MtDNA profile of the

<p>Δ<b><i>Lon1</i></b><b> strain.</b> MtDNA profiles were determined by <i>Hae</i>III digestion of total DNA extracted from mycelium for the indicated dying strains. <i>Hae</i>III digestion of mtDNA from a young wild-type mycelium was used as a control. The arrows indicate the senDNAα multimeric subgenomic molecules present in dying strains (right and left panels). The identity of the senDNAα molecules was assessed by Southern blot analysis using an intron α specific probe (right panel). In addition to senDNAα (2.5 kb), hybridization revealed two <i>Hae</i>III fragments on the intact mitochondrial genome (1.9 and 0.8 kb). Note that these two bands were only detected in young wild-type (Control) and dying Δ<i>Lon1</i> strains.</p

Mitochondrial proteolytic activity of <i>PaLon1</i> mutants.

<p>Values represent the average ratio of mutant/wild type ± standard deviation of three independent experiments. Three incubation times were used for each experiment.</p

The mitochondrial PaLON1 protein.

<p>(A) Schematic representation of the PaLON1 protein outlining the three domains present in both prokaryotes and eukaryotes. The N-domain, which is the most divergent domain between Lon proteins, is followed by the highly conserved ATPase and protease domains. Within the N-domain, the most conserved region is within the C-terminal part (hatched). The line referring to residues 382 to 619 indicates the part of the protein presented in (B). Diamond (S423L), point (L430P), and inverted triangles (Δ514–567) mark changes induced by <i>PaLon1-31</i>, <i>PaLon1-1</i> and <i>PaLon1-f</i>, respectively. (B). Primary sequence and secondary structure of the C-terminal part of the N-domain of <i>B. subtilis</i>, <i>E. coli,</i> and <i>P. anserina</i> Lon proteases. Sequences were aligned using the Clustal W program. Conserved amino acids are boxed in black (identical) and gray (similar). For the <i>P. anserina</i> sequence (PODAN), changes induced by <i>PaLon1</i> mutations are represented by the same symbols as in (A). The GenBank accession numbers for <i>B. subtilis</i> (BACSU) and <i>E. coli</i> (ESCCO) proteins are CAA99540.1 and AAC36871.1, respectively. The Walker A motif of the central ATPase domain is boxed and begins at position 607, 356, and 354 in <i>P. anserina</i>, <i>E. coli</i> and <i>B. subtilis</i> proteins, respectively. The predicted consensus secondary structure of the PaLON1 region was determined on the <a href="mailto:NPS@" target="_blank">NPS@</a> Web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038138#pone.0038138-Combet1" target="_blank">[42]</a> using a combination of available methods. For the same region, the secondary structure information available for <i>E. coli</i> and <i>B. subtilis</i> proteins ends at residue 245 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038138#pone.0038138-Li2" target="_blank">[32]</a> or contains a gap of 36 amino acids (dotted line), respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038138#pone.0038138-Duman1" target="_blank">[33]</a>. For the <i>B. subtilis</i> protein, structure information was not available after the last α helix just before the Walker A motif. Secondary structures are indicated above each sequence as follows: lines, α helices; c letter, random coil (no secondary structure); and question mark (?), ambiguous state.</p

Phenotypic characteristics of <i>PaLon1</i> mutants.

<p>(A) Mycelium phenotype of <i>PaLon1-1</i> germinating ascospores on germination medium at 27°C after 2 days of growth. A cross between <i>PaLon1-1</i> and the wild-type strain gave rise to a progeny of <i>PaLon1-1</i> germinating ascospores with a less dense mycelium (letters: b, c, e, f, h, i, k) than that of the wild type (letters: a, d, g, j, l). (B) Growth phenotype exhibited by the <i>PaLon1</i> mutants. Strains were grown on M2 standard medium for 2 days (27°C and 36°C), 3 days (18°C), or 7 days (11°C). The genotype of each strain is shown in the table, except for the <i>rmp1-1</i> (<i>mat</i>−) and <i>rmp1-2</i> (<i>mat</i>+) alleles that are represented by a gray and white tone, respectively. (C) DASPMI staining of mitochondria. Mitochondria of growing strains (2 days at 27°C on M2) were stained with DASPMI, a vital mitochondrion-specific dye. For each indicated strain, filaments were gently mixed with a drop of DASPMI (25 mg/ml) directly on microscope slides and observed immediately with a fluorescence microscope (450–490/500–550 nm). All panels are at the same magnification and the scale bar corresponds to 5 μm. (D) Hydrogen peroxide (H₂O₂) sensitivity. Nine subcultures of each strain were inoculated in M2 or M2 supplemented with 0.005% (1.47 mM) or 0.01% (2.94 mM) H₂O₂. Growth (cm) was determined by measuring the radius of each thallus after 2 days at 27°C in the dark. Error bars indicate standard deviation. The statistically significant increase in the sensitivity of the <i>PaLon1</i>-f and Δ<i>Lon1</i> strains to 2.94 mM H₂O₂ is marked by an asterisk. In each case, the <i>p</i>-value (0.001) is below 0.05, as determined by the Mann-Whitney test.</p

Detection of PaLON1 mutant proteins.

<p>Mitochondrial extracts (70 μg) purified from the indicated strains were resolved on an SDS-polyacrylamide gel and subjected to immunoblotting. The PaLON1 protein and the β-subunit of mitochondrial ATPase were detected by a <i>P. anserina</i> anti-PaLON antibody and an <i>S. cerevisiae</i> anti-Atp2 antibody, respectively. Mitochondrial extraction and western blotting were repeated at least twice for each strain. The left and right panels correspond to two independent membranes.</p

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

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

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