Maria Patrizia Somma

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.

Feo, the Drosophila Homolog of PRC1, Is Required for Central-Spindle Formation and Cytokinesis

We performed a functional analysis of fascetto (feo), a Drosophila gene that encodes a protein homologous to the Ase1p/PRC1/MAP65 conserved family of microtubule-associated proteins (MAPs). These MAPs are enriched at the spindle midzone in yeast and mammals and at the fragmoplast in plants, and are essential for the organization and function of these microtubule arrays. Here we show that the Feo protein is specifically enriched at the central-spindle midzone and that its depletion either by mutation or by RNAi results in aberrant central spindles. In Feo-depleted cells, late anaphases showed normal overlap of the antiparallel MTs at the cell equator, but telophases displayed thin MT bundles of uniform width instead of robust hourglass-shaped central spindles. These thin central spindles exhibited diffuse localizations of both the Pav and Asp proteins, suggesting that these spindles comprise improperly oriented MTs. Feo-depleted cells also displayed defects in the contractile apparatus that correlated with those in the central spindle; late anaphase cells formed regular contractile structures, but these structures did not constrict during telophase, leading to failures in cytokinesis. The phenotype of Feo-depleted telophases suggests that Feo interacts with the plus ends of central spindle MTs so as to maintain their precise interdigitation during anaphase-telophase MT elongation and antiparallel sliding.

DNA copy number evolution in Drosophila cell lines

BackgroundStructural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles.ResultsOur work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167).ConclusionOur study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.

Characterization of the opposite-strand genes from the mouse bidirectionally transcribed HTF9 locus

The mouse HTF9 locus contains two genes that are bidirectionally transcribed with opposite polarity from a shared CpG-rich island. Both genes were previously shown to be expressed in a housekeeping fashion in mouse. We have now determined the molecular organization of the genes over 12 kb surrounding the island. In addition, we show that the HTF9 locus resides in the proximal region of mouse chromosome 16. We have sequenced the cDNAs corresponding to both divergent transcripts. Both genes appear to code for novel proteins that are structurally unrelated to each other. Finally, we show that both genes are highly conserved and efficiently expressed in human cells.

Drosophila Dgt6 Interacts with Ndc80, Msps/XMAP215, and γ-Tubulin to Promote Kinetochore-Driven MT Formation

In centrosome-containing cells, spindle assembly relies on microtubules (MTs) nucleated from both centrosomes and chromosomes. Recent work has suggested that additional spindle MTs can be nucleated by gamma-tubulin ring complexes (gamma-TuRCs) that associate laterally with preexisting spindle MTs, leading to spindle amplification. It has been proposed that in Drosophila S2 cells, gamma-TuRCs are anchored to the spindle MTs by augmin, a multiprotein complex that contains at least eight subunits. Here we show that the Dgt6 component of augmin is primarily required for kinetochore fiber (k-fiber) formation. An analysis of MT regrowth after cold exposure showed that formation of kinetochore-driven k-fibers is severely impaired in Dgt6-depleted cells. In control cells, these fibers are enriched in Dgt6, gamma-tubulin, and Msps/XMAP215. Consistent with these results, Dgt6 coprecipitates with Msps, D-TACC, gamma-tubulin, Ndc80, and Nuf2. However, RNA interference (RNAi)-mediated inhibition of gamma-tubulin, Msps/XMAP215, or Ndc80/Hec1 reduced but did not abolish k-fiber regrowth. These results indicate that Dgt6 plays a pivotal role in kinetochore-driven k-fiber formation, mediating nucleation and/or initial stabilization of chromosome-induced MTs. We propose that Dgt6 binds and stabilizes nascent chromatin-induced MTs, facilitating their interaction with the Ndc80-Nuf2 complex. Dgt6 may also promote elongation of kinetochore-driven k-fibers through its interaction with gamma-tubulin and Msps.

Bypassing the Greatwall–Endosulfine Pathway: Plasticity of a Pivotal Cell-Cycle Regulatory Module in Drosophila melanogaster and Caenorhabditis elegans

In vertebrates, mitotic and meiotic M phase is facilitated by the kinase Greatwall (Gwl), which phosphorylates a conserved sequence in the effector Endosulfine (Endos). Phosphorylated Endos inactivates the phosphatase PP2A/B55 to stabilize M-phase-specific phosphorylations added to many proteins by cyclin-dependent kinases (CDKs). We show here that this module functions essentially identically in Drosophila melanogaster and is necessary for proper mitotic and meiotic cell division in a wide variety of tissues. Despite the importance and evolutionary conservation of this pathway between insects and vertebrates, it can be bypassed in at least two situations. First, heterozygosity for loss-of-function mutations of twins, which encodes the Drosophila B55 protein, suppresses the effects of endos or gwl mutations. Several types of cell division occur normally in twins heterozygotes in the complete absence of Endos or the near absence of Gwl. Second, this module is nonessential in the nematode Caenorhaditis elegans. The worm genome does not contain an obvious ortholog of gwl, although it encodes a single Endos protein with a surprisingly well-conserved Gwl target site. Deletion of this site from worm Endos has no obvious effects on cell divisions involved in viability or reproduction under normal laboratory conditions. In contrast to these situations, removal of one copy of twins does not completely bypass the requirement for endos or gwl for Drosophila female fertility, although reducing twins dosage reverses the meiotic maturation defects of hypomorphic gwl mutants. These results have interesting implications for the function and evolution of the mechanisms modulating removal of CDK-directed phosphorylations.

A signature inferred from Drosophila mitotic genes predicts survival of breast cancer patients.

Introduction The classification of breast cancer patients into risk groups provides a powerful tool for the identification of patients who will benefit from aggressive systemic therapy. The analysis of microarray data has generated several gene expression signatures that improve diagnosis and allow risk assessment. There is also evidence that cell proliferation-related genes have a high predictive power within these signatures. Methods We thus constructed a gene expression signature (the DM signature) using the human orthologues of 108 Drosophila melanogaster genes required for either the maintenance of chromosome integrity (36 genes) or mitotic division (72 genes). Results The DM signature has minimal overlap with the extant signatures and is highly predictive of survival in 5 large breast cancer datasets. In addition, we show that the DM signature outperforms many widely used breast cancer signatures in predictive power, and performs comparably to other proliferation-based signatures. For most genes of the DM signature, an increased expression is negatively correlated with patient survival. The genes that provide the highest contribution to the predictive power of the DM signature are those involved in cytokinesis. Conclusion This finding highlights cytokinesis as an important marker in breast cancer prognosis and as a possible target for antimitotic therapies.

The relative roles of centrosomal and kinetochore-driven microtubules in Drosophila spindle formation

Mitotic spindle assembly in centrosome-containing cells relies on two main microtubule (MT) nucleation pathways, one based on centrosomes and the other on chromosomes. However, the relative role of these pathways is not well defined. Here we review the studies on spindle formation in Drosophila centrosome-containing cells. Mutants with impaired centrosome function assemble functional anastral spindles in somatic tissues and survive to adulthood. In contrast, mutants defective in chromosome-driven MT formation form highly aberrant mitotic spindles and die at larval stages. The requirements for spindle assembly in Drosophila male meiotic cells are diametrically opposed to those of somatic cells. Spermatocytes assemble morphologically normal spindles in the complete absence of chromosome-induced MTs, but are unable to organize a functional spindle in the absence of centrosomal MTs. Male meiotic spindles are much larger than mitotic spindles as they contain most of the tubulin needed for sperm tail formation. We suggest that the centrosome-based mechanism of spindle assembly in spermatocytes reflects their need for rapid and efficient polymerization of a particularly large amount of tubulin.

A Role for the Twins Protein Phosphatase (PP2A-B55) in the Maintenance of Drosophila Genome Integrity

The protein phosphatase 2A (PP2A) is a conserved heterotrimeric enzyme that regulates several cellular processes including the DNA damage response and mitosis. Consistent with these functions, PP2A is mutated in many types of cancer and acts as a tumor suppressor. In mammalian cells, PP2A inhibition results in DNA double strand breaks (DSBs) and chromosome aberrations (CABs). However, the mechanisms through which PP2A prevents DNA damage are still unclear. Here, we focus on the role of the Drosophila twins (tws) gene in the maintenance of chromosome integrity; tws encodes the B regulatory subunit (B/B55) of PP2A. Mutations in tws cause high frequencies of CABs (0.5 CABs/cell) in Drosophila larval brain cells and lead to an abnormal persistence of γ-H2Av repair foci. However, mutations that disrupt the PP4 phosphatase activity impair foci dissolution but do not cause CABs, suggesting that a delayed foci regression is not clastogenic. We also show that Tws is required for activation of the G2/M DNA damage checkpoint while PP4 is required for checkpoint recovery, a result that points to a conserved function of these phosphatases from flies to humans. Mutations in the ATM-coding gene tefu are strictly epistatic to tws mutations for the CAB phenotype, suggesting that failure to dephosphorylate an ATM substrate(s) impairs DNA DSBs repair. In addition, mutations in the Ku70 gene, which do not cause CABs, completely suppress CAB formation in tws Ku70 double mutants. These results suggest the hypothesis that an improperly phosphorylated Ku70 protein can lead to DNA damage and CABs.

The Drosophila orthologue of the INT6 onco-protein regulates mitotic microtubule growth and kinetochore structure

INT6/eIF3e is a highly conserved component of the translation initiation complex that interacts with both the 26S proteasome and the COP9 signalosome, two complexes implicated in ubiquitin-mediated protein degradation. The INT6 gene was originally identified as the insertion site of the mouse mammary tumor virus (MMTV), and later shown to be involved in human tumorigenesis. Here we show that depletion of the Drosophila orthologue of INT6 (Int6) results in short mitotic spindles and deformed centromeres and kinetochores with low intra-kinetochore distance. Poleward flux of microtubule subunits during metaphase is reduced, although fluorescence recovery after photobleaching (FRAP) demonstrates that microtubules remain dynamic both near the kinetochores and at spindle poles. Mitotic progression is delayed during metaphase due to the activity of the spindle assembly checkpoint (SAC). Interestingly, a deubiquitinated form of the kinesin Klp67A (a putative orthologue of human Kif18A) accumulates near the kinetochores in Int6-depleted cells. Consistent with this finding, Klp67A overexpression mimics the Int6 RNAi phenotype. Furthermore, simultaneous depletion of Int6 and Klp67A results in a phenotype identical to RNAi of just Klp67A, which indicates that Klp67A deficiency is epistatic over Int6 deficiency. We propose that Int6-mediated ubiquitination is required to control the activity of Klp67A. In the absence of this control, excess of Klp67A at the kinetochore suppresses microtubule plus-end polymerization, which in turn results in reduced microtubule flux, spindle shortening, and centromere/kinetochore deformation.