1. Volume 12, Issue 6, Pages 1577-1589 (December 2003)
Partitioning and Plasticity of Repressive Histone Methylation States in Mammalian Chromatin 
Antoine H.F.M. Peters, Stefan Kubicek, Karl Mechtler, Roderick J. O'Sullivan, Alwin A.H.A. Derijck, Laura Perez-Burgos, Alexander Kohlmaier, Susanne Opravil, Makoto Tachibana, Yoichi Shinkai, Joost H.A. Martens, Thomas Jenuwein 
Molecular Cell 
Volume 12, Issue 6, Pages (December 2003)
DOI: /S (03)

2. Figure 1
Protein Blot Analysis of In Vivo Histone Lysine Methylation States
(A) Schematic representation of currently known methylatable lysine positions in the murine histone H3 amino terminus highlighting the common -ARKS- motif around the H3-K9 and H3-K27 positions. The histone-fold domain containing the methylatable H3-K79 residue is indicated by two brackets. The 2× branched 11- and 12-mer peptide antigens against which the H3-K9 and H3-K27 methylation-specific antibodies were raised are shown.
(B) Protein blot analyses of nuclear extracts (0.75 μg, 3 μg, and 12 μg) from wt, Suv39h dn, and G9a-deficient ES cells with H3-K9 (top panels) and H3-K27 (middle panels) methylation-specific antibodies. Nuclear extracts were calibrated using H3 and H4 modification-insensitive antibodies (bottom panels).
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
Quantitative Mass Spectrometry of H3-K9 and H3-K27 Methylation States
(A) Principle of the propionylation procedure. Schematic diagram of the murine H3 N terminus showing a hypothetical combination of several in vivo lysine modifications. Other lysine residues that are known to carry in vivo methyl or acetyl marks are indicated by a dot. In untreated samples, trypsin cleaves proteins after arginine residues and after lysine residues that are either unmodified or monomethylated (but not di- or trimethylated or acetylated), resulting in a population of peptides with variable lengths. In vitro propionylation of unmodified or monomethylated lysine residues prevents tryptic cleavage after such lysine residues. As a consequence, H3 proteins are more uniformly cleaved by trypsin after each arginine, thereby enabling a quantitative analysis of methylation states with comparable peptide fragments.
(B) Comparative analysis of H3-K9 mono-, di-, and trimethylation levels in H3 (aa 9–17) peptide fragments isolated from wt, Suv39h dn, and G9a-deficient ES cells. The relative level of a particular H3-K9 methylation state is indicated with respect to the sum of all H3-K9 modifications present in the H3 (aa 9–17) peptide population. Levels were normalized for different ionization potentials as determined by measuring the relative recovery of known amounts of chemically synthesized and modified peptide standards. For wt, Suv39h dn, and G9a-deficient samples, we analyzed the H3-K9 methylation levels in two cell lines in duplicate. For G9a+/+ ES cells, we analyzed one cell line in duplicate.
(C) Comparative analysis of H3-K27 mono-, di-, and trimethylation levels in H3 (aa 27–40) peptides isolated from Suv39h dn, G9a-deficient, and their wt control ES cells. Relative quantifications were performed as described in (B).
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 H3-K9 and H3-K27 Methylation States in Interphase Chromatin
(A and B) Indirect immunofluorescence of interphase chromatin from wt, Suv39h dn and G9a-deficient male ES cells with H3-K9 (A) and H3-K27 (B) methylation-specific antibodies. In Suv39h dn cells, arrows indicate altered H3-K9 monomethylation and H3-K27 trimethylation at pericentric heterochromatin.
(C) Statistical evaluation of the relative distribution of H3-K9 and H3-K27 mono-, di-, and trimethylation states at pericentric heterochromatin versus euchromatin in wt and mutant ES nuclei. For example, in wt ES cells the signals of H3-K9 monomethylation at pericentric heterochromatin are underrepresented (hypomethylated) relative to euchromatin in approximately 80% of nuclei. In the remaining 20% of cells, pericentric and euchromatic domains display similar degrees of staining. In contrast, 60% of Suv39h dn nuclei show comparable H3-K9 monomethylation levels at pericentric heterochromatin and euchromatin while 40% of these nuclei have enriched signals at pericentric heterochromatin (hypermethylated). For Suv39h wt and dn and for G9a-deficient ES cells, we analyzed 200–300 nuclei from two independent cell lines. For G9a+/+ ES cells, we scored around 150 cells from one cell line. Staining patterns were analyzed by epifluorescence microscopy.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 H3-K9 and H3-K27 Methylation Patterns on Mitotic Chromosomes
Indirect immunofluorescence of mitotic chromosomes prepared from wt and mutant ES cells with H3-K9 (A) and H3-K27 (B) methylation-specific antibodies. In G9a-deficient cells, arrowheads point at chromosomes with H3-K27 trimethylated pericentric heterochromatin.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
ChIP Analysis for H3-K9 and H3-K27 Methylation States at Constitutive Heterochromatin
(A) Schematic diagram of a mitotic mouse chromosome illustrating position and organization of major (pericentric) and minor (centromeric) satellite repeats. Primers used to amplify the repeat sequences and corresponding PCR products are indicated.
(B and C) Occupancy of major (B) and minor (C) satellite chromatin by H3-K9 and H3-K27 mono-, di-, and trimethylation in wild-type, Suv39h dn, G9a+/+, and G9a-deficient ES cells as assessed by chromatin immunoprecipitation. The final DNA extractions were amplified both by standard PCR (gel images in top panels) and by real-time PCR (bottom panels). For Suv39h wt and dn and for G9a-deficient ES cells, we analyzed histone lysine methylation levels in two cell lines in duplicate. For G9a+/+ ES cells, we analyzed one cell line in triplicate.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6 Partitioning of the Genome by Histone Lysine Methylation
Closed rectangles represent classically defined chromatin domains such as euchromatin, facultative heterochromatin (the inactive X chromosome, Xi), and constitutive (pericentric) heterochromatin. In euchromatin, G9a appears as the major HMTase directing H3-K9 mono- and dimethylation. In addition, all three H3-K27 methylation states are broadly present. At the Xi, Xist mediates H3-K27 trimethylation presumably via recruitment of the Ezh2/Eed complex. H3-K9 dimethylation can also be detected. Pericentric heterochromatin is characterized by Suv39h-dependent H3-K9 trimethylation and by Suv39h-independent H3-K27 monomethylation. A transient H3-K9 monomethylation state is predicted to function as a substrate for the Suv39h HMTases. In Suv39h dn ES cells (dashed rectangle), there is a conversion of pericentric methylation marks toward H3-K9 mono- and H3-K27 trimethylation—a signature that partially resembles facultative heterochromatin. The accumulation of H3-K27 trimethylation may maintain the silent state and rescue the biological functions of compromised heterochromatin in Suv39h dn cells. Constitutive heterochromatin is depicted by more compact nucleosomal arrays, indicating that in addition to H3-K9 trimethylation a distinct higher-order structure may be present at this chromosomal domain (Maison et al., 2002).
Molecular Cell  , DOI: ( /S (03) )