Distinct Contributions of Histone H3 Lysine 9 and 27 Methylation to Locus-Specific Stability of Polycomb Complexes  Leonie Ringrose, Heidi Ehret, Renato Paro  Molecular Cell  Volume 16, Issue 4, Pages 641-653 (November 2004) DOI: 10.1016/j.molcel.2004.10.015

Figure 1 Colocalization of Polycomb and Methylated Histones on Polytene Chromosomes (A–L) Double stainings with anti-PC antibody (red, [B], [F], and [J]) and either anti-H3K27Me2 at 1:2.5 dilution (C), H3K27Me3 at 1:75 dilution (G), or H3K9Me3 at 1:500 dilution (K) (green in all panels). Signals from PC and each methyl histone are merged in (D), (H), and (L). (M–Q) Each antibody was incubated with dot blots carrying peptides at different concentrations as indicated. H31-13 and H3 21-33 are unmethylated. (M), H3K27Me2, 1:50; (N), H3K27Me3, 1:1,500; (O), H3K9Me3, 1:10,000. Blots (N and O) were quantified (P and Q). Peptide bound (y axis) represents fraction of maximum binding. (P) K27Me3 antibody. Solid line, K27Me3 peptide; dotted line, K9Me3 peptide. (Q) K9Me3 antibody. Solid line, K9Me3 peptide; dotted line, K27Me3 peptide. Molecular Cell 2004 16, 641-653DOI: (10.1016/j.molcel.2004.10.015)

Figure 2 Summary of Polycomb and Trithorax Group Binding, Sites of H3K9 and H3K27 Trimethylation, and Gene Expression (A) All sites of PC binding and histone methylation (H3K27Me3 or H3K9Me3) were cytologically mapped using immunostained polytene chromosomes and are shown as colored bars above ideograms for each chromosome. Blue, H3K27Me3; yellow, H3K9Me3; solid red bars, PC; stippled red bars, other published PcG protein binding sites at which no PC signal is detected (PSC, PH, SU(Z)2); green, published binding sites for TRX and ASH1. Cytological map positions are shown below each chromosome arm. Thin vertical lines show predicted PREs from Ringrose et al. (2003). The top two rows of bars above the figure show summary of competition experiment (see Figure 6). Red bars, sites from which PC was not competed; gray bars, sites from which PC was lost upon peptide competition. Top row, H3K27Me3 peptide; second from top, H3K9Me3 peptide. (B) Number of bands mapped for PC (red) H3K9Me3 (yellow) and H3K27Me3 (blue). (C) Real-time PCR analysis of expression levels in salivary glands of genes associated with predicted PREs. The graph shows relative quantities in cDNA prepared from larval salivary glands of 38 transcripts compared to the level of Tubulin cDNA. Mean and standard deviation of three to six experiments are shown for each gene. PCR products after 32 cycles are shown below the graph. Gene names include cytological location for comparison to genome map (A). See also Figure 4 and legend. (D) Comparison of expression level with methylation status. Genes are divided into three classes based on percentage of Tubulin level as shown. For a given class, the proportion of genes with a given methylation status (none, one, or both of K9Me3 and K27Me3) is expressed as a percentage of the total number in that class. Expected distribution was calculated from methylation status of all PC bound sites (A). χ2 values were calculated using absolute numbers. For 2 degrees of freedom, the three methylation classes shown were considered. For 1 degree of freedom, two classes, with and without methylation, were considered. Molecular Cell 2004 16, 641-653DOI: (10.1016/j.molcel.2004.10.015)

Figure 3 H3K9 and K27 Methylation of a Transgene Carrying a PRE and Promoter from the Bithorax Complex (A–D) The endogenous Bithorax complex locus at 89E is shown (white arrowhead). Polytene chromosomes were stained for Pc ([A], red), H3K27Me3 ([B], blue), H3K9Me3 ([C], yellow), or DAPI. (D) H3K9Me3 from (C) is colored red and merged with DAPI. (E–N) The bxd14 transgene carrying the bxd PRE (positions 207,560–222,061 of the BX-C, accession number U31961) fused to the Ubx promoter driving a LacZ reporter gene (N), inserted at 62A on chromosome 3L, was analyzed for Pc binding (E–G), H3K27 methylation (H–J), and H3K9 methylation (K–M). In each case, the 62A region of a control strain lacking the transgene is shown (wt, [E], [H], and [K]) compared to the 85-39 strain that carries the bxd 14 transgene (F, I, and L). Merging with DAPI-stained chromosomes (G, J, and M) indicated an additional band at 62A in all cases (white arrowhead on [F], [G], [I], [J], [L], and [M]). Molecular Cell 2004 16, 641-653DOI: (10.1016/j.molcel.2004.10.015)

Figure 4 Polycomb Binding, Histone Methylation, and Gene Expression at Individual PREs (A–D) Enrichment of PC (A), H3K27Me3 (B), H3K9Me3 (C), and GAGA factor (D) on 1 kb fragments for predicted PREs was determined by chromatin IP from Drosophila Schneider cells. Similar results were obtained in at least three independent experiments for each antibody; the figure shows the mean and standard deviation of three experiments. Neg: mean enrichments for 43 control non-PRE fragments. Inset in each panel shows linear correlation coefficient of each data set with others as indicated, based on means. For the PREs of the BX-C, gene names are shown in italics. (E) Real-time PCR analysis of expression levels in Schneider cells, calculated as indicated in Figure 2 legend. PCR products after 32 cycles are shown below the graph: upper panel, Schneider cells; middle panel, embryos as control for primer function. Lower panel, −RT control (Schneider cells). Numbers below embryo panel indicate primer efficiencies relative to Tubulin, calculated as described in Supplemental Data. (F) RT-PCR products after 32 cycles for each transcript in SL2 cells (top), embryos (middle), and −RT controls (bottom). Molecular Cell 2004 16, 641-653DOI: (10.1016/j.molcel.2004.10.015)

Figure 5 Mapping of Polycomb Binding and Histone Methylation across a Section of the Bithorax Complex Enrichments of PC (A), H3K27Me3 (B), and H3K9Me3 (C) on the BX-C in SL2 cells determined by ChIP. The figure shows the mean and standard deviations of three independent experiments. Hybridizations of immunoprecipitated material to genomic DNA controls were used to set equivalent vertical scales in (A)–(C). The four sections of the BX-C analyzed are shown schematically above the figure. Slanted double lines indicate discontinuities in the sequences analyzed. Coordinates from U31961: iab-4, 118,576–146,018; iab-3-abdA, 150,921–153,965; bxd, 209,717–229,344; Ubx, 240,095–243,057. Gray boxes indicate published PREs or promoters. Solid black box: minimal 1.6 kb bxd PRE. The transcription start sites of the abdA and Ubx genes are indicated by arrows. Molecular Cell 2004 16, 641-653DOI: (10.1016/j.molcel.2004.10.015)

Figure 6 H3K27 and H3K9 Methylated Peptides Compete for Polycomb Binding (A–J) Salivary glands were dissected from third instar larvae, permeabilized, and incubated for 6 min in a 0.5 mM solution of each of the histone H3 peptides indicated in (J). Glands were then fixed, squashed, and double stained with anti-PC (red, [C], [F], and [I]) and with anti-Modulo (green, [B], [E], and [H]). DAPI staining is also shown (A, D, and G). For each peptide, between 25 and 61 chromosome spreads were identified that showed robust Modulo staining throughout the arms, and, of these, the percentage was calculated that showed partial loss of PC staining (J). Sites of PC loss upon H3K9Me3 or H3K27Me3 competition were cytologically mapped and are shown in Figure 2. (K) Comparison of competition with methylation status. Loci are divided into three classes based on response to competition with H3K9Me3 and H3K27Me3 peptides as indicated. For a given class, the proportion of genes with a given methylation status is expressed as a percentage of the total number in that class. Expected distribution was calculated from methylation status of all PC bound sites (Figure 2). χ2 values were calculated using absolute numbers. For 2 degrees of freedom, the three methylation classes shown were considered. For 1 degree of freedom, two classes, with and without methylation, were considered. (L) Comparison of competition with expression level, axes as for (K). Molecular Cell 2004 16, 641-653DOI: (10.1016/j.molcel.2004.10.015)

Figure 7 Mathematical Modeling of Peptide Competition (A) For input to mathematical models, the concentration of PC in salivary glands and Schneider (SL2) cells was measured by comparing known quantities of the purified antigen used to generate PC antibody, with known numbers of cells of each type on Western blots probed with PC antibody. 20 salivary glands are estimated to contain 2000 cells. Both lower and upper Pc bands seen in cells and salivary glands are specific: the lower band corresponds to the expected molecular weight of monomeric PC; the upper band appears to be a modified form. Quantification of blots from three independent salivary gland and cell preparations yielded 8.7 (± 3.2) × 10−4 ng PC per cell in salivary glands (= 1.45 [±0.53] × 107 molecules per cell) and 4.7 (±2.1) × 10−5 ng PC per cell in SL2 cells (= 7.8 [± 4.6] × 105 molecules per cell). (B and C) Schematic model for PC binding to methylated PREs. Gray circles, PC; yellow triangles, H3 tails trimethylated at K9; blue circles, H3 tails trimethylated at K27; red circles, other proteins bound directly to PRE, e.g., GAGA, Zeste, PHO. Forward (kn) and backward (k − n) rate constants for each interaction are shown. Black arrows, constants derived from measured binding data (see main text and Supplemental Data); red arrows, unknown constants for which different values are explored in (D)–(I). (C) Higher order complexes between PC bound to chromatin via K27 methylated tails, or PRE bound proteins. (D–I) Simulations of binding and peptide competition using the model shown in (B) and (C). Vertical scale shows the absolute number of PC molecules that are bound to the PRE, ×103; thus, approximately 10,000 molecules are bound at equilibrium in (D). At 5 min (red arrow), the effects of addition of 0.5 mM of either K9Me3 (dashed line) or K27Me3 peptide (solid line) were examined. Molecular Cell 2004 16, 641-653DOI: (10.1016/j.molcel.2004.10.015)