Analyse à haute resolution de la chromatine pendant la reparation des cassures Doubles Brins de l'ADN – ChIP'ing breaks

DNA Double Strand Breaks (DSBs) are highly harmful lesions since they can lead to mutations and chromosome rearrangements, themselves promoting tumour progression. Eucaryotic cells have thus developed efficient DNA repair processes, mainly homologous recombination (HR), and Non Homologous End Joining (NHEJ), to face this danger. However DNA packaging into chromatin creates a significant barrier to DSBs detection and repair, and therefore, major chromatin remodelling events accompany DNA repair. Due to the lack of convenient tools, the chromatin changes induced by and around DSBs, together with their functions, still remains largely unknown, especially in mammalians cells. We have developed a new experimental system, based on the use of a restriction enzyme fused to the ligand binding domain of the oestrogen receptor (AsiSI-ER), which permits a tightly controlled induction of sequence-specific DSBs (at known loci) throughout the genome, in human cells. This new DSB-inducible system, combined with high throughput genome wide technologies (such as ChIP-chip, or GCC), provides now the unique opportunity to study specific chromatin changes during the repair of DSBs, since it enables high resolution profiling of any DSB induced chromatin modification and DNA repair proteins around breaks. Using this system, we have already established the first high resolution map of a DSB-induced chromatin modification (gammaH2AX), and have investigated its spreading properties (manuscript submitted). With such a system in hand, we propose to proceed in an investigation of the relationships between chromatin structure and DSB repair. First of all, we plan to map, by ChIP-chip, various chromatin modifications and DNA repair proteins, around DSBs. Such a thorough description of the chromatin landscape(s) before and after DSBs induction should give insights concerning chromatin modification events that occur during DNA repair. In addition, by monitoring the state of chromatin before DNA damage, we should be able to characterize the influence of chromatin structure, on DNA repair efficiency (specifically concerning the choice between NHEJ and HR). To further gain insights in the influence of chromatin structure on DNA repair, we will also utilize a model already extensively used to study the role of chromatin in transcription: the drosophila 'dosage compensation' process. Indeed, in drosophila, the male X chromosome exihibit very specific chromatin features, to ensure proper equalisation of X genes transcription between male and female, through a process called 'dosage compensation'. We want to take advantage of this different chromatin structure between X chromosome and autosomes, to study the influence of chromatin on DSB repair. We plan to perform these studies in male (SL2) and female (Kc) drosophila cell lines, stably transfected with the AsiSI-ER fusion enzyme. We will also examine the tri-dimensional genome reorganisation upon DSB induction, using GCC (Genome Conformation Capture), a very new technique developed by Dr O'Sullivan (Rodley, C.D.F et al, submitted), in order to better understand the clustering of DSB that occur in the whole nucleus and the function of such clustering events. Finally we will develop whole organism models (transgenic flies and mice) expressing the AsiSI-ER fusion enzyme, in a spatially and temporally controlled manner, in order to dissect, at a molecular level, DSB repair in vivo in various cell types and across differentiation (nervous system, germ cells, stem cells...) The completion of this project will undoubtedly lead to important breakthroughs in our understanding of the role of chromatin in DSB repair. Since the impairment of DNA repair processes is involved in many human diseases such as cancer, neurodegenerative diseases, ageing and sterility, a better understanding of the molecular processes involved in DNA repair will assist in the design of new therapeutic strategies targeting such diseases. An additional benefit of the successful completion of the proposed analyses stems from our desire to deposit all data and computational methodology into the public domain. Both information about chromatin structure and its relationship to DNA damage repair as well as methods used to harness the massive amounts of data, will be invaluable to researchers in many fields including DNA repair, transcription and chromatin structure.