ESR5: “Mechanisms underlying ubiquitin-mediated DNA damage responses to telomere deprotection”

NKI (J. Jacobs group): Mechanisms underlying ubiquitin-mediated DNA damage responses to telomere deprotection

i. Objective of research: Evaluate how DNA repair at deprotected telomeres is controlled by ubiquitylation.

ii. Current state of the art: Maintenance of genome integrity requires appropriate detection, timely and error-free repair of DNA lesions, as well as inhibition of DDR and DNA repair at natural chromosome ends. The latter is governed by telomeres, specialized nucleoprotein structures at chromosome ends that help distinguish these from broken DNA to allow proliferation and prevent repair activities from causing genome instability, which would otherwise contribute to premature aging and cancer. Ubiquitylation has been recognized as playing important regulatory roles in the DDR, also at telomeres, but much of the underlying mechanisms and proteins involved remains unknown. To better understand the mechanisms underlying DNA repair control at telomeres we previously performed genetic screens to identify factors with critical roles in telomere-driven genomic instability, called TIGIRs (for telomere induced genomic instability regulators). This approach, using temperature-dependent TRF2 inactivation, allowed us to identify multiple new factors controlling NHEJ at telomeres, including proteins involved in ubiquitylation. Our follow-up work identified important contributions by ubiquitin E2 and E3 enzymes previously linked to the protein quality control pathway or H2B-ubiquitination, but how these processes are important to NHEJ at telomeres is unclear. Here, we aim to obtain further insights in how these factors, called ubiquitin-TIGIRs, contribute to DNA repair control.

iii. Research methodology and approach: We will focus on elucidating the roles of our recently identified ubiquitin-TIGIR factors in controlling NHEJ at uncapped telomeres, and potentially elsewhere. We will address whether they control the recognition of uncapped telomeres by DDR proteins or their processing by DNA repair proteins. For this we will make use of well controllable telomere uncapping systems in MEFs and human cells, such as fast temperature-dependent inactivation of TRF2. We will also address whether these TIGIR factors act in a unique manner at telomeres or affect DNA repair similarly at DNA lesions elsewhere. Furthermore, we will examine if these factors control NHEJ at telomeres through their previously reported roles in protein quality control or H2B ubiquitination or whether they act in a different manner. We aim to reveal through which ubiquitination events these TIGIR factors operate, based on the alterations in DDR and DNA repair observed upon their inactivation and facilitated by recent diGLy-proteomics data that we generated on ubiquitome changes upon telomere uncapping.

iv. Originality and innovative aspects of the ESR project: Our novel implication of several ubiquitin system components in telomere uncapping responses, incorporating our recent diGly proteomics data and a unique telomere uncapping system, will allow us to obtain important new insights in how ubiquitylation contributes to telomere uncapping responses.

v. Integration of the ESR project to the overall research programme: Our ESR will closely work with the Lingner group on dissecting the functional relevance of ubiquitination in telomere uncapping, with the Mailand group on mass spectrometry data analysis and with Norgenotech on the development of assays for the detection of NHEJ-related DNA lesions.