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Dna Repair In Transposon Elements And Retroelements

Reference : Phd Evi SOUTOGLOU

Offer publication : April 6, 2016

DNA double strand breaks (DSBs) are among the rarest but also the most cytotoxic lesions affecting DNA integrity and their efficient repair is essential, as they are the origin of genome instability, chromosomal translocations, cellular transformation and cancer. Cells respond to DSBs by initiating the DNA damage response (DDR), a signaling cascade, which leads to the activation of cell cycle checkpoints and DNA repair by two main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). HR allows faithful repair and takes place in the replicative and post-replicative stages of the cell cycle (S/G2), when sister chromatids are present. In addition to these, other back up DNA repair pathways have been described: Alternative End Joining (AEJ), Break Induced Replication (BIR) and sister chromatid annealing (SSA). All these pathways are highly mutagenic and are usually predominate when the main pathways are perturbed.

Therefore, controlling the balance between these different DNA repair pathways is very important to suppress mutagenic events and limit their oncogenic potential. Although recent studies suggest that histone pos-translational modifications influence the choice in DNA repair pathway at a specific genomic location, the mechanisms involved in repairing DSBs in repressive chromatin environments remain elusive. Moreover, it is not clear how the different types of repressive chromatin (facultative, null
chromatin, the different types of constitutive heterochromatin) regulate this choice. 40% of the mammalian genome is comprised of heterochromatic interspersed repeats, the retroelements fall into two major classes: long and short interspersed nuclear elements (LINEs and SINEs; 27%) and
endogenous retroviruses (ERVs; 10%). ERVs are silenced during the first few days of embryogenesis by the transcriptional corepressor KAP1 that induces heterochromatinization and repressive histone modifications like H3K9me3 and Heterochromatin protein HP1.

To further investigate DDR and DNA repair in different heterochromatin domains, we use the system described above to induce breaks at retroelements in mESCs. To this end, we
will utilize the CRISP/Cas 9 system to induce specifically DSBs in these retroelements. We have designed guide RNAs that match sequences of known ERVs (IAP1) and LINE1 sequences and will assess recruitment of DDR and DNA repair factors by ChIP or Immnuno FISH at these sites.

Subsequently, we will test whether induction of a break influences chromatin status of the retroelement and as a result its expression and therefore its ability to “jump” and reintegrate. To this end, ChIP experiments will be performed using different antibodies for heterochromatin markers and histone
modifications and expression pattern of specific retroelements will be checked by qPCR and “reintegration” assays. These results will have tremendous implications in the genomic instability caused by reactivation of retroelements.

- WISHED SKILLS : Molecular biology skills

- EXPERTISES WHICH WILL BE ACQUIRED DURING THE TRAINING : DNA repair, CRISP/Cas9, chromatin, microscopy, superresolution microscopy,biochemistry.

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Application Deadline : Dec. 31, 2016

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Université de Strasbourg

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