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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - RAGES (Molecular determination of Rif1-Associated Genomic Elements and their function in regulating genome activity and integrity)

Teaser

Summary

DNA double-strand breaks (DSBs) are highly toxic and must usually be accurately repaired to prevent oncogenic mutations. However, DSBs also represent necessary intermediates of recombination events required to create genetic diversity in immune repertoires and the germline. These distinct cellular contexts require that DSBs are differentially metabolised to achieve the required genetic outcome. Thus a complex system has evolved to regulate DSB repair. Rif1 is a critical regulator of DSB repair, whose recruitment to chromatin at DSBs by the 53BP1 chromatin reader plays a critical role in protecting the integrity of DNA ends during DSB break repair by the non-homologous end joining pathway. However, little is known about how 53BP1-RIF1 protein complexes regulate chromatin structure at DNA damage sites, and how this impacts on the molecular mechanisms that underpin DNA repair. This proposal aimed to discover the mechanism in which Rif1 regulates DSB repair.

Understanding the molecular function of the RIF1 protein is paramount, as mis-regulation of DSB repair at the level of Rif1/53BP1-deficiency manifests in primary immunodeficiency in mammals. Conversely, an inability to counteract Rif1/53BP1- dependent activities during DNA repair is associated with genomic instability that drives carcinogenesis. Interestingly, evidence suggests that Rif1 may also mediate gene-repression, raising the possibility that common Rif1-dependent mechanisms may regulate gene transcription and DNA repair.

In examining: (1) the chromosomal contexts in which Rif1 operates; (2) the factors with which Rif1 cooperates; and (3) the phenotypic consequences of Rif1 loss; this proposal set out to test the overarching hypothesis that a common activity exerted by the Rif1 protein in chromatin plays a vital role in regulating both gene silencing and DNA repair activities. Thus successful completion of this project aims to discover vital, yet hitherto undefined mechanism that may link genome activity and stability.

Work performed

In contingency of initial failed attempts to tag the endogenous Rif1 gene in mammalian cells for downstream immunofluorescence microscopy and proteomics approaches, the RIF1 gene was deleted in the human breast cancer cell-line MCF-7 using CRISPR/Cas9 technology. In preliminary experiments, Rif1-/- MCF-7 were found to recapitulate the reported phenotypes in Rif1-/- mouse embryonic fibroblast cell-lines, namely, chronic basal activation of the p21 cell-cycle inhibitor, reduced S phase index and slow cellular growth. In downstream experiments, a series of Rif1 domain-deletion transgenes, expressed from an inducible, tetracycline-responsive promoter, were complemented into Rif1-/- in view of enabling the mapping of the minimal sequence requirements for Rif1 localisation to sites of IR-induced DNA damage. This revealed that sequences located within the amino- and carboxyl- terminus of Rif1 are required for its association with chromatin at DNA damage sites. The carboxyl-terminus of Rif1 comprises both reported oligomerisation and BLM interacting sequences, indicates that protein oligomerisation and/or BLM interactions may facilitate Rif1 recruitment to sites of DNA damage. Future studies in which the independent functions and protein interactions mediated by the Rif1 N- and C- terminal domains will enable for a better understanding of Rif1â€™s function in DNA repair.

In parallel, collaborative published work performed as part of the RAGES MSCIF action contributed to a better understanding of cell-cycle control by the Rif1-interacting protein 53BP1, its upstream regulator during DNA repair, via its function as a p53-regulatory protein (doi: 10.1016/j.molcel.2016.08.002.).

Final results

State-of-the-art cell-line tools were generated that are likely to facilitate the downstream functional characterisation of the vital genome regulator Rif1. Results from potential future work will reveal mechanisms that are essential for the regulation of genome organisation and the mechanisms that maintain genome stability. Given Rif1\'s role in mammalian adaptive immunity and tumourigenesis, these results may enable the better understanding of human physiology and disease.