SLY regulates genes involved in chromatin remodeling and interacts with TBL1XR1 during sperm differentiation

Sperm differentiation requires unique transcriptional regulation and chromatin remodeling after meiosis to ensure proper compaction and protection of the paternal genome. Abnormal sperm chromatin remodeling can induce sperm DNA damage, embryo lethality and male infertility, yet, little is known about the factors which regulate this process. Deficiency in Sly, a mouse Y chromosome-encoded gene expressed only in postmeiotic male germ cells, has been shown to result in the deregulation of hundreds of sex chromosome-encoded genes associated with multiple sperm differentiation defects and subsequent male infertility. The underlying mechanism remained, to date, unknown. Here, we show that SLY binds to the promoter of sex chromosome-encoded and autosomal genes highly expressed postmeiotically and involved in chromatin regulation. Specifically, we demonstrate that Sly knockdown directly induces the deregulation of sex chromosome-encoded H2A variants and of the H3K79 methyltransferase DOT1L. The modifications prompted by loss of Sly alter the postmeiotic chromatin structure and ultimately result in abnormal sperm chromatin remodeling with negative consequences on the sperm genome integrity. Altogether our results show that SLY is a regulator of sperm chromatin remodeling. Finally we identified that SMRT/N-CoR repressor complex is involved in gene regulation during sperm differentiation since members of this complex, in particular TBL1XR1, interact with SLY in postmeiotic male germ cells.This work was supported by Inserm (Institut National de la Sante et de la Recherche Medicale), the Agence Nationale de la Recherche program ANR-12–JSV2-0005–01 (to JC), Labex ‘Who am I?’(ANR-11- LABX-0071 under program ANR-11-IDEX-0005-01) and a Marie Curie fellowship FP7-PEOPLE-2010-IEF-273143 (to JC

Budding yeast ATM/ATR control meiotic double-strand break (DSB) levels by down-regulating Rec114, an essential component of the DSB-machinery

An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks

The CAF-1 and Hir Histone Chaperones Associate with Sites of Meiotic Double-Strand Breaks in Budding Yeast.

In the meiotic prophase, programmed DNA double-strand breaks (DSB) are introduced along chromosomes to promote homolog pairing and recombination. Although meiotic DSBs usually occur in nucleosome-depleted, accessible regions of chromatin, their repair by homologous recombination takes place in a nucleosomal environment. Nucleosomes may represent an obstacle for the recombination machinery and their timely eviction and reincorporation into chromatin may influence the outcome of recombination, for instance by stabilizing recombination intermediates. Here we show in budding yeast that nucleosomes flanking a meiotic DSB are transiently lost during recombination, and that specific histone H3 chaperones, CAF-1 and Hir, are mobilized at meiotic DSBs. However, the absence of these chaperones has no effect on meiotic recombination, suggesting that timely histone reincorporation following their eviction has no influence on the recombination outcome, or that redundant pathways are activated. This study is the first example of the involvement of histone H3 chaperones at naturally occurring, developmentally programmed DNA double-strand breaks

Differential Association of the Conserved SUMO Ligase Zip3 with Meiotic Double-Strand Break Sites Reveals Regional Variations in the Outcome of Meiotic Recombination

<div><p>During the first meiotic prophase, programmed DNA double-strand breaks (DSBs) are distributed non randomly at hotspots along chromosomes, to initiate recombination. In all organisms, more DSBs are formed than crossovers (CO), the repair product that creates a physical link between homologs and allows their correct segregation. It is not known whether all DSB hotspots are also CO hotspots or if the CO/DSB ratio varies with the chromosomal location. Here, we investigated the variations in the CO/DSB ratio by mapping genome-wide the binding sites of the Zip3 protein during budding yeast meiosis. We show that Zip3 associates with DSB sites that are engaged in repair by CO, and Zip3 enrichment at DSBs reflects the DSB tendency to be repaired by CO. Moreover, the relative amount of Zip3 per DSB varies with the chromosomal location, and specific chromosomal features are associated with high or low Zip3 per DSB. This work shows that DSB hotspots are not necessarily CO hotspots and suggests that different categories of DSB sites may fulfill different functions.</p></div

Recombination in CAF-1 mutants does not rely more of the class II crossover pathway.

<p>DSB formation and CO frequency at <i>HIS4LEU2</i> in <i>mms4 slx4 yen1</i> triple mutant (VBD1444) and in <i>mms4 slx4 yen1 cac1Δ</i> (VBD1443) monitored by Southern blot. The graph shows DSB and CO quantification from the same time-courses.</p

The CAF-1 large subunit is recruited to Spo11 and VDE meiotic DSBs.

<p>(A) Cac1-3HA association with two Spo11 DSB sites, DSB1 (<i>GAT1</i> promoter) and DSB2 (<i>BUD23</i> promoter). Enrichment at each site is measured relative to that at a negative control site. ChIP from WT (VBD1098) and from <i>spo11Y135F</i> (VBD1101) meiotic time course at the indicated times in meiosis. DSB formation during VBD1098 time course was monitored at the <i>BUD23</i> promoter by Southern Blot and quantified (upper panel). (B) Cac1-3HA association with the VDE DSB site inserted at <i>ARE1</i> and with one Spo11 DSB site (<i>GAT1</i> promoter). ChIP from VBD1115 (VRS::<i>ARE1/</i>VRS mut::<i>ARE1</i>), VBD1161 (VRS mut::<i>ARE1</i>/VRS mut::<i>ARE1</i>) and VBD1211 (VRS::<i>ARE1</i>/VRS mut::<i>ARE1 spo11Δ</i>) meiotic time courses. VDE DSB formation during VBD1115 time course was monitored by Southern blot and quantified. (C) Cac1-3HA association around the VDE DSB site, using qPCR primers located at different distances from the VDE break. ChIP samples are from a +4h time-point from VBD1115 (VRS::<i>ARE1/</i>VRS mut::<i>ARE1</i>) and from VBD1161 (VRS mut::<i>ARE1/</i>VRS mut::<i>ARE1</i>) meiotic time courses.</p

Mutation of the Zip3 consensus phosphorylation sites by Mec1/Tel1 kinases alters its association with DSB sites and decreases crossover levels.

<p>(A) Meiotic progression of wild-type (ORD9670) or <i>zip3-4AQ</i> mutant (VBD1094) cells. Nuclear divisions were monitored by DAPI staining. (B) Zip3 association in the time-courses shown in (A) was monitored by ChIP with anti-Flag antibodies and revealed by qPCR with primer pairs covering the indicated regions. (C) Genetic distance in the <i>EST3-FAA3</i> interval on chromosome IX measured in the <i>ZIP3-</i>Flag (VBD1229) and <i>zip3-4AQ-</i>Flag (VDB1113) strains (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003416#pgen.1003416.s016" target="_blank">Table S2</a>). The configuration of the hemizygous resistance markers used to measure the genetic distances is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003416#pgen.1003416.s008" target="_blank">Figure S8</a>. (D) Genetic distances determined for nine intervals distributed on three chromosomes. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003416#pgen.1003416.s015" target="_blank">Table S1</a>. <i>ZIP3</i>-Flag: VBH334/VBH335 strain; <i>zip3-4AQ</i>-Flag: VBH332/VBH331 strain. (E) Physical analysis of COs in the <i>EST3-FAA3</i> interval in <i>ZIP3</i>, <i>zip3-4AQ</i> and <i>mus81Δ</i> mutants. Genomic DNA was extracted at the indicated times of synchronous meiosis and digested with <i>Bsp</i>EI and <i>Bss</i>HII. The parental (P1 and P2) and CO (R1 and R2) bands are indicated. The R2 band was quantitated and expressed as % of total DNA. <i>ZIP3</i>: VBD1229; <i>zip3-4AQ</i>: VBD1113; <i>ZIP3 mus81Δ</i>: VBD1244; <i>zip3-4AQ mus81Δ</i>: VBD1245. The graph indicates the mean of two independent experiments. Error bars represent standard deviation.</p

Comparison of the ChIP–chip enriched peaks between pairs of experiments.

<p>The name of each experiment is indicated, as well as the number of peaks in common between the two experiments and the percentage of the peaks of the first experiment. Pcorr assesses the linear Pearson's correlation coefficient between the profiles of the two experiments after denoising and smoothing with a 2 kb window.</p

Zip3 SUMO ligase activity is required for Zip3 association with centromeres, axes, and meiotic double-strand break sites.

<p>(A) DSB formation in a wild-type (ORD9670) meiotic time-course at the DSB site in the <i>BUD23</i> promoter (DSB1), also monitored by ChIP in (E). The graph shows the quantification of DSB formation at DSB1. (B) Zip3-Flag expression was monitored by western blotting with an anti-Flag antibody in strains containing Flag-tagged wild-type <i>ZIP3</i> (ORD9670), <i>zip3H80A</i> (VBD1072) or <i>zip3I96K</i> (VBD1073) alleles. The asterisk indicates the presumed sumoylated forms of Zip3 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003416#pgen.1003416-Cheng1" target="_blank">[18]</a>. Pgk1 served as loading control. (C) Schematic representation of the position of the different regions assessed by qPCR for Zip3 binding. (D) Meiotic progression in the three Zip3 strains, monitored by nuclear division. (E) ChIP monitoring of Zip3-Flag association with the indicated regions during the same time-courses in cells with wild-type (ORD9670), <i>H80A</i> (VBD1072) or <i>I96K</i> (VDB1073) <i>ZIP3</i> alleles. Below is the control experiment performed by ChIP with the anti-Flag antibody in an untagged strain (ORD7339).</p