MOESM5 of ASF1 is required to load histones on the HIRA complex in preparation of paternal chromatin assembly at fertilization

Additional file 5: Fig. S4. ASF1::V5 is not detected in the decondensing male pronucleus. a: An egg in metaphase of meiosis II from a g-asf1::V5 transgenic female stained with anti-V5 antibodies. Scale bar: 50 μm. b: Pronuclear migration. Scale bar: 10 μm. c: ASF1::V5 is incorporated in both pronuclei at the onset of DNA replication. Scale bar: 10 μm. d: Pronuclei stained with anti-V5 antibodies during apposition. Scale bar: 10 μm

MOESM4 of ASF1 is required to load histones on the HIRA complex in preparation of paternal chromatin assembly at fertilization

Additional file 4: Fig. S3. asf1 KD embryos lack paternal chromosomes. Late control and asf1 KD embryos fathered by GFP::cid transgenic males. Zygotic expression of the paternal centromeric marker is only detected in nuclei of control embryos (arrows in left inset). n = 140 for control embryos and n = 40 for asf1 KD embryos. Scale bar: 50 μm

Histone post-translational modifications associated with Drosophila transposable elements.

<p>Heatmap of ChIP-qPCR average fold enrichment for all TEs analyzed. Fold enrichment for each wild type strain is normalized by input, hence copy number, and also by <i>actin,</i> allowing comparison of species and srains. Each row represents a wild type strain of either <i>D. melanogaster</i> (DM) or <i>D. simulans</i> (DS). Each column represents a different antibody used (H3K4me2, H3K27me3 and H3K9me2). Location of primers used for ChIP-qPCR amplification is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044253#pone-0044253-g001" target="_blank">Figure 1</a>.</p

Expression of transposable element families in <i>D. melanogaster</i> (blue) and <i>D. simulans</i> (red).

<p>Mann Whitney P-values are shown with asterisks (p-value *<0.05).</p

Cartoon of the four retrotransposons studied (not to scale).

<p>Colored boxes represent open reading frames (ORF) and white boxes long terminal repeats (LTR). Arrows represent the quantitative PCR primers used for ChIP-qPCR (black arrows) and expression analysis (red arrows). The size of each canonical element is given.</p

YEM is essential for chromatin assembly in the male pronucleus at fertilization.

<p>(A) Confocal images of fertilized eggs in telophase of meiosis II stained for DNA (red) and acetylated histone H4 (H4act, green). In wild-type eggs (WT), the male pronucleus (arrow) is brightly and specifically stained with anti-H4act antibodies. In <i>yem</i> mutant eggs, H4act incorporation in the male nucleus is very weak or absent. (B) Eggs at the pronuclear apposition stage. In <i>yem¹</i> and <i>yem²</i> mutant eggs, the male pronucleus (arrowheads) fails to decondense (compare with wild-type) and contains very low or undetectable levels of H4act. Note that paternal chromatin assembly is partially restored in eggs laid by weakly fertile <i>yem-flag^HPF1/+; yem¹/Df(3R)3450</i> females (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003285#pgen-1003285-t001" target="_blank">Table 1</a>). (C) Cycle 1 embryos in metaphase (top) and telophase (bottom), stained as in (A) and (B). In <i>yem</i> mutant eggs, the male nucleus (arrowheads) is excluded from the first zygotic division. (D) <i>yem¹/Df(3R)3450</i> females produce gynogenetic haploid embryos. Left: the male nucleus (red) is still detected (arrowhead) among the haploid cleavage nuclei containing chromosomes of maternal origin. Right: close-up of cleavage nuclei in metaphase from wild-type (diploid) and <i>yem</i> (haploid) embryos.</p

Female sterility associated with <i>yem</i> mutations.

<p>All <i>yem</i> mutant alleles are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003285#pgen-1003285-g001" target="_blank">Figure 1</a>. <i>Df(3R)3450</i> is a large deficiency covering the <i>yem</i> locus. <i>yem-flag^HPF1</i> and <i>yem-flag^HPF16</i> are two independent insertions of the same transgene.</p

YEM and HIRA are interdependent for their localization in the germinal vesicle.

<p>(A) HIRA and YEM interact <i>in vivo</i>. Ovary extracts were prepared from <i>Hira-flag</i> transgenic flies and immunoprecipitated with either YEM AS2 polyclonal antibody or anti-Flag monoclonal antibody in the conditions described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003285#s4" target="_blank">Materials and Methods</a> section. The mock immunoprecipitation was performed with a rabbit pre-immune serum. The protein extracts (PE), the flowthrough (FL) and the immunoprecipitated (IP) fractions were submitted to Western blot analysis. HIRA and YEM were revealed respectively with anti-Flag (1∶1000) and AS2 (1∶100) antibodies. Rabbit polyclonal antibody against CHD1 was used at a 1∶250 dilution. Note that YEM and HIRA were found in the same immune complex whereas CHD1 was recovered in the FL fraction. In the mock experiment, HIRA was recovered in the FL fraction, which assesses the specificity of the immunoprecipitation reactions. (B) YEM and HIRA colocalize in the GV (arrowheads) throughout oogenesis. Wild-type ovaries dissected from <i>Hira-Flag</i> transgenic females were stained with anti-Flag (green), anti-YEM antibodies (red) and DAPI (blue). Bar: 20 µm. (C) YEM and HIRA proteins are interdependent for their localization in the GV. Ovaries from wild-type (wt), <i>yem¹/Df(3R)3450</i> or <i>yem²/Df(3R)3450</i> females bearing one copy of the <i>Hira-Flag</i> transgene were stained to visualize DNA (blue) and Flag (green). HIRA accumulation in the GV (arrowheads) is not affected in <i>yem¹/Df(3R)3450</i> but is abolished in <i>yem²/Df(3R)3450</i> ovaries. Conversely, YEM-Flag GV localization was not significantly affected in <i>Hira^ssm</i> ovaries, but appeared highly variable in <i>Hira^HR1</i> ovaries. Percentages indicate the number of egg chambers with positive staining in the GV, equal or superior to the background fluorescence. At least 60 egg chambers were observed for each experiment. (D) Western-blot analysis of HIRA-Flag (left) and YEM-Flag (right) from protein extracts of ovaries of the indicated genotypes. α-Tubulin was used as a loading control.</p

<i>yem</i> is required for H3.3 deposition in the male nucleus.

<p>(A) At pronuclear apposition in wild-type eggs, the male nucleus (arrowhead) contains high levels of maternally expressed H3.3-Flag whereas the female pronucleus incorporates low levels of H3.3-Flag, presumably during S phase. In <i>yem¹/Df(3R)3450</i> mutant eggs, only the weak incorporation of H3.3-Flag in the female pronucleus is detected. (B) The monoclonal anti-H3.3 antibody specifically stains the male nucleus (arrowhead) at fertilization in wild-type eggs. Maternal chromosomes are in anaphase of the second meiotic division. (C) H3.3 is still detected in paternal chromosomes (arrowheads) during the first zygotic cycle in wild-type eggs but not in the male nucleus on cycle 1 <i>yem</i> or <i>Hira</i> mutant embryos. Bars: 10 µm.</p

HIRA and YEM are interdependent for their recruitment to the male pronucleus.

<p>(A) YEM localizes to the decondensing male nucleus. Confocal images of eggs from <i>yem-flag^HPF3 yem²</i> homozygous females (upper panels) with the male nucleus (arrows) magnified in lower panels. YEM-Flag (green) is detected throughout the decondensing male nucleus but also accumulates in a small number of nuclear foci (arrowheads). Note that YEM-Flag is no longer detected at pronuclear apposition (right panels). Bar: 10 µm. (B) <i>Hira</i> mutations affect the general distribution of YEM-Flag in the male nucleus. Eggs from females of the indicated genotype were stained with anti-Flag antibodies and imaged as in (A). For each male nucleus, the presence of YEM-Flag (whole nucleus and/or foci) was evaluated and each category is represented as a percentage of the total number (n) of observed pronuclei. (C) YEM-Flag foci in the male nucleus do not colocalize with telomeres or centromeres. Upper panels: Nuclear foci of maternally expressed YEM-Flag (red arrow) do not localize to the cluster of telomeres marked with the paternal telomere marker GFP-K81 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003285#pgen.1003285-Dubruille1" target="_blank">[69]</a> (green arrow). Lower panels: YEM-Flag nuclear foci (red arrows) do not colocalize with paternal centromeres (green arrows) in the male nucleus. (D) Confocal sections of male pronuclei in eggs laid by females of the indicated genotype (HIRA-Flag is shown in green, DNA in red) (n>20). In wild-type eggs, HIRA-Flag accumulates in the decondensing male nucleus. In eggs from <i>yem</i> mutant females however, HIRA-Flag is not detected.</p