Volume 19, Issue 6, Pages 849-856 (September 2005) Molecular Regulation of Histone H3 Trimethylation by COMPASS and the Regulation of Gene Expression  Jessica Schneider, Adam Wood, Jung-Shin Lee, Rebecca Schuster, Jeff Dueker, Courtney Maguire, Selene K. Swanson, Laurence Florens, Michael P. Washburn, Ali Shilatifard  Molecular Cell  Volume 19, Issue 6, Pages 849-856 (September 2005) DOI: 10.1016/j.molcel.2005.07.024 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Multiple Subunits of COMPASS Are Required for Histone H3 Lysine 4 Trimethylation (A and B) Extracts were made from each of the nonessential gene deletion mutants of the S. cerevisiae genome. In GPS, the extracts from the collection were resolved by SDS-PAGE and tested for the presence of dimethylated (A) or trimethylated (B) lysine 4 of histone H3. Red arrows at positions A2 and H3 indicate empty wells as place markers. Blue arrows indicate the position of the CPS60 deletion mutant at plate location B10. (C and D) Dimethylated (C) or trimethylated (D) lysine 4 of histone H3. Blue arrows indicate the position of the CPS40 deletion mutant at plate location B9. (E) The level of mono-, di-, and trimethylated H3K4 as the result of deletion of the subunits of COMPASS. Extracts deleted for each of the components of COMPASS were subjected to SDS-PAGE and tested for the presence of monomethylated, dimethylated, or trimethylated lysine 4 of histone H3. The extracts were probed with an antibody against actin to serve as a loading control. Molecular Cell 2005 19, 849-856DOI: (10.1016/j.molcel.2005.07.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Upstream Regulators of COMPASS Activity Affect Di- and Trimethylation of Histone H3 (A) A titration analysis was performed by the application of extracts from null strains for either cps40 or cps60. Each extract was analyzed by its application to SDS-PAGE and tested for the presence of modified H3K4 with appropriate antibodies. As load controls, antibodies to either unmodified or modified histone H3 and actin were also used. (B) The ratio of each modified histone over its appropriate load control as described in the Experimental Procedures is shown in a bar graph. (C) Extracts from strains lacking Bre1 and Rad6, as well as a strain containing a point mutant in histone H2B (K123R) were resolved by SDS-PAGE and tested for the presence of mono-, di-, and trimethylated lysine 4 of histone H3. The extracts were probed with an antibody against unmodified histone H3 and actin to serve as loading controls. (D) Extracts with deletions for the Leo1, Paf1, and Rtf1 subunits of the Paf1 complex were subjected to SDS-PAGE and tested for the presence of mono-, di-, and trimethylated lysine 4 of histone H3. Molecular Cell 2005 19, 849-856DOI: (10.1016/j.molcel.2005.07.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 COMPASS’s Stability Is Not Affected in the Absence of Cps40 and Cps60 (A and B) COMPASS composition is stable in the absence of Cps60 and Cps40. (A) Extracts from either wild-type (wt) strains carrying a Set1::HA tag or the same parental strains with CPS60 deleted were applied to size-exclusion chromatography. The elution of Set1::HA was monitored by Western analysis with antibody specific for the HA tag. (B) Determination of subunit stoichiometry within COMPASS based on tandem mass spectrometry in the presence or absence of Cps40. The number of MS/MS spectra matching peptides unique to COMPASS subunits was divided by each subunit molecular weight and scaled up by an arbitrary factor of 500,000 to define a spectral abundance factor (SAF). To compare independently acquired datasets, SAFs were normalized to the total spectral count for the entire complex (normalized SAF, NSAF). Average NSAF values and standard deviations were calculated for subunits detected in Cps60-TAP pull-downs digested with trypsin and independently analyzed by MudPIT. (C–E) Recruitment of Set1 to chromatin is not reduced by the loss of Cps60. (C) Extracts were prepared from Set1::HA, and Set1::HA Δcps60 strains were tested for the presence of trimethylated lysine 4. Because we and others previously demonstrated that Set1/COMPASS is associated with the early elongating RNA polymerase II (Krogan et al., 2003; Ng et al., 2003), the same strains as in (A) and (C) were used for analysis of the localization of Set1 to the promoter or early body of the PMA1 gene (D) and ADH1 gene (E). Primers for PMA1 or ADH1 were used to amplify the immunoprecipitated DNA. The primer sets used are graphically represented in the lower panel and span base pairs 165–372 of the PMA1 gene and −235 to −13 of the ADH1 gene. Molecular Cell 2005 19, 849-856DOI: (10.1016/j.molcel.2005.07.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Analysis of Histone Methyltransferase Activity of COMPASS In Vitro (A) Purified COMPASS from wt or cps60 null strains were analyzed for histone methyltransferase activity toward histone H3 in vitro. The levels of methylated H3K4 were tested by the subjection of the reaction mixtures to SDS-PAGE and Western blotting with appropriate antibodies. An asterisk indicates nonspecific signal obtained by the H3K4 mono- and dimethyl antibody. (B) To determine the direct in vivo role of Cps60 in histone H3K4 trimethylation, a complementation experiment was performed by cloning the DNA encoding the CPS60 gene under the Gal promoter and introducing it into a cps60 null strain. The level of histone methylation was tested in the presence or absence of Cps60. (C) Mono- and dimethylation of histone H3, but not trimethylation, appear to be required for telomeric silencing. Each of the subunits of COMPASS was deleted in a wt strain containing URA3, a reporter of telomeric gene silencing, inserted into the telomere of chromosome 7. The mutants were scored for growth in the presence of 5-FOA. Wt cells silence expression of the telomere-associated URA3 gene and are resistant to 5-FOA. Cells defective for telomeric gene silencing have increased expression of URA3 and are sensitive to 5-FOA. Molecular Cell 2005 19, 849-856DOI: (10.1016/j.molcel.2005.07.024) Copyright © 2005 Elsevier Inc. Terms and Conditions