MOESM13 of Expression and epigenomic landscape of the sex chromosomes in mouse post-meiotic male germ cells

Additional file 13. a RPKM threshold determination. Reads were mapped to Ensembl Genes (in blue) and to intergenic regions (in red). By comparing the expression levels of exons and intergenic regions, the intersection of the density plots was used to determine the threshold value to consider a gene as expressed (in our study= 0.22). b The true number of expressed genes in each bin (black) was estimated from the observed numbers for Ensembl genes (blue, same as a) by multiplication of the latter by the false discovery rate. This estimate was converted to cumulative amount, and the false negative rate was estimated as a function of expression level using the formula described in [48]. c. Bins were converted to cumulative amounts of genes expressed above the expression levels for genes (cum_genes; blue) and controls (cum_background; red). A false discovery rate fdr (green) was calculated at each expression level as described in [48]

The combination of shSLX and shSLY transgenes produces an efficient knockdown of <i>Slx/Slxl1</i> and <i>Sly</i> genes.

<p>A–B) Real time PCR quantification of <i>Slx</i>/<i>Slxl1</i> (<i>Slx-all</i> primers) (A) and <i>Sly</i> (<i>Sly1</i> and <i>Sly2</i> variants) (B) transcript levels in WT, shSLX1, shSLY and shSLX1shSLY round spermatids. The y-axis indicates the level of expression compared to WT after normalization with <i>Acrv1</i> (2^ΔΔCt ± standard errors). The reduction in <i>Slx/Slxl1</i> transcript level was similar in shSLX1shSLY males and in shSLX1 siblings. As observed before <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002900#pgen.1002900-Cocquet1" target="_blank">[19]</a>, <i>Slx/Slxl1</i> transcript level was found increased in shSLY males. One asterisk indicates significant difference from WT (p<0.05; t test on ΔΔCt values). <i>Sly</i> knockdown was even stronger in shSLX1shSLY males compared to shSLY siblings [two asterisks indicate significant difference between shSLX1shSLY and shSLY (p = 0.02; t test on ΔΔCt values)]. C–E) Western blot detection of SLY1, SLX and SLXL1 proteins in nuclear and cytoplasmic fractions from WT, shSLY, shSLX1 and shSLX1shSLY round spermatids. LAMIN B1 and ACTIN detection were used as loading controls for nuclear and cytoplasmic fractions, respectively. No SLY1 protein could be detected in shSLY or in shSLX1shSLY samples.</p

Model presenting how SLX/SLXL1 and SLY proteins have antagonistic effects in the spermatid nucleus and cytoplasm.

<p>In the spermatids, SLX/SLXL1 (yellow lozenges) and SLY (pink triangles) proteins have antagonistic effects i) in the nucleus, on the expression of XY genes and of a few autosomal genes, such as <i>Speer</i> (group 1 genes); ii) in the cytoplasm, on the regulation of metabolic processes which secondarily causes a deregulation of ∼100 autosomal genes (group 2 genes). i) In WT, SLY proteins are located in both the nucleus and cytoplasm, while SLX/SLXL1 proteins are almost exclusively in the cytoplasm. The nuclear fraction of SLY proteins colocalizes with the sex chromosomes and the autosomal <i>Speer</i> gene cluster, and represses their expression. A very small fraction of SLX/SLXL1 proteins also appears to colocalize with the sex chromatin. In <i>Sly</i>-deficient spermatids (shSLY), SLX/SLXL1 proteins relocate to the nuclear sites (both sex-linked and autosomal) vacated by SLY proteins; however, SLX/SLXL1 proteins have an opposite effect to that of SLY, and activate XY gene expression. This is associated with a reduction in the repressive epigenetic mark H3K9me3 on the sex chromatin (purple octagon), and produces sperm differentiation defects such as spermhead abnormalities, shedding delay, motility defects and subsequent male infertility. In <i>Slx/Slxl1</i>-deficient spermatids (shSLX), the absence of SLX/SLXL1 nuclear proteins has only minor effect on gene regulation, since it does not change SLY localization profile. There is only a slight reduction in XY transcription, congruent with the idea that SLX/SLXL1 is a transcription activator sharing the targets of SLY when present in the nucleus. In the double knock-down (shSLXshSLY), <i>Slx/Slxl1</i> deficiency almost fully abrogates the effects of <i>Sly</i> knockdown: in shSLXshSLY spermatids, group 1 gene expression and repressive epigenetic marks are close to WT values. This is correlated with a rescue of SLY-dependent sperm differentiation defects. In sum, in the nucleus, the experimental observations indicate that SLX/SLXL1 competes with SLY at the level of sex chromatin regulation: SLY acts as a repressor while SLX/SLXL1 acts as a positive regulator. ii) <i>Slx/Slxl1</i> deficiency induces various spermiogenic defects (such as spermatid elongation delay and apoptosis, reduced sperm count, abnormal head to tail connections of the spermatozoa and subsequent male infertility) associated with an up-regulation of ∼100 autosomal genes which code for proteins of the cytoskeleton, the extracellular matrix, or implicated in various metabolic processes (i.e. group 2 genes). Since SLX/SLXL1 proteins are predominantly cytoplasmic in WT spermatids, we propose that this gene deregulation is a manifestation of the spermiogenesis defects occasioned by <i>Slx/Slxl1</i> deficiency, and not a direct effect of SLX/SLXL1 proteins on autosomal gene transcription. In the case of <i>Sly</i> deficiency, group 2 gene expression is unaffected; however, in the double knock-down, <i>Sly</i> deficiency corrects SLX/SLXL1-dependent phenotypes which abrogates the subsequent group 2 gene up-regulation. This means that SLY protein has a cytoplasmic role, opposing that of SLX/SLXL1. This antagonism could be mediated <i>via</i> interaction with (a) common partner(s) in the cytoplasm; the absence of competition between SLX/SLXL1 and SLY proteins in the dual knockdown model would explain the absence of defects. In sum, SLX/SLXL1 and SLY proteins apparently compete in the cytoplasm for the regulation of spermiogenic processes. The functional role of SLX/SLXL1 could be to prevent the access of SLY to cytoplasmic proteins that are necessary for spermiogenesis.</p

SLX/SLXL1 and SLY have opposite effects on gene expression and on the recruitment/maintenance of H3K9me3 on the sex chromatin.

<p>A) Representation of the microarray results obtained for <i>Slx/Slxl1</i>-deficient (shSLX1), <i>Sly</i>-deficient (shSLY) and <i>Slx/y</i>-deficient (shSLX1shSLY) compared to WT spermatids. B) Real time PCR quantification showed that transcript levels of X-encoded (<i>Actrt1</i> and <i>1700008I05Rik</i>) and of Y-encoded genes (<i>Ssty1</i> and <i>Zfy2</i>) were lower in shSLX1shSLY than in shSLY spermatids. Transcript levels were also lower in shSLX1 spermatids compared to WT spermatids. The y-axis indicates the level of expression compared to WT after normalization with <i>Acrv1</i> (2^ΔΔCt ± standard errors). One asterisk indicates significant difference from corresponding shSLY or WT value (t test on ΔΔCt values; p<0.05). C) Graphic representation of the expression ratio relative to WT for the 222 genes showing greater than 1.5 fold-change in shSLY. Most genes affected by shSLY are sex-linked and up-regulated <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002900#pgen.1002900-Cocquet1" target="_blank">[19]</a>. The majority of them (196/222) were corrected to some degree by addition of the shSLX transgene (in shSLX1shSLY). For 46 of these genes, the difference between shSLY and shSLX1shSLY was itself statistically significant. D) Immunofluorescence detection of H3K9me3 (green) in WT, shSLY and shSLX1/2shSLY round spermatid nuclei. DAPI (white or blue) was used to stain nuclei. The round DAPI-dense structure is the chromocenter. The less DAPI-dense structure at the periphery of the chromocenter is the postmeiotic sex chromatin (PMSC) and is indicated by an arrow. Pictures were taken using the same image capture parameters. Note the decreased H3K9me3 signal on the sex chromatin of <i>Sly</i>-deficient spermatids; this is almost completely restored by <i>Slx/Slxl1</i> deficiency (in shSLX1/2shSLY spermatids). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002900#pgen.1002900.s004" target="_blank">Figure S4</a>. E) Graphic representation of the expression ratio relative to WT for the 115 genes showing greater than 1.5 fold-change in shSLX1. Most genes affected by shSLX1 are autosomal and up-regulated <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002900#pgen.1002900-Cocquet2" target="_blank">[28]</a>. Note that almost all of them (111/115) have a lower fold change in shSLX1shSLY than in shSLX1. For 91 of these genes, the difference between shSLX1 and shSLX1shSLY was itself statistically significant.</p

Analysis of the reproductive parameters of <i>Slx/y</i>-deficient mice compared to controls.

*<p>significant difference from WT (t-test,p<0.02);</p>a<p>significant difference from shSLY (t-test,p<0.03);</p>b<p>significant difference from shSLX1 (t-test,p<0.02);</p>c<p>significant difference from shSLX1/2 (t-test,p<0.03).</p

Model comparing <i>Slx:Sly</i> copy number imbalance in natural and laboratory mouse strains to <i>Slx:Sly</i> gene expression imbalance in shRNA knockdown models.

<p>A. A model for how <i>Slx/Slxl1</i>:<i>Sly</i> imbalance affects sperm shape, offspring sex ratio and fertility. B. Approximate copy number ratio of <i>Slx/Slxl1</i> and <i>Sly</i> in the reciprocal crosses studied by Good et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002900#pgen.1002900-Good1" target="_blank">[37]</a> based on an estimate of ∼100 copies of each gene in <i>musculus</i> and ∼50 in <i>domesticus</i>, in the WT laboratory strain MF1 Y^RIII which has a <i>domesticus</i> X and autosomes but a <i>musculus</i>-derived Y <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002900#pgen.1002900-Yalcin1" target="_blank">[40]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002900#pgen.1002900-Mardon1" target="_blank">[42]</a>, and in the two natural mutants from the same background studied by us and others <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002900#pgen.1002900-Scavetta1" target="_blank">[18]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002900#pgen.1002900-Ellis1" target="_blank">[20]</a>. C. The relative magnitude of Group 1 and Group 2 transcriptional responses seen in the various shRNA/deletion models on the MF1 Y^RIII background. The double and triple shRNA models show a partial Group 1 response, but no Group 2 response. Importantly, in this model, the shSLX1 and shSLX1/2 phenotypes are expected to fall outside the range of variation seen in the natural mutant and reciprocal cross males, since they are on a background which has already a deficiency in <i>Slx/Slxl1</i> copy number compared to <i>Sly</i> (50∶100). We emphasise that the effects of <i>Slx/Slxl1</i>:<i>Sly</i> imbalance are only one contributor to hybrid sterility: sperm shape and testis size QTLs on the <i>musculus</i> X map to distinct locations and show different interactions with the <i>domesticus</i> autosomes and Y chromosome <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002900#pgen.1002900-Campbell1" target="_blank">[50]</a>.</p

<i>Slx/y</i>-deficient males have fewer spermiogenesis defects than males that are deficient for either <i>Sly</i> or <i>Slx/Slxl1</i> alone.

<p>A) Bar graph representing the percentage of tubules containing apoptotic elongating spermatids measured by TUNEL assay (in grey) and the percentage of tubules containing delayed elongating spermatids (in black). Hatch symbol (#) indicates significant difference from WT (ANOVA, p<0.0001). One asterisk indicates significant difference between values obtained for <i>Slx/y</i>-deficient males (shSLX1shSLY or shSLX1/2shSLY) and <i>Slx/Slxl1</i>-deficient siblings (shSLX1 or shSLX1/2) (ANOVA, p<0.00001). B) Representative black and white TUNEL pictures of WT, shSLX1/2 and shSLX1/2shSLY testicular sections. Note the presence of apoptotic cells (TUNEL+ cells in white) in most shSLX1/2 seminiferous tubules, while in WT and in shSLX1/2shSLY they are largely restricted to stage XII tubules (and correspond to metaphasic cells undergoing normal apoptosis). Scale bar represents 200 µm. C) Bar graph representing the percentage of sperm head abnormalities in <i>Slx/Slxl1</i>-deficient, <i>Sly</i>-deficient and <i>Slx/y</i>-deficient males (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002900#pgen.1002900.s006" target="_blank">Figure S6</a>). The percentage of total abnormal sperm heads is significantly lower in <i>Slx/y</i>-deficient males (shSLX1shSLY and shSLX1/2shSLY) than in <i>Sly</i>-deficient males (shSLY). In particular, the number of grossly flattened spermheads – which are specifically observed in <i>Sly</i>-deficient males – is reduced (ANOVA, p<0.001). ShSLX1/2shSLY males also show a significant decrease in this percentage compared to shSLX1shSLY males (ANOVA, p<0.03). The number of slightly abnormal sperm heads – which are specifically observed in <i>Slx/Slxl1</i>-deficient males – is also reduced in <i>Slx/y</i>-deficient males compared to <i>Slx/Slxl1</i>-deficient males (ANOVA, p<0.0008). For this category of sperm abnormalities, <i>Slx/y</i>-deficient males did not significantly differ from WT.</p

SLX/SLXL1 proteins behave similarly to SLY in its absence.

<p>A) Immunofluorescence detection of SLX/SLXL1 protein (green) in wild-type (WT) and <i>Sly</i>-deficient (shSLY) testicular sections. DAPI (blue) was used to stain nuclei and lectin-PNA (red) was used to stain acrosomes in order to determine tubular stage. The inset represents a 3× magnification. Pictures were taken using the same image capture parameters. Scale bar indicates 10 µm. B) Western blot detection of SLX/SLXL1 proteins in nuclear extracts from shSLY and WT round spermatids. SLY1 antibody was used on the same extracts to confirm the absence of SLY protein in the shSLY nuclear fraction. <i>Sly</i> gene encodes two alternative splice variants (<i>Sly1</i> and <i>Sly2</i>) which are predicted to be translated into a long and a short protein isoform (SLY1 and SLY2), but only SLY1 proteins have been detected so far and it remains unclear whether <i>Sly2</i> transcripts are translated <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002900#pgen.1002900-Reynard1" target="_blank">[21]</a>. LAMIN-B1 detection was used as a loading control. C) Immunofluorescence detection of SLX/SLXL1 protein (green) in shSLY and WT round spermatid nuclei. DAPI (blue) was used to stain nuclei. X and Y chromosome painting were performed sequentially. A strong SLX/SLXL1 signal was observed in the majority of shSLY spermatid nuclei (76%). This signal colocalized with either sex chromosome in 96.5% of the cases. No signal could be detected in the majority of WT round spermatid nuclei (84%). D) Immunofluorescence detection of SLY1 (pink) or SLX/SLXL1 (green) protein in WT or shSLY round spermatids. Hybridization with a DNA probe detecting <i>Speer</i> gene cluster was subsequently performed, followed by Y chromosome painting. DAPI (white or blue) was used to stain nuclei. SLY1 protein colocalized with <i>Speer</i> gene cluster in 78.5% of WT spermatids while SLX/SLXL1 proteins colocalized with <i>Speer</i> gene cluster in 73% of shSLY spermatids.</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

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