MEIOSIS&DEVELOPMENT · Control of meiosis and oocyte development by DNA damage checkpoints in Drosophila melanogaster
FP7 — People (Marie Curie Actions)
- Duration
- 2009-08-01 → 2013-07-31
- EU contribution
- €100,000
- Participants
- 1
- Scheme
- MC-IRG
Lines connect the coordinator with its partners.
Results in brief
Control of meiosis and oocyte development by DNA damage checkpoints in Drosophila melanogaster.
Our work was focused on two major areas. One related to chromatin surveillance during meiosis and germinal vesicle development, and another concerning the activation of the DNA damage response (DDR) late in oogenesis due to deficient function of genes related to the PIWI-interacting germ line-specific family of non-coding RNAs, piRNAs. In Attachement, the two manuscripts in preparation. SilvaEtAl referes to the dPds5 function in germinal vesicle architecture. LaosEtAl referes to the piRNA screen. Chromatin Surveillance: A relatively long prophase I arrest is conserved among animals. Following its condensation into a karysome the Drosophila meiotic chromatin appears inert until metaphase I. We show that the cohesion protein dPDS5 localizes in foci specifically in the germinal vesicle. These foci change stereotypically in number and never touch the karyosome. We identified the dPDS5 foci as insulator bodies as they co-localize with the insulator proteins CP190, Mod(mdg4)2.2 and BEAF32. CP190 and dPDS5 physically interact mainly in the oocyte as the interaction is mostly abolished in ovarian extracts from egalitarian mutants. Moreover, loss of dPds5 causes over-accumulation of CP190 protein in large bodies, which occur in the GV and never in equally mutant nurse cells. Although not monitored by the dATR pathway dPDS5 is under checkpoint surveillance. One of the two peaks of normal oocyte-specific transcription during Prophase I arrest is delayed due to checkpoint activation. Our results suggest that GVs are not arrested in prophase I. We propose a new insulator -dependent karyosome model. Genome protection: Piwi associated small RNAs – piRNAs – function in animal germline to protect genome against mobilization of transposable elements (TEs). In Drosophila, failure of such protection activates a CHK2-mediated checkpoint in the germ line during mid oogenesis generating ribonucleoproteic clumps in the germ cell cytoplasm. To identify piRNA genes we screened a collection of eggshell ventralized mutantions using a clump marker the cargo linker protein BicD. Of the 121 mutations screened, 24 were clump-forming. Clump morphology and validation by testing both TE upregulation and checkpoint activation revealed two major classes. The larger class contains 15 mutations, which consist of alleles of the known piRNA genes krimper, cutoff, tejas and rhino as well as three new loci. Mutants in the second class neither upregulate TEs nor activate CHK2 and form a large clump in the middle of germ cell cysts resembling the phenotype of rab6 mutants. We discuss the efficiency of our screening method and potential role of the new candidates in the pathway.
Data: CORDIS, © European Union
Project objective
DNA damage response (DDR) monitors chromosome status to ensure correct homologous recombination, genomic integrity and chromosome segregation. Thus the effect of DDR activation upon exogenous DNA breaks has been intensely studied. Less is known about the effects of such mechanisms due to endogenous breaks. In multicellular organisms meiosis is a classical example of double strand break (DSB) production and repair occurring concomitantly with gamete development. In Drosophila persistent meiotic DSBs activates the ATR/Mei-41 checkpoint, delays progression through meiosis and causes defects in DNA condensation in the oocyte nucleus. Checkpoint activation has also been linked to decreased levels of the TGFα-like molecule Gurken (GRK), which controls normal eggshell patterning. I used this easy scorable eggshell phenotype in a germ line mosaic screen to identify new genes affecting meiotic progression, DNA condensation and GRK signalling. My work on these mutants challenged the hypothesis that checkpoint activation upon persistent DSBs is exclusively mediated by ATR kinase and instead reveals a more complex network of interactions that link DSB formation, checkpoint activation, meiotic delay, DNA condensation and GRK synthesis. To understand how multicellular organisms monitor chromatin integrity during meiosis and how these surveillance mechanisms affect development I propose the study of the cohesion gene dPds5 in early and late meiosis, the characterization of two rasiRNA genes montecristo and sancho-panza in transposon silencing during oogenesis and the identification of genes downstream of checkpoint pathway mediating eggshell polarity response upon checkpoint activation. I discuss two major new ideas explored in this project: 1. cohesion is required for eggshell polarity, and 2. various branches of the DDR monitor aspects of chromatin independently of DSB.
Original text from CORDIS.
Participants
- FUNDACAO CALOUSTE GULBENKIAN · LisboaCoordinatorPortugal
Links
Data: CORDIS, © European Union