the inactive X chromosome in human females.Chromosoma. 1996; 105: 303-309Crossref PubMed Scopus (72) Google Scholar], and incorporation of a variant histone macroH2A [8Costanzi C. Pehrson J.R. Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals.Nature. 1998; 393: 599-601Crossref PubMed Scopus (455) Google Scholar, 9Chadwick B.P. Willard H.F. Histone H2A variants and the inactive X chromosome: identification of a second macroH2A variant.Hum. Mol. Genet. 2001; 10: 1101-1113Crossref PubMed Scopus (133) Google Scholar, 10Costanzi C. Pehrson J.R. MacroH2A2, a new member of the macroH2A core histone family.J. Biol. Chem. 2001; 276: 21776-21784Crossref PubMed Scopus (117) Google Scholar]. X inactivation is initiated by a cis-acting RNA molecule, the X-inactive specific transcript (Xist), which coats the chromosome [11Brown C.J. Hendrich B.D. Rupert J.L. Lafreniere R.G. Grompe M. Tololrenzi R. Willard F. A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome.Nature. 1991; 349: 38-44Crossref PubMed Scopus (1078) Google Scholar, 12Brown C.J. Hendrich B.D. Rupert J.L. Lafreniere R.G. Xing Y. Lawrence J. Willard H.F. The human XIST gene: Analysis of a 17kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus.Cell. 1992; 71: 527-542Abstract Full Text PDF PubMed Scopus (940) Google Scholar, 13Brockdorff N. Ashworth A. Kay G.F. McCabe V.M. Norris D.P. Cooper P.J. Swift S. Rastan S. The product of the mouse Xist gene is a 15kb inactive X-specific transcript containing no conserved ORF and located in the nucleus.Cell. 1992; 71: 515-526Abstract Full Text PDF PubMed Scopus (781) Google Scholar, 14Penny G.D. Kay G.F. Sheardown S.A. Rastan S. Brockdorff N. Requirement for Xist in X chromosome inactivation.Nature. 1996; 379: 131-137Crossref PubMed Scopus (932) Google Scholar, 15Lee J.T. Strauss W.M. Dausman J.A. Jaenisch R. A 450 kb transgene displays properties of the mammalian X inactivation center.Cell. 1996; 86: 83-94Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 16Clemson C.M. McNeil J.A. Willard H.F. Lawrence J.B. XIST RNA paints the inactive X chromosome at interphase: Evidence for a novel RNA involved in nuclear chromosome structure.J. Cell Biol. 1996; 132: 259-275Crossref PubMed Scopus (599) Google Scholar, 17Wutz A. Jaenisch R. A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation.Mol. Cell. 2000; 5: 695-705Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar]. However, the mechanism by which Xist induces chromosome silencing is poorly understood. An important approach towards answering this question has been to determine the temporal order of epigenetic chromatin modifications in an in vitro model system, differentiating XX embryonic stem (ES) cells, and thereby to identify candidate targets for Xist RNA [18Kay G.F. Penny G.D. Patel D. Ashworth A. Brockdorff N. Rastan S. Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation.Cell. 1993; 72: 171-182Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 19Keohane A.M. O'Neill L.P. Belyaev N.D. Lavender J.S. Turner B.M. X-Inactivation and histone H4 acetylation in embryonic stem cells.Dev. Biol. 1996; 180: 618-630Crossref PubMed Scopus (207) Google Scholar, 20Sheardown S.A. Duthie S.M. Johnston C.M. Newall A.E.T. Formstone E.J. Arkell R.M. Nesterova T.B. Alghisi G.-C. Rastan S. Brockdorff N. Stabilisation of Xist RNA mediates initiation of X chromosome inactivation.Cell. 1997; 91: 99-107Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 21Mermoud J.E. Costanzi C. Pehrson J.R. Brockdorff N. Histone macroH2A1.2 relocates to the inactive X chromosome after initiation and propagation of X inactivation.J. Cell Biol. 1999; 147: 1399-1408Crossref PubMed Scopus (132) Google Scholar]. To date, these studies have demonstrated that, following accumulation of Xist RNA [18Kay G.F. Penny G.D. Patel D. Ashworth A. Brockdorff N. Rastan S. Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation.Cell. 1993; 72: 171-182Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 20Sheardown S.A. Duthie S.M. Johnston C.M. Newall A.E.T. Formstone E.J. Arkell R.M. Nesterova T.B. Alghisi G.-C. Rastan S. Brockdorff N. Stabilisation of Xist RNA mediates initiation of X chromosome inactivation.Cell. 1997; 91: 99-107Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar], the transition to late replication of the X chromosome is the earliest detectable event [19Keohane A.M. O'Neill L.P. Belyaev N.D. Lavender J.S. Turner B.M. X-Inactivation and histone H4 acetylation in embryonic stem cells.Dev. Biol. 1996; 180: 618-630Crossref PubMed Scopus (207) Google Scholar]. H4 hypoacetylation [19Keohane A.M. O'Neill L.P. Belyaev N.D. Lavender J.S. Turner B.M. X-Inactivation and histone H4 acetylation in embryonic stem cells.Dev. Biol. 1996; 180: 618-630Crossref PubMed Scopus (207) Google Scholar], macroH2A1.2 incorporation [21Mermoud J.E. Costanzi C. Pehrson J.R. Brockdorff N. Histone macroH2A1.2 relocates to the inactive X chromosome after initiation and propagation of X inactivation.J. Cell Biol. 1999; 147: 1399-1408Crossref PubMed Scopus (132) Google Scholar], and DNA methylation [19Keohane A.M. O'Neill L.P. Belyaev N.D. Lavender J.S. Turner B.M. X-Inactivation and histone H4 acetylation in embryonic stem cells.Dev. Biol. 1996; 180: 618-630Crossref PubMed Scopus (207) Google Scholar] all occur subsequently. Recently, it has been shown that chromatin of the inactive X is also characterized by methylation of histone H3 at lysine 9 (H3-K9) [22Peters A.H.F.M. Mermoud J.E. O'Carroll D. Pagani M. Schweizer D. Brockdorff N. Jenuwein T. Histone H3 Lysine 9 methylation is an epigenetic imprint for facultative heterochromatin at the inactive X chromosome.Nat. Genet. 2001; 30 (Published online December 10, 2001. 10.1038/ng789): 77-80Crossref PubMed Scopus (388) Google Scholar, 23Boggs B.A. Cheung P. Heard E. Spector D.L. Chinault A.C. Allis C.D. Differentially methylated forms of histone H3 show unique association patterns with inactive human X chromosomes.Nat. Genet. 2001; 30 (Published online December 10, 2001. 10.1038/ng787): 73-76Crossref PubMed Scopus (295) Google Scholar]. Here we show that H3-K9 methylation is a very early event in the process of X inactivation, which closely parallels the onset of Xist RNA accumulation. In female mammals, a single X chromosome is stably and heritably silenced early in embryogenesis [1Lyon M.F. Gene action in the X chromosome of the mouse (Mus musculus L.).Nature. 1961; 190: 372-373Crossref PubMed Scopus (2452) Google Scholar]. The inactive X is characterized by asynchronous DNA replication [2Priest J.H. Heady J.E. Priest R.E. Delayed onset of replication of human X chromosomes.J. Cell Biol. 1967; 35: 483-487Crossref PubMed Scopus (44) Google Scholar, 3Takagi N. Differentiation of the X chromosome in early female mouse embryos.Exp. Cell Res. 1974; 86: 127-135Crossref PubMed Scopus (83) Google Scholar] and epigenetic chromatin modifications, including DNA methylation [4Mohandas T. Sparkes R.S. Shapiro L.J. Reactivation of an inactive X chromosome: Evidence for X-inactivation by DNA methylation.Science. 1981; 211: 393-396Crossref PubMed Scopus (467) Google Scholar], histone H3/H4 hypoacetylation [5Jeppesen P. Turner B.M. The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene-expression.Cell. 1993; 74: 281-289Abstract Full Text PDF PubMed Scopus (579) Google Scholar, 6Belyaev N.D. Keohane A.M. Turner B.M. Differential underacetylation of histones H2A, H3 and H4 on the inactive X chromosome in human female cells.Hum. Genet. 1996; 97: 573-578Crossref PubMed Scopus (97) Google Scholar, 7Boggs B.A. Connors B. Sobel R.E. Chinault A.C. Allis C.D. Reduced levels of histone H3 acetylation on the inactive X chromosome in human females.Chromosoma. 1996; 105: 303-309Crossref PubMed Scopus (72) Google Scholar], and incorporation of a variant histone macroH2A [8Costanzi C. Pehrson J.R. Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals.Nature. 1998; 393: 599-601Crossref PubMed Scopus (455) Google Scholar, 9Chadwick B.P. Willard H.F. Histone H2A variants and the inactive X chromosome: identification of a second macroH2A variant.Hum. Mol. Genet. 2001; 10: 1101-1113Crossref PubMed Scopus (133) Google Scholar, 10Costanzi C. Pehrson J.R. MacroH2A2, a new member of the macroH2A core histone family.J. Biol. Chem. 2001; 276: 21776-21784Crossref PubMed Scopus (117) Google Scholar]. X inactivation is initiated by a cis-acting RNA molecule, the X-inactive specific transcript (Xist), which coats the chromosome [11Brown C.J. Hendrich B.D. Rupert J.L. Lafreniere R.G. Grompe M. Tololrenzi R. Willard F. A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome.Nature. 1991; 349: 38-44Crossref PubMed Scopus (1078) Google Scholar, 12Brown C.J. Hendrich B.D. Rupert J.L. Lafreniere R.G. Xing Y. Lawrence J. Willard H.F. The human XIST gene: Analysis of a 17kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus.Cell. 1992; 71: 527-542Abstract Full Text PDF PubMed Scopus (940) Google Scholar, 13Brockdorff N. Ashworth A. Kay G.F. McCabe V.M. Norris D.P. Cooper P.J. Swift S. Rastan S. The product of the mouse Xist gene is a 15kb inactive X-specific transcript containing no conserved ORF and located in the nucleus.Cell. 1992; 71: 515-526Abstract Full Text PDF PubMed Scopus (781) Google Scholar, 14Penny G.D. Kay G.F. Sheardown S.A. Rastan S. Brockdorff N. Requirement for Xist in X chromosome inactivation.Nature. 1996; 379: 131-137Crossref PubMed Scopus (932) Google Scholar, 15Lee J.T. Strauss W.M. Dausman J.A. Jaenisch R. A 450 kb transgene displays properties of the mammalian X inactivation center.Cell. 1996; 86: 83-94Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 16Clemson C.M. McNeil J.A. Willard H.F. Lawrence J.B. XIST RNA paints the inactive X chromosome at interphase: Evidence for a novel RNA involved in nuclear chromosome structure.J. Cell Biol. 1996; 132: 259-275Crossref PubMed Scopus (599) Google Scholar, 17Wutz A. Jaenisch R. A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation.Mol. Cell. 2000; 5: 695-705Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar]. However, the mechanism by which Xist induces chromosome silencing is poorly understood. An important approach towards answering this question has been to determine the temporal order of epigenetic chromatin modifications in an in vitro model system, differentiating XX embryonic stem (ES) cells, and thereby to identify candidate targets for Xist RNA [18Kay G.F. Penny G.D. Patel D. Ashworth A. Brockdorff N. Rastan S. Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation.Cell. 1993; 72: 171-182Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 19Keohane A.M. O'Neill L.P. Belyaev N.D. Lavender J.S. Turner B.M. X-Inactivation and histone H4 acetylation in embryonic stem cells.Dev. Biol. 1996; 180: 618-630Crossref PubMed Scopus (207) Google Scholar, 20Sheardown S.A. Duthie S.M. Johnston C.M. Newall A.E.T. Formstone E.J. Arkell R.M. Nesterova T.B. Alghisi G.-C. Rastan S. Brockdorff N. Stabilisation of Xist RNA mediates initiation of X chromosome inactivation.Cell. 1997; 91: 99-107Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 21Mermoud J.E. Costanzi C. Pehrson J.R. Brockdorff N. Histone macroH2A1.2 relocates to the inactive X chromosome after initiation and propagation of X inactivation.J. Cell Biol. 1999; 147: 1399-1408Crossref PubMed Scopus (132) Google Scholar]. To date, these studies have demonstrated that, following accumulation of Xist RNA [18Kay G.F. Penny G.D. Patel D. Ashworth A. Brockdorff N. Rastan S. Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation.Cell. 1993; 72: 171-182Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 20Sheardown S.A. Duthie S.M. Johnston C.M. Newall A.E.T. Formstone E.J. Arkell R.M. Nesterova T.B. Alghisi G.-C. Rastan S. Brockdorff N. Stabilisation of Xist RNA mediates initiation of X chromosome inactivation.Cell. 1997; 91: 99-107Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar], the transition to late replication of the X chromosome is the earliest detectable event [19Keohane A.M. O'Neill L.P. Belyaev N.D. Lavender J.S. Turner B.M. X-Inactivation and histone H4 acetylation in embryonic stem cells.Dev. Biol. 1996; 180: 618-630Crossref PubMed Scopus (207) Google Scholar]. H4 hypoacetylation [19Keohane A.M. O'Neill L.P. Belyaev N.D. Lavender J.S. Turner B.M. X-Inactivation and histone H4 acetylation in embryonic stem cells.Dev. Biol. 1996; 180: 618-630Crossref PubMed Scopus (207) Google Scholar], macroH2A1.2 incorporation [21Mermoud J.E. Costanzi C. Pehrson J.R. Brockdorff N. Histone macroH2A1.2 relocates to the inactive X chromosome after initiation and propagation of X inactivation.J. Cell Biol. 1999; 147: 1399-1408Crossref PubMed Scopus (132) Google Scholar], and DNA methylation [19Keohane A.M. O'Neill L.P. Belyaev N.D. Lavender J.S. Turner B.M. X-Inactivation and histone H4 acetylation in embryonic stem cells.Dev. Biol. 1996; 180: 618-630Crossref PubMed Scopus (207) Google Scholar] all occur subsequently. Recently, it has been shown that chromatin of the inactive X is also characterized by methylation of histone H3 at lysine 9 (H3-K9) [22Peters A.H.F.M. Mermoud J.E. O'Carroll D. Pagani M. Schweizer D. Brockdorff N. Jenuwein T. Histone H3 Lysine 9 methylation is an epigenetic imprint for facultative heterochromatin at the inactive X chromosome.Nat. Genet. 2001; 30 (Published online December 10, 2001. 10.1038/ng789): 77-80Crossref PubMed Scopus (388) Google Scholar, 23Boggs B.A. Cheung P. Heard E. Spector D.L. Chinault A.C. Allis C.D. Differentially methylated forms of histone H3 show unique association patterns with inactive human X chromosomes.Nat. Genet. 2001; 30 (Published online December 10, 2001. 10.1038/ng787): 73-76Crossref PubMed Scopus (295) Google Scholar]. Here we show that H3-K9 methylation is a very early event in the process of X inactivation, which closely parallels the onset of Xist RNA accumulation. Chromatin structure plays a key role in setting up heritable states of gene activation and silencing. An important level at which this is achieved is through modifications of key residues at the N termini of the core histones. Recent attention has focused on histone methylation. H3 lysine 9 (H3-K9) methylation has been shown to be a feature of constitutive centromeric heterochromatin [24Peters A.H.F.M. O'Carroll D. Scherthan H. Mechtler K. Sauer S. Schofer C. Weipoltshammer K. Pagani M. Lachner M. Kohlmaier A. et al.Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability.Cell. 2001; 107: 323-337Abstract Full Text Full Text PDF PubMed Scopus (1265) Google Scholar] and also to occur in silent chromatin domains at the silent mating type locus in S. pombe[25Noma K. Allis C.D. Grewal S.I. Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries.Science. 2001; 293: 1150-1155Crossref PubMed Scopus (575) Google Scholar] and in a developmentally regulated manner at the chicken β-globin locus [26Litt M.D. Simpson M. Gaszner M. Allis D. Felsenfeld G. Correlation between histone lysine methylation and developmental changes at the chicken β-globin locus.Science. 2001; 293: 2453-2455Crossref PubMed Scopus (503) Google Scholar]. Methylation of H3 lysine 4 (H3-K4) shows a reciprocal relationship, correlating with acetylated histones H3 and H4 in active chromatin domains [25Noma K. Allis C.D. Grewal S.I. Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries.Science. 2001; 293: 1150-1155Crossref PubMed Scopus (575) Google Scholar, 26Litt M.D. Simpson M. Gaszner M. Allis D. Felsenfeld G. Correlation between histone lysine methylation and developmental changes at the chicken β-globin locus.Science. 2001; 293: 2453-2455Crossref PubMed Scopus (503) Google Scholar]. More recently, H3-K9 methylation has been found to be present on facultative heterochromatin of the inactive X chromosome [22Peters A.H.F.M. Mermoud J.E. O'Carroll D. Pagani M. Schweizer D. Brockdorff N. Jenuwein T. Histone H3 Lysine 9 methylation is an epigenetic imprint for facultative heterochromatin at the inactive X chromosome.Nat. Genet. 2001; 30 (Published online December 10, 2001. 10.1038/ng789): 77-80Crossref PubMed Scopus (388) Google Scholar, 23Boggs B.A. Cheung P. Heard E. Spector D.L. Chinault A.C. Allis C.D. Differentially methylated forms of histone H3 show unique association patterns with inactive human X chromosomes.Nat. Genet. 2001; 30 (Published online December 10, 2001. 10.1038/ng787): 73-76Crossref PubMed Scopus (295) Google Scholar]. To better understand the role of this modification in X inactivation, we set out to determine the timing of H3-K9 methylation on the inactive X chromosome, using differentiating XX ES cells as a model system. In undifferentiated XX ES cells, both X chromosomes are active. X inactivation proceeds in a stepwise fashion as cells undergo differentiation in culture. We used an antibody raised against a branched peptide containing four K9 dimethylated H3 amino termini [24Peters A.H.F.M. O'Carroll D. Scherthan H. Mechtler K. Sauer S. Schofer C. Weipoltshammer K. Pagani M. Lachner M. Kohlmaier A. et al.Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability.Cell. 2001; 107: 323-337Abstract Full Text Full Text PDF PubMed Scopus (1265) Google Scholar] in indirect immunofluorescence studies. This allowed us to analyze temporal changes and to assess the extent of methylation over the whole chromosome. Metaphase spreads were prepared from undifferentiated PGK12.1 XX ES cells and at various times after induction of differentiation. Efc-1 XY ES cells were used as a control. In undifferentiated XX ES cells there was staining of pericentromeric regions of the chromosomes (Figure 1A), consistent with analyses in different cell types [24Peters A.H.F.M. O'Carroll D. Scherthan H. Mechtler K. Sauer S. Schofer C. Weipoltshammer K. Pagani M. Lachner M. Kohlmaier A. et al.Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability.Cell. 2001; 107: 323-337Abstract Full Text Full Text PDF PubMed Scopus (1265) Google Scholar]. A similar pattern was seen in XY ES cells. Additionally, a single small chromosome was strongly stained in the majority of metaphase spreads (Figure 1B). DNA FISH analysis demonstrated that this was the Y chromosome. Methylation of the Y was also seen in differentiated Efc-1 cells (Figure 2A), indicating that it is not a developmentally regulated feature. The Y chromosome is largely heterochromatic, and H3-K9 methylation may simply reflect this. However, in mouse bone marrow cells [22Peters A.H.F.M. Mermoud J.E. O'Carroll D. Pagani M. Schweizer D. Brockdorff N. Jenuwein T. Histone H3 Lysine 9 methylation is an epigenetic imprint for facultative heterochromatin at the inactive X chromosome.Nat. Genet. 2001; 30 (Published online December 10, 2001. 10.1038/ng789): 77-80Crossref PubMed Scopus (388) Google Scholar] and also in an XY somatic cell line (data not shown), H3-K9 methylation occurs only on the Y chromosome short arm. The reason for this difference is unknown.Figure 2Quantitation of H3-K9 Methylation of the X and Y Chromosomes during ES Cell DifferentiationShow full captionSummary of data indicating the percentage of X or Y methylated and the standard deviation as calculated from scoring individual slides and independent experiments. The table below indicates the total number (n) of metaphases scored for each time point. (A) Scoring for a fully methylated Y chromosome in Efc-1 cells differentiated for 0, 12, and 15 days. (B) Scoring for H3-K9 methylation of a single X chromosome in XX PGK12.1 ES cells differentiated for 0, 4, 8, and 12 days. Only metaphases with two X chromosomes were included in the analysis. (C) Scoring for H3-K9 methylation on one of the two X chromosomes in XX PGK12.1 cells differentiated for 1, 2, 3, 4, and 8 days using optimized conditions for immunofluorescence (see legend for Figure 1). Mouse XX fibroblast cell cultures (somatic) were analyzed to provide a positive control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Summary of data indicating the percentage of X or Y methylated and the standard deviation as calculated from scoring individual slides and independent experiments. The table below indicates the total number (n) of metaphases scored for each time point. (A) Scoring for a fully methylated Y chromosome in Efc-1 cells differentiated for 0, 12, and 15 days. (B) Scoring for H3-K9 methylation of a single X chromosome in XX PGK12.1 ES cells differentiated for 0, 4, 8, and 12 days. Only metaphases with two X chromosomes were included in the analysis. (C) Scoring for H3-K9 methylation on one of the two X chromosomes in XX PGK12.1 cells differentiated for 1, 2, 3, 4, and 8 days using optimized conditions for immunofluorescence (see legend for Figure 1). Mouse XX fibroblast cell cultures (somatic) were analyzed to provide a positive control. For analysis of methylation during X inactivation, we initially scored metaphases from cells differentiated for 0, 4, 8, and 12 days. These time points broadly encompass transitions for the other epigenetic markers of the inactive X observed in previous studies (see Figure 3 and associated text below for further detail). For each time point, data were compiled from a number of individual slides and in most cases from independent experiments carried out on different days. DNA FISH was used to verify that both X chromosomes were present. In undifferentiated XX ES cells, H3-K9 methylation of the X chromosome was undetectable in 99.5% of metaphase spreads, consistent with both X chromosomes being active. At day 4 of differentiation, 14% of spreads exhibited H3-K9 methylation of a single X chromosome. At later time points, day 8 and day 12, we observed levels of 44% and 68%, respectively. An example from day 8 differentiated cells is shown in Figure 1C. The scoring data are summarized in Figure 2B. These results demonstrate that there is a progressive methylation of H3-K9 on the inactive X chromosome in differentiating XX ES cells. To determine exactly when H3-K9 methylation of the X chromosome begins, we analyzed time points between 1 and 4 days of differentiation. At day 1, methylation of the X chromosome was undetectable in 98.2% of metaphase spreads. This is not significantly different from the frequency in undifferentiated cells and therefore indicates that H3-K9 methylation has not begun at this stage. In marked contrast, by day 2 of differentiation there was a large increase, with 30% of spreads having H3-K9 methylation of the X chromosome (an example is illustrated in Figure 1D). Further increases to 48%, 46%, and 82%, respectively, were seen at days 3, 4, and 8. These data, summarized in Figure 2C, demonstrate that H3-K9 methylation is a very early event in the establishment of the inactive state. In the latter experiment we used a modified immunofluorescence method with enhanced sensitivity (see legend to Figure 1). For this reason, the proportion of cells with H3-K9 methylation at days 4 and 8 was much higher than in our initial experiment (compare Figures 2B and 2C). In fact, the value at day 8 is similar to that seen in XX somatic cells (Figure 2C). We interpret this as signifying that within individual cells there is a progressive increase in total levels of X chromosome H3-K9 methylation during the differentiation time course. Thus, H3-K9 methylation could occur initially at a limited number of sites and then spread progressively to include the entire chromosome. This would be similar to the proposed spreading mechanism of H3-K9 methylation mediated by Suv39h and HP1 proteins in pericentromeric heterochromatin [27Jenuwein T. Re-SET-ting heterochromatin by histone methyltransferases.Trends Cell Biol. 2001; 11: 266-273Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 28Lachner M. O'Carroll D. Rea S. Mechtler K. Jenuwein T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins.Nature. 2001; 410: 116-120Crossref PubMed Scopus (2073) Google Scholar, 29Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromodomain.Nature. 2001; 410: 120-124Crossref PubMed Scopus (2055) Google Scholar]. It should be noted that the progressive increase in the proportion of cells that score positive could also, in part, be attributable to asynchronicity in the onset of H3-K9 methylation in the differentiating cell population. Indeed, the frequency of cells with accumulated Xist RNA during early stages of ES cell differentiation indicates that this is likely to be the case (see Figure 3 and associated text below). Figure 3 summarizes our findings in the context of other characteristics of X chromosome inactivation. The initiating event, Xist RNA accumulation, begins at day 1.3 and reaches maximal levels by day 6 [20Sheardown S.A. Duthie S.M. Johnston C.M. Newall A.E.T. Formstone E.J. Arkell R.M. Nesterova T.B. Alghisi G.-C. Rastan S. Brockdorff N. Stabilisation of Xist RNA mediates initiation of X chromosome inactivation.Cell. 1997; 91: 99-107Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar]. The transition to asynchronous replication of the inactive X chromosome begins at day 2 of differentiation and reaches maximal levels by day 7. Transcriptional silencing of X-linked genes was also reported to occur between days 2 and 4 [19Keohane A.M. O'Neill L.P. Belyaev N.D. Lavender J.S. Turner B.M. X-Inactivation and histone H4 acetylation in embryonic stem cells.Dev. Biol. 1996; 180: 618-630Crossref PubMed Scopus (207) Google Scholar]. In this study, we observe accumulation of H3-K9 methylation at day 2, synchronous with the latter two events, increasing progressively throughout the differentiation time course. Hypoacetylation of histone H4 begins later at day 4 of differentiation and reaches maximal levels after day 7 [19Keohane A.M. O'Neill L.P. Belyaev N.D. Lavender J.S. Turner B.M. X-Inactivation and histone H4 acetylation in embryonic stem cells.Dev. Biol. 1996; 180: 618-630Crossref PubMed Scopus (207) Google Scholar]. Similar kinetics have been observed for hypoacetylation of H3 lysine 14 (H. Spotswood and B.M. Turner p.communication). Incorporation of macroH2A1.2 begins much later at day 7 and reaches a maximal level by day 9 [21Mermoud J.E. Costanzi C. Pehrson J.R. Brockdorff N. Histone macroH2A1.2 relocates to the inactive X chromosome after initiation and propagation of X inactivation.J. Cell Biol. 1999; 147: 1399-1408Crossref PubMed Scopus (132) Google Scholar]. Thus, there is a sequential and possibly interdependent relationship between different chromatin modifications on the inactive X. The frequency and kinetics of H3-K9 methylation of the inactive X closely mirrors the accumulation of Xist RNA observed in a previous study [20Sheardown S.A. Duthie S.M. Johnston C.M. Newall A.E.T. Formstone E.J. Arkell R.M. Nesterova T.B. Alghisi G.-C. Rastan S. Brockdorff N. Stabilisation of Xist RNA mediates initiation of X chromosome inactivation.Cell. 1997; 91: 99-107Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar]. This is shown in Figure 3 (bottom). The close correlation is consistent with a scenario whereby the enzyme(s) responsible for methylating H3-K9 on the inactive X chromosome may be directly recruited by Xist RNA. Based on prevailing models that propose that H3-K9 methylation stabilizes the hypoacetylated state [27Jenuwein T. Re-SET-ting heterochromatin by histone methyltransferases.Trends Cell Biol. 2001; 11: 266-273Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar], it is likely that hypoacetylation, specifically at H3-K9, occurs prior to methylation. Thus, Xist RNA may target a complex comprised of H3-K9 deacetylase and methyltransferase activities. It should be noted that the Suv39h histone methyltransferase enzymes which catalyze H3-K9 methylation of centromeric heterochromatin, are not involved in X inactivation and that there are as yet unidentified enzymes that carry out H3-K9 methylation of facultative heterochromatin [22Peters A.H.F.M. Mermoud J.E. O'Carroll D. Pagani M. Schweizer D. Brockdorff N. Jenuwein T. Histone H3 Lysine 9 methylation is an epigenetic imprint for facultative heterochromatin at the inactive X chromosome.Nat. Genet. 2001; 30 (Published online December 10, 2001. 10.1038/ng789): 77-80Crossref PubMed Scopus (388) Google Scholar, 24Peters A.H.F.M. O'Carroll D. Scherthan H. Mechtler K. Sauer S. Schofer C. Weipoltshammer K. Pagani M. Lachner M. Kohlmaier A. et al.Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability.Cell. 2001; 107: 323-337Abstract Full Text Full Text PDF PubMed Scopus (1265) Google Scholar, 30Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Regulation of chromatin structure by site-specific histone H3 methyltransferases.Nature. 2000; 406: 593-599Crossref PubMed Scopus (2047) Google Scholar]. Discovery of the enzyme(s) responsible for this activity should allow the biochemical interactions between Xist RNA and chromatin silencing components to be elucidated. We thank members of the X-Inactivation Group and Ros John for support and comments on the manuscript. This work was supported by the Medical Research Council, UK (J.E.M. and N.B.) and by the IMP through Boehringer Ingelheim and the Austrian Research Promotion Fund and the Vienna Economy Promotion Fund to T.J.