Dot1 is required for <i>zip1</i>-induced localization and activation of the Hop1 meiotic checkpoint adaptor.

<p>(A) Immunofluorescence of meiotic chromosome spreads stained with DAPI (blue), anti-Hop1 (green) and anti-myc (red) antibodies. Representative nuclei are shown. The same exposure time was used to capture the signal from the different strains. Spreads were prepared 24 h after meiotic induction of <i>ndt80</i> cells. Strains are: DP848 (<i>zip1</i>) and DP849 (<i>zip1 dot1</i>). (B) Quantification of the Hop1 staining pattern (left) and the number of Mek1 foci (right) on spread chromosomes analyzed as in (A). 14 and 21 nuclei were scored for <i>zip1</i> and <i>zip1 dot1</i>, respectively. (C) Representative images of <i>ndt80</i>-arrested cells expressing <i>HOP1-GFP</i> in <i>zip1</i> (DP964) and <i>zip1 dot1</i> (DP965) captured after 24 h in meiosis. (D) Quantification of the Hop1-GFP signal intensity on fluorescence images (a.u., arbitrary units). 300 individual nuclei were analyzed for each strain. (E) Dot1 is required for Hop1 phosphorylation. Western blot analysis of Hop1 in cell extracts obtained 24 h after meiotic induction in <i>ndt80</i> cells. The middle panel corresponds to an overexposure (OEx) of the blot shown in the upper panel. PGK was used as a loading control. Strains are: DP428 (<i>zip1</i>), DP655 (<i>zip1 dot1</i>), DP680 (<i>zip1 mec1</i>) and DP674 (<i>zip1 mek1</i>). Means, standard deviations and <i>P-</i>values are shown in (B) and (D).</p

The <i>H3-K79R</i> and <i>H3-K79A</i> mutations recapitulate <i>dot1</i> defects in checkpoint-induced Mek1 and Hop1 phosphorylation and localization.

<p>(A) Immunofluorescence of meiotic chromosome spreads stained with DAPI (blue), anti-Hop1 (green) and anti-GFP (red) antibodies. Representative nuclei are shown. The same exposure time was used to capture the signal from the different strains. (B) and (C) Representative images of meiotic cells expressing <i>MEK1-GFP</i> and <i>HOP1-GFP</i>, respectively. The scattered plots represent the quantification of the Mek1-GFP focal signal (B) and total Hop1-GFP signal intensity (C) on fluorescence images (a.u., arbitrary units). Error bars represent the median with interquartile range. 300 individual nuclei were analyzed for each strain. (D) Western blot analysis of <i>zip1</i>-induced Mek1-GFP phosphorylation in a Phos-tag gel using anti-Mek1 antibodies. The basal Mek1-GFP form (line), and the forms resulting from Mec1/Tel1-dependent phosphorylation (black arrowhead) and autophosphorylation (white arrowheads) are indicated. Ponceau S staining of the membrane is shown as a loading control. (E) Western blot analysis of <i>zip1</i>-induced Hop1-GFP phosphorylation using anti-GFP antibodies. Tubulin is shown as a loading control. Strains in (A), (B) and (D) are: DP1046 (<i>zip1</i>), DP1049 (<i>zip1 dot1</i>), DP1048 (<i>zip1 H3-K79R</i>) and DP1047 (<i>zip1 H3-K79A</i>). Strains in (C) and (E) are: DP1042 (<i>zip1</i>), DP1045 (<i>zip1 dot1</i>), DP1044 (<i>zip1 H3-K79R</i>) and DP1043 (<i>zip1 H3-K79A</i>). In all cases (A–E), spreads were made, GFP images were captured and cell extracts were prepared after 24 h of meiotic induction in <i>ndt80</i> strains.</p

Dot1-Dependent Histone H3K79 Methylation Promotes Activation of the Mek1 Meiotic Checkpoint Effector Kinase by Regulating the Hop1 Adaptor

<div><p>During meiosis, accurate chromosome segregation relies on the proper interaction between homologous chromosomes, including synapsis and recombination. The meiotic recombination checkpoint is a quality control mechanism that monitors those crucial events. In response to defects in synapsis and/or recombination, this checkpoint blocks or delays progression of meiosis, preventing the formation of aberrant gametes. Meiotic recombination occurs in the context of chromatin and histone modifications, which play crucial roles in the maintenance of genomic integrity. Here, we unveil the role of Dot1-dependent histone H3 methylation at lysine 79 (H3K79me) in this meiotic surveillance mechanism. We demonstrate that the meiotic checkpoint function of Dot1 relies on H3K79me because, like the <em>dot1</em> deletion, <em>H3-K79A</em> or <em>H3-K79R</em> mutations suppress the checkpoint-imposed meiotic delay of a synapsis-defective <em>zip1</em> mutant. Moreover, by genetically manipulating Dot1 catalytic activity, we find that the status of H3K79me modulates the meiotic checkpoint response. We also define the phosphorylation events involving activation of the meiotic checkpoint effector Mek1 kinase. Dot1 is required for Mek1 autophosphorylation, but not for its Mec1/Tel1-dependent phosphorylation. Dot1-dependent H3K79me also promotes Hop1 activation and its proper distribution along <em>zip1</em> meiotic chromosomes, at least in part, by regulating Pch2 localization. Furthermore, <em>HOP1</em> overexpression bypasses the Dot1 requirement for checkpoint activation. We propose that chromatin remodeling resulting from unrepaired meiotic DSBs and/or faulty interhomolog interactions allows Dot1-mediated H3K79-me to exclude Pch2 from the chromosomes, thus driving localization of Hop1 along chromosome axes and enabling Mek1 full activation to trigger downstream responses, such as meiotic arrest.</p> </div

<i>Saccharomyces cerevisiae</i> strains.

*<p>All strains are isogenic diploids homozygous for the indicated markers.</p

Dot1 contributes to Mek1 activation by autophosphorylation.

<p>(A) Whole cell extracts (WCE) from a <i>zip1 ndt80</i> culture at 24 h in meiosis were incubated in the presence (+) or absence (−) of lambda phosphatase (λPPase). (B), (C) and (D) Detection of different phosphorylated forms of Mek1 in <i>ndt80</i>-arrested cells after 24 h in meiosis using high-resolution Phos-tag gels. Basal Mek1 (line) and several phosphorylated forms (black and white arrowheads) are indicated; see text for explanation. PGK or Ponceau S staining were used as loading controls. Asterisk in (D) marks a weak non-specific band. (E) Schematic representation of a model for the sequential phosphorylation events leading to Mek1 activation and the relevant mutations analyzed above. (1) Priming phosphorylation by Mec1/Tel1 (black arrowhead in B, C, D) is followed by (2) autophosphorylation of Mek1 (white arrowheads in B, C, D) leading to its full activation and (3) the checkpoint response. H3K79 methylation by Dot1 contributes to Mek1 autophosphorylation. Strains were: (A); DP428 (<i>zip1</i>). (B); DP428 (<i>zip1</i>), DP701 (<i>zip1 hop1</i>) and DP655 (<i>zip1 dot1</i>). (C); DP428 (<i>zip1</i>), DP655 (<i>zip1 dot1</i>), DP680 (<i>zip1 mec1</i>), DP861 (<i>zip1 mec1 tel1</i>), DP877 (<i>zip1 rad24 tel1</i>), DP728 (<i>zip1 spo11</i>) and DP674 (<i>zip1 mek1Δ</i>). (D); DP885 (<i>zip1</i>), DP890 (<i>zip1 dot1</i>), DP886 (<i>zip1 mek1-T327A</i>), DP887 (<i>zip1 mek1-T331A</i>), DP888 (<i>zip1 mek1-K199R</i>), DP674 (<i>zip1 mek1Δ</i>), DP680 (<i>zip1 mec1</i>) and DP861 (<i>zip1 mec1 tel1</i>).</p

Dot1 is required for checkpoint-promoted localization and activation of Mek1.

<p>(A) Formation of <i>zip1</i>-induced Mek1 foci is defective in the absence of Dot1. Representative images of Ddc2-GFP and Mek1-GFP foci in <i>zip1</i> and <i>zip1 dot1</i> cells after 24 h in meiosis. Strains are DP460 (<i>zip1 DDC2-GFP</i>), DP579 (<i>zip1 dot1 DDC2-GFP</i>), DP582 (<i>zip1 MEK1-GFP</i>) and DP583 (<i>zip1 dot1 MEK1-GFP</i>). All strains are <i>ndt80</i>-arrested at pachytene. The graphs show the quantification of Ddc2 and Mek1 foci formation from the same samples determined as the intensity of the total focal GFP signal relative to total nuclear signal (a.u., arbitrary units). Error bars represent the median with interquartile range. Each spot in the plot represents the foci intensity of every nucleus measured. 175 and 150 nuclei were analyzed for Ddc2-GFP and Mek1-GFP, respectively. (B) Western blot analysis of Mek1 activation by phosphorylation and Cdc5 production throughout meiosis in wild type (DP421), <i>zip1</i> (DP422), <i>zip1 dot1</i> (DP555), <i>zip1 dot1 GST-MEK1</i> (DP785) and <i>zip1 GST-MEK1</i> (DP792) using Phos-tag gels. PGK was used as a loading control. (C) Analysis of Mek1 activation in <i>ndt80</i>-arrested cells. Strains are DP424 (wild type), DP428 (<i>zip1</i>) and DP655 (<i>zip1 dot1</i>). (D) Time course of meiotic nuclear divisions; the percentage of cells containing more than two nuclei is represented. Strains are: DP421 (wild type), DP422 (<i>zip1</i>), DP555 (<i>zip1 dot1</i>), DP785 (<i>zip1 dot1 GST-MEK1</i>), DP783 (<i>zip1 dot1 GST-mek1-K199R</i>), DP784 (<i>zip1 dot1 GST-mek1-T327A</i>), DP792 (<i>zip1 GST-MEK1</i>), DP790 (<i>zip1 GST-mek1-K199R</i>) and DP791 (<i>zip1 GST-mek1-T327A</i>).</p

H3K79me controls Hop1 localization by excluding Pch2 from chromosomes.

<p>(A) H3K79me is required to prevent Pch2 localization outside of the rDNA. Immunofluorescence of meiotic chromosome spreads stained with DAPI (blue), anti-HA (red) and anti-Red1 (green) antibodies. Strains are: DP1050 (<i>zip1</i>), DP1053 (<i>zip1 dot1</i>), DP1052 (<i>zip1 H3-K79R</i>) and DP1051 (<i>zip1 H3-K79A</i>). (B–D) The absence of Pch2 partially restores Hop1 chromosomal abundance in <i>zip1 dot1</i>. (B) Immunofluorescence of meiotic chromosome spreads stained with DAPI (blue) and anti-Hop1 antibody (red). Strains are: DP428 (<i>zip1</i>), DP655 (<i>zip1 dot1</i>) and DP1054 (<i>zip1 dot1 pch2</i>). (C) Representative images of cells expressing <i>HOP1-GFP</i> in wild type (DP963), <i>dot1</i> (DP966), <i>zip1</i> (DP964), <i>zip1 dot1</i> (DP965) and <i>zip1 dot1 pch2</i> (DP1027). (D) Quantification of the Hop1-GFP signal intensity on fluorescence images (a.u., arbitrary units). 300 individual nuclei were analyzed for each strain. Each spot in the plot represents the fluorescence intensity of every nucleus measured. Error bars represent the median with interquartile range. <i>P</i><0.01 in pairwise comparisons. In all cases (A–C), spreads were prepared and GFP images were taken 24 h after meiotic induction in <i>ndt80</i> strains. (E, F) The absence of Pch2 does not restore the pachytene checkpoint response in <i>zip1 dot1</i>. (E) Time course of meiotic nuclear divisions; the percentage of cells containing more than two nuclei is represented. Strains are: DP421 (wild type), DP422 (<i>zip1</i>), DP555 (<i>zip1 dot1</i>), DP1029 (<i>zip1 pch2</i>) and DP1041 (<i>zip1 dot1 pch2</i>). (F) Western blot analysis of <i>zip1</i>-induced Mek1 phosphorylation in <i>ndt80</i> strains. PGK was used as a loading control. The asterisk marks a presumed non-specific band (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003262#pgen-1003262-g003" target="_blank">Figure 3D</a>). Strains are: DP428 (<i>zip1</i>), DP655 (<i>zip1 dot1</i>), DP881 (<i>zip1 pch2</i>) and DP1054 (<i>zip1 dot1 pch2</i>).</p

Model for Dot1 function in the meiotic recombination checkpoint.

<p>See text for details.</p

p21 and SUMO-1 colocalize in the disrupted nucleolus upon DNA damage.

<p>A) Immunodetection of endogenous SUMO-1 (green) using anti-SUMO-1 mouse antibody and p21 (red) using anti-p21 rabbit antibody in control (CTL) or treated with Adr for 48 hours (Adr) HCT116 cells. Scale bar: 5μm. B) Immunogold electronic microscopy of SUMO-1 showing the presence of SUMO-1 in the INoB HCT116 cells treated with Adr for 24 hours. Scale Bar: 0.5μm. C) Immunostaining of endogenous p21 (red) in GFP-SUMO-1 (two representative cells are shown) or GFP-SUMO-1ΔGly-Gly transfected cells treated 24h with Adr. Scale bar: 5μm. D) Immunostaining of p21 (red) and UBC9 (green) (rabbit polyclonal antibody) in 24-h Adr-treated HCT116 cells. Scale bar: 5μm. E) Immunostaining of SUMO-1 (green) and UBC9 (red) (rabbit monoclonal antibody) of 24-h Adr-treated HCT116 cells transfected with non-targeting (siNT) or UBC9 (siUBC9). Scale bar: 5 μm.</p

INoB growth is dependent on p21 whereas nucleolar disruption does not.

<p><b>A)</b> Immunostaining of UBF (green) and Fibrillarin (red) to analyse nucleolar disruption HCT116 cells transfected with non-targeting (siNT), p21 (sip21) siRNAs not treated (CTL) or treated with Adr for 48 hours (Adr). Ph.C.: Phase contrast. Scale bar: 5μm. <b>B)</b> Immunostaining of UBF (green) and Fibrillarin (red) to analyse nucleolar disruption in HCT116 p21KO cells after Adr treatment for 48 hours versus control (CTL). Ph.C.: Phase contrast. Scale bar: 5μm. <b>C)</b> Box-plot graphs showing the INoB size (μm) in HCT116 cells versus HCT116 p21KO cells (left), in HCT116 cells transfected with non-targeting (siNT) versus p21 (sip21) siRNA (middle); and in HCT116 cells transfected with pSUPER-puro-EGFP (shNT) versus pSUPER-puro-EGFP-p21 (shp21) (right). All cells were treated with Adr for 48 hours. Box shows Median and first quartiles, and whiskers show Min and Max. Number of cells (n) for each condition is shown. Example of how INoB size is quantified is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178925#pone.0178925.s005" target="_blank">S5A Fig</a>.</p