Identification of Drosophila Mitotic Genes by Combining Co-Expression Analysis and RNA Interference

RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression–based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression–based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins

Chromosome condensation defects in barren RNA-interfered Drosophila cells.

Barren, the Drosophila homolog of XCAP-H, is one of three non-SMC subunits of condensin, a conserved 13S multiprotein complex required for chromosome condensation. Mutations in barren (barr) were originally shown to affect sister-chromatid separation during mitosis 16 of the Drosophila embryo, whereas condensation defects were not detected. In contrast, mutations in yeast homologs of barren result in defective mitotic chromosome condensation as well as irregular chromatid separation. We have used double-stranded RNA-mediated interference (RNAi) to deplete Barren in Drosophila S2 cells. Our analyses indicate that inactivation of barr leads to extensive chromosome condensation and disrupts chromatid segregation

A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters.

Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion-"X-shredding" and "X-poisoning"-and dissected their target-site requirements and repair dynamics. X-shredding resulted in sex distortion when Cas9 endonuclease activity occurred during the meiotic stages of spermatogenesis but not when Cas9 was expressed from the stem cell stages onwards. Our results suggest that X-shredding is counteracted by the NHEJ DNA repair pathway and can operate on a single repeat cluster of non-essential sequences, although the targeting of a number of such repeats had no effect on the sex ratio. X-poisoning by contrast, i.e. targeting putative haplolethal genes on the X chromosome, induced a high bias towards males (>92%) when we directed Cas9 cleavage to the X-linked ribosomal target gene RpS6. In the case of X-poisoning sex distortion was coupled to a loss in reproductive output, although a dominant-negative effect appeared to drive the mechanism of female lethality. These model systems will guide the study and the application of sex distorters to medically or agriculturally important insect target species

The <em>Drosophila</em> Mi-2 Chromatin-Remodeling Factor Regulates Higher-Order Chromatin Structure and Cohesin Dynamics <em>In Vivo</em>

<div><p>dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in <em>Drosophila</em>. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells.</p> </div

Analysis of GAL4-regulated transgenes.

<p>Data are shown for progeny of the following crosses:</p>(a)<p><i>dMi-2⁴/TM6B</i>, <i>Tb</i> males X <i>w; da-GAL4</i> females;</p>(b)<p><i>w; dMi-2⁴/TM6B</i>, <i>Tb</i> males X <i>w; dMi-2⁴ da-GAL4/TM6B</i>, <i>Tb</i> females;</p>(c)<p><i>w; P[w⁺, UAS-dMi-2</i>^{Δ<i>932-1158</i>}]6-5 males X <i>w; da-GAL4</i> females;</p>(d)<p><i>w; P[w⁺, UAS-dMi-2</i>^{Δ<i>932-1158</i>}]6-5; dMi-2^f08103/TM6B, <i>Tb</i> males X <i>w; da-GAL4</i> females;</p>(e)<p><i>w; P[w+, UAS-dMi-2</i>^{Δ<i>932-1158</i>}]6-5; dMi-2⁴/TM6B, <i>Tb</i> males X <i>w; da-GAL4</i> females;</p>(f)<p><i>w; P[w⁺, UAS-dMi-2⁺]3-3; dMi-2⁴/TM6B</i>, <i>Tb</i> males X <i>w; dMi-2⁴ da-GAL4/TM6B</i>, <i>Tb</i> females;</p>(g)<p><i>w; P[w⁺, UAS-dMi-2⁺]3-3</i> males X <i>w; da-GAL4</i> females;</p>(h)<p><i>w; P[w⁺, UAS-dMi-2⁺]3-3; dMi-2⁴/TM6B</i>, <i>Tb</i> males X <i>w; da-GAL4</i> females.</p

dMi-2 colocalizes with cohesin.

<p>(A–C) Magnified images of salivary gland polytene chromosomes stained with antibodies against dMi-2 (red) and Pol II Ser2 (A, green), stromalin (B, green) and Nipped B (C, green). Note the extensive overlap between the chromosomal distributions of the four proteins. (D) Reducing <i>dMi-2</i> gene dosage suppresses the small wing blade phenotype of individuals heterozygous for the <i>Nipped-B⁴⁰⁷</i> null allele. A minimum of twenty adult male wing blade areas was measured for each of the indicated genotypes, and the distributions of blade areas are presented as box-plots. For each genotype, the chromosome to the left of the separator (/) came from the male parent, and the chromosome to the right came from the female parent. The P57B chromosome is the wild-type chromosome in which the <i>Nipped-B⁴⁰⁷</i> mutation was induced by γ rays <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002878#pgen.1002878-Rollins1" target="_blank">[39]</a>. For the +/<i>Nipped-B⁴⁰⁷</i>, +/<i>dMi-2⁴</i> and +/P57B genotypes, the wild-type chromosomes came from an Oregon R male parent.</p

The loss of dMi-2 function alters chromosome structure.

<p>Salivary gland polytene chromosomes of late third-instar <i>UAS-LacZ/+; da-GAL4/+</i> control larvae (A and C) or larvae expressing the dominant-negative dMi-2^{Δ<i>932-1158</i>} protein (<i>UAS-dMi-2</i>^{Δ<i>932-1158</i>} 6-5/+; da-GAL4/+) (B and D). (A–D) Squashes of fixed polytene chromosomes stained with DAPI. (E–H) Live analysis of chromosomes of larvae expressing His2Av-GFP. The expression of <i>dMi-2^Δ932-1158 reduces the size of polytene chromosomes and disrupts their banding pattern. (Compare C and D to A and B, and G and H to E and F). A, B, E and F scale bars are 10 µm.</i></p

dMi-2 regulates chromosome cohesion in the larval salivary gland.

<p>(A) Overview of LacI-GFP/LacO tethering assay. The GFP-tagged LacI fusion protein (LacI-GFP) binds 256 tandem LacO sequences inserted at position 60F on the second chromosome allowing the detection of the locus in living cells. The <i>HS83</i> heat-shock promoter (hs) that drives <i>LacI-GFP</i> is activated at 37°C. (B-E) Live analysis of the LacI-GFP signal at 60F in the polytene chromosomes of third-instar larvae. <i>UAS-dMi-2⁺ 3-3/HS83-LacI-GFP LacO</i>; <i>UAS-dMi-2⁺ 15-1/da-GAL4 GAL80^ts</i> individuals were cultured at 18°C until the middle of the third larval instar, maintained at 18°C (B and D) or shifted to 29°C (C and E) for 27 hours, and heat-shocked at 37°C for 1 hour to induce LacI-GFP expression. Both longitudinal (B and C) and transverse (D and E) views of the lacO array are shown.</p

The over-expression of dMi-2 does not decrease cohesin levels.

<p>(A) Protein blot of salivary gland chromatin extracted from <i>UAS-dMi-2⁺ 3-3/+; UAS-dMi-2⁺ 15-1/da-GAL4 GAL80^ts</i> individuals raised at 18°C until the late third-instar stage and then shifted to 29°C for 24 hours to induce <i>UAS-Mi-2⁺</i> expression. The blot was probed with antibodies against Smc1 and histone H3 as a control. (B, C and D) RT-PCR analysis of Smc1, SA, and Rad21 RNA levels in the salivary glands of <i>UAS-LacZ/+</i>; <i>da-GAL4/+</i> (<i>UAS-LacZ</i>) control larvae and <i>UAS-dMi-2⁺ 3-3/+</i>; <i>UAS-dMi-2⁺ 15-1/da-GAL4</i> (<i>UAS-Mi2⁺</i>) larvae raised at 29°C. Histone H1 RNA levels are shown as a control.</p