In the <i>set1_sid</i> mutant, Spp1 is still important to maintain H3K4me3 levels.

<p>(A) Histone H3K4 methylation levels in vegetatively growing cells detected by Western blot. Anti-Spp1, anti H3K4me2, anti-H3K4me3 or anti-Pgk1 antibodies were used, as indicated. A representative experiment is shown. WT: ORT4601; <i>set1∆</i>: ORT4784; <i>spp1∆</i>: VBH152; <i>set1_sid</i>: VBH1881; <i>set1_sid spp1∆</i>: VBH1972; <i>spp1W45A</i>: VBH1419; <i>set1_sid spp1W45A</i>: VBH2021. The bar graph on the right indicates histone modification levels normalized to Pgk1 levels and relative to the WT strain. Values are the mean ± S.E.M. of the normalized relative levels from 3 to 6 replicates for each strain, except for <i>spp1W45A</i>, where only 2 replicates are available and the error bars indicate the range. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.s012" target="_blank">S4 Table</a>. (B) Histone H3K4me3 levels in vegetatively growing cells detected by ChIP at the highly transcribed <i>ADH1</i> gene. Same strains as in A. Values are expressed as % of input DNA, and are the mean ± S.E.M. of six independent experiments.</p

MOESM4 of Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

Additional file 4. Details of the SRM transitions for each signature peptide. SRM assay parameters including precursor and fragment ion type, charge state, elution time as well as raw data are provided in Suppl. data. (*) Indicates peptides monitored only in their endogenous form

MOESM5 of Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

Additional file 5. Composition of the mixture of standard peptides

Spp1 interacts with the Set1 complex and the Mer2 DSB protein in meiotic cells.

<p>(A) ChIP-qPCR of Spp1-TAP during meiosis, showing its association with the chromosome axis at 3–4 hr, the expected time for DSB formation. Strain: VBD1266. Sites used for negative control (<i>NFT1</i> gene) and axis (chr 3, nt 232942 to 233010) are the same in all figures. (B) Silver-stained gel of TAP eluates performed at 3.5 hr in meiosis in untagged (ORD7339) or Spp1-TAP (VBD1266). (C) Mass spectrometry analysis of proteins pulled down by Spp1-TAP in meiosis (t = 3.5 hr). <i>SPP1-TAP</i>: VBD1266 –no tag: ORD7339. The volcano plot indicates in red (Mer2) and in blue (Set1 complex subunits) the proteins that significantly co-purify with Spp1 (log2(Fold change)>3 and log10(p-value)<4). The experiment was done in three biological replicates, and average Fold Change values over the untagged control are presented with their corresponding p-value (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#sec015" target="_blank">Methods</a>).</p

Spp1 binds distinct Set1- and Mer2-dependent sites in meiotic cells.

<p>(A) Comparison of Spp1 binding profile with that of RNA polymerase II, in vegetative cells, or in meiosis (t = 3 hr), in wild-type or <i>mer2∆</i> mutant. Spp1-Myc and RNA pol II binding data are from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.ref018" target="_blank">18</a>]. Spp1-Myc <i>mer2∆</i>: VBD1220. ChIPchip (Chromatin immunoprecipitation on chip) profiles are shown for chromosome 10. Centromeres are indicated by green dots, and ratios are plotted after smoothing with a 1 kb window. (B) Mean mRNA levels at the 100 strongest Spp1 peaks or in the whole genome. Spp1 peaks were determined from the experiments shown in A (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#sec015" target="_blank">Methods</a>). mRNA levels are from published data in SK1 diploid vegetative cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.ref057" target="_blank">57</a>] (upper panel) or SK1 meiotic cells at t = 4 hr [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.ref058" target="_blank">58</a>] (lower panel). Error bars: S.E.M. (C) Spp1 and Rec8 binding profile in <i>SET1</i> or <i>set1∆</i> cells in meiosis (t = 3 hr). Spp1-Myc <i>set1∆</i>: VBD1209. <i>SET1</i> Spp1-Myc and Rec8 binding data are from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.ref018" target="_blank">18</a>]. ChIPchip profiles are shown for chromosome 10. Centromere is indicated as a green dot, and ratios are plotted after smoothing with a 1 kb window. (D) qPCR analysis of Spp1 binding to an axis site and two highly transcribed genes in meiosis, <i>ACS1</i> and <i>CIT2</i> (t = 3 hr). No tag: ORD7339; WT: VBD1187; <i>mer2∆</i>: VBD1220; <i>set1∆</i>: VBD1209. Each dot represents a biological replicate and the bar indicates the mean.</p

MOESM9 of Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

Additional file 9. Rules used to select or reject peptides using their transition profiles. The validation of the best transitions was performed using a signal-to-noise ratio (> 5) and a perfect co-elution of the heavy standard peptide with the endogenous peptide. Three fragment ions (F1, F2, and F3) are represented for the heavy and the endogenous peptides. a All fragment ions can be integrated because the heavy and endogenous fragment ions co-elute in the same intensity order. b In that case, only F2 can be integrated because the ratio heavy/endogenous is different for F1 and F3. c The fragment F2 is contaminated by another analyte eluting at a slightly later time; it has to be excluded from the analysis. d Here, the signal-to-noise ratio is below five, no fragment ion can be integrated. e. The endogenous peptide traces do not co-elute with the heavy peptide traces

A <i>mer2</i> point mutant affected for its interaction with Spp1 mimics the meiotic phenotype of <i>spp1∆</i>.

<p>(A) Scheme of the Mer2 protein, with its predicted coil-coil structures (Hhpred) and the Spp1 interacting domain (165–232) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.ref017" target="_blank">17</a>]. Below is the Mer2 protein sequence in the predicted coiled-coiled heptad structure. Buried positions required for coiled-coil pairing are underlined. Amino acids conserved and predicted to be surface-exposed are colored. The <i>mer2_sid</i> mutation (V195D) is indicated by an arrow. (B) 2-hybrid assays for the interaction between Mer2 and Spp1. Growth on the–His medium indicates an interaction between the two tested proteins. (C) Association of Mer2 and Mer2_sid mutant proteins with chromosome axis during meiosis (t = 3 hr) monitored by ChIP. No tag: ORD7339; Mer2-Flag: VBD1251; Mer2_sid Flag: VBD1843. Each dot represents a biological replicate and the bar indicates the mean. (D) Interaction of Spp1 with Mer2 and the Set1 complex subunit Swd1 in meiotic cells examined by Western blot. Proteins pulled down by Spp1-TAP were released in the eluate after Tev cleavage of the Tap tag. Anti-HA, anti-Flag and anti-TAP antibodies were used as indicated. <i>MER2-FLAG SPP1-TAP SWD1-HA</i>: VBD1745; <i>mer2_sid-FLAG SPP1-TAP SWD1-HA</i>: VBD1852; <i>SPP1-TAP SWD1-HA</i>: VBD1742. (E) Association of Spp1 during meiosis (t = 3 hr) monitored by ChIP. No tag: ORD7339; WT: VBD1187; <i>mer2∆</i>: VBD1220; <i>mer2_sid</i>: VBD1924. Each dot represents a biological replicate and the bar indicates the mean. (F) Meiotic DSB formation monitored in <i>dmc1∆</i> cells by Southern blot at <i>CYS3</i> and <i>DEP1</i> DSB (upper panel), or at the <i>spp1∆</i>-specific <i>PES4</i> DSB. DSB sites are indicated by arrows. WT: ORD7354; <i>mer2_sid</i>: VBD1879; <i>spp1∆</i>: VBD1748. Graph shows the DSB quantification relative to the level in WT (for <i>CYS3</i>, <i>DEP1</i>) or <i>spp1∆</i> cells (for <i>PES4</i> site). DSB were quantified at the 5 hr time point. Each dot represents a biological replicate and the bar indicates the mean. (G) Meiotic progression as assessed by DAPI staining of strains with the indicated genotype. WT: ORD7339; <i>mer2_sid</i>: VBD1880; <i>spp1∆</i>: VBD1769; <i>spo11Y135F</i>: VBD1291; <i>spo11Y135F spp1∆</i>: VBD1233. Representative experiments are shown. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.s008" target="_blank">S8 Fig</a>.</p

Illustration of the different functions of Spp1 for H3K4me3 and meiotic DSB formation.

<p>1) in the Set1 complex, Spp1 has a role to allow catalysis of H3K4 trimethylation by Set1, but this function is not essential, since the <i>set1-sid</i> mutant still maintains high levels of H3K4me3. 2) In addition, Spp1 maintains H3K4me3 levels, not by stimulating Set1 catalytic activity, but likely by binding H3K4me3 with its PHD finger. This can take place without interaction with the Set1 complex. We propose this may protect H3K4me3 from active demethylation, by the Jhd2 enzyme. Other possible explanations are described in the text. 3) Finally, the simultaneous binding of Spp1 to H3K4me3 and to the axis-associated Mer2 protein is essential to promote efficient DSB formation by Spo11. It has to be noted that a PHD finger mutant of Spp1 (W45A) is still able to bind Mer2 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.ref018" target="_blank">18</a>], so recognition of H3K4me3 by Spp1 PHD finger is not a prerequisite for its subsequent binding to Mer2. NDR: nucleosome-depleted region; Black circle: first nucleosome of genes; H3R2: arginine 2 of histone H3, in its non-asymmetrically methylated form. The blue square represents H3K4me3.</p

Set1 complex Set1 and Swd1 subunits associate in meiosis with highly transcribed genes, but not with chromosome axis sites.

<p>(A) ChIP-qPCR of Set1, Swd1 and Spp1 during meiosis (t = 3 hr) comparing their association with axis and two highly transcribed genes. HA-Set1: VBD1378; Swd1-HA: VBD1399; Spp1-Myc: VBD1187. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.s002" target="_blank">S2 Fig</a>. Each dot represents a biological replicate and the bar indicates the mean. (B) Chromosome profiles of Swd1 and RNA Pol II binding in meiosis (t = 3 hr). RNA Pol II: data are from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.ref018" target="_blank">18</a>]. Swd1: strain VBD1399 at t = 3 hr. ChIPchip profiles are shown for chromosome 10. Centromere is indicated as a green dot, and ratios are plotted after smoothing with a 1 kb window. (C) ChIPchip signal at the indicated features. The mean signal at the 200 strongest Red1 (axis) and DSB peaks (DSB) is represented, as defined in Methods and in ref [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.ref056" target="_blank">56</a>]. Red1 and DSB data are from previously published studies [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.ref023" target="_blank">23</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.ref059" target="_blank">59</a>]. Ratios after smoothing with a 2 kb window are plotted. Boxplots indicate median (line), 25th–75th percentile (box) 61.5 times the interquartile range (whiskers). Non-overlapping notches of two boxes are indicative that medians are statistically different. (D) mean mRNA levels at the 100 strongest Swd1 peaks or in the whole genome. mRNA levels are from SK1 diploid meiotic cells at t = 4 hr [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007223#pgen.1007223.ref058" target="_blank">58</a>]. Error bars: S.E.M. (E) Co-immunoprecipitation by the core Set1 complex Swd1 protein from cells at 3.5 hr in meiosis analyzed by western blot using anti-HA, anti-Myc or anti-Flag antibody as indicated. Swd1-HA Spp1-Myc Mer2-Flag: VBD1401; Spp1-Myc Mer2-Flag: VBD1395. IP: immunoprecipitation. The asterisk indicates non-specific cross-hybridizing band. (F) Co-immunoprecipitation by the DSB protein Mer2 from cells at 3.5 hr in meiosis. Same antibodies as in (E). Swd1-HA Spp1-Myc Mer2-Flag: VBD1401; Spp1-Myc Swd1-HA: VBD1400. The asterisks indicates non-specific cross-hybridizing bands unrelated to Swd1-HA.</p

Bdf1 is required for the transcription of <i>NDT80</i> and middle sporulation genes.

<p>(A) List of over-represented transcription factors with binding sites in the promoters of the genes which are differentially downregulated at 4h and 8h when Bdf1 bromodomains are mutated (fold change < -2, adjusted p value < 0.05). A transcription factor was considered significantly over-represented when the p-value describing its enrichment was below 0.001. On the right, the normalized counts present the expression levels for <i>NDT80</i>, <i>CUP9</i>, <i>CUP2</i>, <i>FKH1</i> and <i>ROX1</i>. (B) Pie chart representing the proportion of genes which are downregulated in the <i>bdf1-Y187F-Y354F</i> strain at 4 and 8h of sporulation induction (833 genes in total) and with putative Ndt80 binding sites in their promoter (531 genes, their list is available in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006541#pgen.1006541.s012" target="_blank">S5 Table</a>). (C) ROC-AUC plot representing the enrichment of the Ndt80 binding sequence in the promoter of the genes downregulated in the <i>bdf1-Y187F-Y354F</i> mutant strain. (D) Venn diagram illustrating the overlap between genes differentially downregulated in <i>bdf1</i> bromodomain mutant at 4 and 8h of sporulation induction (833 genes) and genes previously identified as Ndt80-regulated in Chu <i>et al</i>. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006541#pgen.1006541.ref035" target="_blank">35</a>]. (E) Sporulation efficiency upon ectopic expression of <i>NDT80</i> in WT and <i>bdf1-Y187F-Y354F</i> strains during sporulation. Here, the endogenous <i>NDT80</i> promoter was replaced by an oestrogen-dependent promoter and its expression activated by the addition of oestradiol 6 hours after sporulation induction [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006541#pgen.1006541.ref044" target="_blank">44</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006541#pgen.1006541.ref045" target="_blank">45</a>]. (F) Bdf1 chromatin immunoprecipitation. Bdf1 occupancy was monitored in the meiotic-specific promoters of the early genes <i>IME1</i> and <i>IME2</i> and middle genes <i>AMA1</i> and <i>NDT80</i> during sporulation. Mutation of Bdf1 bromodomains impairs the recruitment of Bdf1 to chromatin. IntV is an intergenic and transcriptionally inactive region of chromosome V between coordinates 9,762 and 9,812. (G) Sporulation efficiency when Bdf1 bromodomain mutations are combined with Sum1 and Hst1 deletions, two known repressors of <i>NDT80</i> activation [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006541#pgen.1006541.ref041" target="_blank">41</a>].</p