Genome-wide analysis of gene regulation mechanisms during Drosophila spermatogenesis

Background During Drosophila spermatogenesis, testis-specific meiotic arrest complex (tMAC) and testis-specific TBP-associated factors (tTAF) contribute to activation of hundreds of genes required for meiosis and spermiogenesis. Intriguingly, tMAC is paralogous to the broadly expressed complex Myb-MuvB (MMB)/dREAM and Mip40 protein is shared by both complexes. tMAC acts as a gene activator in spermatocytes, while MMB/dREAM was shown to repress gene activity in many cell types. Results Our study addresses the intricate interplay between tMAC, tTAF, and MMB/dREAM during spermatogenesis. We used cell type-specific DamID to build the DNA-binding profiles of Cookie monster (tMAC), Cannonball (tTAF), and Mip40 (MMB/dREAM and tMAC) proteins in male germline cells. Incorporating the whole transcriptome analysis, we characterized the regulatory effects of these proteins and identified their gene targets. This analysis revealed that tTAFs complex is involved in activation of achi, vis, and topi meiosis arrest genes, implying that tTAFs may indirectly contribute to the regulation of Achi, Vis, and Topi targets. To understand the relationship between tMAC and MMB/dREAM, we performed Mip40 DamID in tTAF- and tMAC-deficient mutants demonstrating meiosis arrest phenotype. DamID profiles of Mip40 were highly dynamic across the stages of spermatogenesis and demonstrated a strong dependence on tMAC in spermatocytes. Integrative analysis of our data indicated that MMB/dREAM represses genes that are not expressed in spermatogenesis, whereas tMAC recruits Mip40 for subsequent gene activation in spermatocytes. Conclusions Discovered interdependencies allow to formulate a renewed model for tMAC and tTAFs action in Drosophila spermatogenesis demonstrating how tissue-specific genes are regulated

Functional dissection of Drosophila melanogaster SUUR protein influence on H3K27me3 profile

Abstract Background In eukaryotes, heterochromatin replicates late in S phase of the cell cycle and contains specific covalent modifications of histones. SuUR mutation found in Drosophila makes heterochromatin replicate earlier than in wild type and reduces the level of repressive histone modifications. SUUR protein was shown to be associated with moving replication forks, apparently through the interaction with PCNA. The biological process underlying the effects of SUUR on replication and composition of heterochromatin remains unknown. Results Here we performed a functional dissection of SUUR protein effects on H3K27me3 level. Using hidden Markow model-based algorithm we revealed SuUR-sensitive chromosomal regions that demonstrated unusual characteristics: They do not contain Polycomb and require SUUR function to sustain H3K27me3 level. We tested the role of SUUR protein in the mechanisms that could affect H3K27me3 histone levels in these regions. We found that SUUR does not affect the initial H3K27me3 pattern formation in embryogenesis or Polycomb distribution in the chromosomes. We also ruled out the possible effect of SUUR on histone genes expression and its involvement in DSB repair. Conclusions Obtained results support the idea that SUUR protein contributes to the heterochromatin maintenance during the chromosome replication. A model that explains major SUUR-associated phenotypes is proposed

The Drosophila Su(var)3–7 Gene Is Required for Oogenesis and Female Fertility, Genetically Interacts with piwi and aubergine, but Impacts Only Weakly Transposon Silencing

Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3–7 protein in Drosophila ovaries. We present evidences that Su(var)3–7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3–7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3–7 is required for genome integrity. Females homozygous for Su(var)3–7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3–7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3–7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3–7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3–7 and piwi or aubergine controls important developmental processes independently of transposon silencing

<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

<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> 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

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> 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