Additional file 1: of Drosophila melanogaster positive transcriptional elongation factors regulate metabolic and sex-biased expression in adults

Summary of gene expression and k-means clustering. (XLS 10240 kb

<i>Su(var)3</i>–<i>7</i> does not interact with <i>piwi</i> and <i>aubergine</i> for transposon silencing in ovaries.

<p>Quantitative RT-PCR analysis on the indicated transposons in (<b>A</b>) <i>piwi²</i>/+; <i>Su(var)3</i>–<i>7^R2a8</i> and (<b>B</b>) <i>aub^QC42</i>/+; <i>Su(var)3</i>–<i>7^R2a8</i> ovaries. Bars represent the fold changes in RNA levels relative to <i>Su(var)3</i>–<i>7^R2a8</i> siblings (n = 3; ** : p<0,01).</p

Effect of <i>Su(var)3</i>–<i>7</i> mutations on <i>P</i>-element repression elicited by <i>P</i> copies inserted in subtelomeric heterochromatin.

<p>The <i>P</i> repression capacities of the female progeny having inherited or not a <i>Sb</i> chromosome are shown. The mean GD percentage calculated on the basis of all replicates is given with the standard deviation among replicates (in parenthesis). “n” indicates the number of replicates performed. F: female; M: male.</p

<i>Su(var)3</i>–<i>7</i> genetically interact with <i>piwi</i> and <i>aubergine</i> for female fertility.

a<p>Percentage of female giving no progeny when crossed with WT male.</p>b<p>Adult progeny obtained from a period of 8 days egg laying at 25°C.</p

<i>Su(var)3</i>–<i>7</i> has a weak impact on transposon silencing, but regulates piRNA clusters transcription.

<p>(<b>A</b>) Quantitative RT-PCR analysis on 22 retrotransposons in <i>Su(var)3</i>–<i>7^R2a8</i> homozygous mutant ovaries (grey bars) and female carcasses (black bars). Histograms represent the fold changes in RNA levels relative to <i>Su(var)3</i>–<i>7^R2a8</i>/<i>TM6</i> siblings; error bars indicate the standard deviation of triplicate samples (n = 3). Differences in the fold changes were tested by a Welch t-test (* : p<0,05; ** : p<0,01). (<b>B</b>) Quantitative RT-PCR analysis of <i>cluster 2</i> and <i>flamenco</i> from control (<i>w¹¹¹⁸</i>) and <i>Su(var)3</i>–<i>7^R2a8</i> heterozygote and homozygote mutant ovaries. Shown are the fold changes in RNA levels relative to the control (n = 3; * : p<0,05). The position of the primer sets used for qRT-PCR are indicated by bars named 1, 2 and 3 along the map above. Coordinates of the clusters along the <i>X</i> chromosome are indicated in Mb. Boxes indicate protein coding genes (blue) and transposon fragments in sense (black) and antisense (red) orientation. (<b>C</b>) Quantitative RT-PCR analysis of <i>cluster1</i> from control (<i>w¹¹¹⁸</i>) and <i>Su(var)3</i>–<i>7^R2a8</i> heterozygote and homozygote mutants. Shown are the fold changes in RNA levels relative to the control (n = 3; * : p<0,05). A map of the <i>cluster1</i>/<i>42AB</i> locus with position of the qPCR primer sets 4 and 5 is shown above. (<b>D</b>) Histograms show the log2 fold ratios of normalized ovarian piRNAs mapping antisense to transposons (left) and uniquely mapping piRNAs (sense plus antisense) over piRNA clusters (right) between homozygous and heterozygous <i>Su(var)3</i>–<i>7</i> mutants. Up to 1 mismatch was allowed between reads and transposon sequences.</p

<i>Su(var)3</i>–<i>7</i> is expressed in somatic and germline cells of wild type ovaries and colocalizes with HP1.

<p>(<b>A</b>–<b>C</b>) Confocal images of wild type ovary stained with anti-Su(var)3–7 (green) and DAPI (blue) for DNA visualisation. Same procedure on <i>Su(var)3</i>–<i>7^R2a8</i> homozygous ovaries shows complete absence of Su(var)3–7 staining (not shown). (<b>D</b>–<b>F</b>) Focus on somatic follicular cells and (<b>G</b>–<b>I</b>) germline cells co-stained with anti Su(var)3–7 (green) and anti-HP1 (red) antibodies. Arrows indicate the germinal vesicle with the karyosome. (<b>J</b>–<b>M</b>) Polytene chromosomes from <i>otu¹¹</i> pseudonurse cells labelled with anti-Su(var)3–7 (green) and DAPI (blue). <i>otu</i> mutation causes polytenization of nurse cells chromosomes allowing mapping of chromosome-associated proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096802#pone.0096802-King1" target="_blank">[59]</a>. Su(var)3–7 binds centromeric heterochromatin (bracket) and several euchromatic sites scattered along the chromosome arms (arrowheads).</p

Rescue of <i>Su(var)3</i>–<i>7^R2a8</i> ovarian phenotypes by expression of <i>P[HA:Su(var)3</i>–<i>7]</i>.

<p>n indicates the number of examined egg chambers.</p

<i>Su(var)3</i>–<i>7</i> is required for oogenesis, embryogenesis and female fertility.

<p>(<b>A</b>) Fertility test of control (<i>w¹¹¹⁸</i>) and <i>Su(var)3</i>–<i>7^R2a8</i> and <i>Su(var)3</i>–<i>7⁹</i> homozygous mutant females. Bars represent the rate of laid eggs per female and the viability at pupal and adult stages. Error bars indicate the standard deviation, n = 40. (<b>B</b>) DAPI staining of a 3 days old <i>Su(var)3</i>–<i>7^R2a8</i> mutant ovary. Arrows indicate degenerated egg chambers. (<b>C</b>) Confocal pictures of stage 5 egg chambers labelled with anti-H3K14ac antibody (green), DNA was visualized by DAPI (blue) staining. The round-shaped oocyte nucleus (karyosome) observed in the control (<i>w¹¹¹⁸</i>, left panel) is altered in <i>Su(var)3</i>–<i>7</i> mutant ovary (right panel). (<b>D</b>) Phase-contrast images of mature eggs produced by control (<i>w¹¹¹⁸</i>) and <i>Su(var)3</i>–<i>7^R2a8</i> mutant females. (<b>E</b>) <i>Su(var)3</i>–<i>7</i> loss-of-function causes meiosis defects and embryonic development arrest. (<b>a</b>–<b>e</b>) Confocal images of (<b>a</b>) control (<i>w¹¹¹⁸</i>) and (<b>b</b>–<b>e</b>) <i>Su(var)3</i>–<i>7^R2a8</i> mutant embryos stained with an anti-H3S10P (green) used as mitotic marker and anti-core histone proteins (red) antibodies. Embryos were examined 60′ to 120′ AED to ensure that control embryos have reach and exceeded mitotic cycle 6. (<b>a</b>) Mitotic cycle 7/8 control embryo. The nuclei are uniformly distributed within the embryo and the three female polar bodies are assembled into a single rosette. (<b>b</b>) Mutant embryo arrested at mitotic cycle 1. The rosette is misassembled and fragmented. (<b>c</b>) Mutant embryo arrested at mitotic cycle 3. The nuclei remain localized in the anterior part of the embryo and some nuclei contain a single set of chromosomes (1 n) suggesting cases of haploid mitotic cycles. (<b>d</b>) Mutant embryo arrested at mitotic cycle 4. The nuclei divided asynchronously. (<b>e</b>) Mitotic cycle 6 mutant embryo. Arrowheads point damaged mitotic nuclei. R, rosette.</p