We first successfully set up laser microirradiation and subsequent time-lapse imaging with a new spinning disk confocal microscope (available at the in-house microscopy core facility). Using this system, we characterized the relative mobility of the entire hnRNP family at DSBs, both in space and time. We then proceeded to identify the determinants of hnRNP exclusion. This end, we assessed whether inhibition of key components of early, medium and late DSB response affects hnRNP localization to laser microirradiation-induced DSBs, also studied the impact of transcription activation and blockage on hnRNPs mobility at DSBs, and mapped relevant domains on a model hnRNP factor. Together, the results from these experiments provided new insights into the signalling mechanisms that coordinate hnRNPs exclusion at DNA damage sites.
In addition, we also performed experiments to assess the functional significance of hnRNP exclusion for DNA repair. To this end, we generated hnRNP transgenes that can be stably tethered to DNA breaks and assessed the impact of hnRNP tethering on DSB signalling and repair. Our results showed that the exclusion of hnRNPs from damaged chromatin is essential for proper activation of the DSB response.
To identify the protein complexes associated with excluded hnRNPs at DSB sites, we made use of the TurboID system, a technology based on the expression of a fusion between a bait protein (here hnRNPs) and the E.coli biotin biotin ligase mutant TurboID. In the presence of biotin, this mutant biotinylates proteins that are near neighbours of the fusion proteins (about within a radius of 10 nm) thus allowing for the efficient isolation of biotinylated proteins and subsequent identification by mass spectrometry (MS). To date, we have generated HEK293 cells expressing TurboID-hnRNPF and performed MS analysis. We are currently extending this MS setup to other hnRNP family members. Ultimately, we anticipate that the MS results will be critical to decipher how the hnRNPs interact with each other and with other proteins at DSBs, and how the different complexes are integrated into the overall DSB response.
