Final Report Summary - ERC-ID (Excision Repair and chromatin interaction dynamics)
The genetic information (DNA) is continuously damaged by cellular metabolites, environmental agents and radiation, which disturbs cellular processes and induces mutations. Different repair systems remove DNA damage, nucleotide excision repair (NER) is likely the most versatile process as it removes many different lesions. Inherited NER-defects has severe clinical consequences, such as strong cancer-susceptibility and premature aging. Detailed insight into this process (aim of this project) is highly relevant, since cancer and age-related discomfort are among the major clinical problems in our aging society. In this project we used an integrated multi-disciplinary approach to dissect NER, and obtained significant new insights on how NER functions in vivo; highlights are summarized below.
1) Transcription-coupled NER (TC-NER), removes DNA damage that block gene transcription. TC-NER-deficiency causes the premature aging Cockayne syndrome (CS). We developed a sensitive TC-NER assay (>20 x more sensitive than previous), allowing TC-NER quantification at physiological low doses of DNA damage (PMID:28088761).
2) We developed a new research-approach by combining live-cell imaging with targeted isolation of cellular proteins; interacting proteins can be discerned by Mass-Spectrometry (MS). Cells were engineered to express fluorescently-marked (GFP) NER-proteins. This GFP-mark allows NER tracking in living cells, and to specifically isolate NER-interacting proteins. The function of newly identified interactors was directly tested in living cells, using the same GFP-NER protein expressing cells. With this procedure we identified novel interactors of the TC-NER factor CSA, that are required to properly shape CSA (PMID:29531219), others are under investigation.
3) We found a new role for the TC-NER factor CSB in removing oxidative DNA lesions (PMID:29955842), resolving a long-standing controversy in the field on its involvement in removing these lesions.
5) We found that specific XPF mutations that cause XP and CS, induced persistent DNA binding of NER that disturbs all DNA-associated processes. This provided, for the first time, direct insight into the genotype-phenotype relationship of XPF mutations (PMID:30165384).
Together these findings, disclosed important new insight into the vital TC-NER process.
6) We identified a novel DNA damage signaling pathway, induced by lesion-blocked transcription which triggered non-canonical ATM (well-known DDR signaling kinase) activation (PMID:26106861). This observation is highly relevant to understand the thus far unexplained neurologic features in ATM-deficient patients.
7) DNA is compacted by wrapping around histone proteins, designated chromatin. To allow DNA repair, chromatin needs to be remodeled. We identified a specific chromatin-remodeler that facilitates TC-NER (PMID:24990377).
8) We found a specific DNA damage-induced chemical modification of histones (acetylation) (PMID:30104204) and identified the HUWE1 enzyme that modifies histones with the ubiquitin protein (PMID:29127375). Both processes control DNA damage signaling and revealed that chromatin-modification is involved in this response, one of the main project’s research goals.
10) We found that TC-NER and base excision repair (next to previously known other repair pathways) are required to remove DNA lesions induced by Platinum-based chemotherapeutics (PMID:30137419). This finding is important for stratifying tumors for their vulnerability to chemotherapeutic drugs.
11) We showed that affecting the stability of TFIIH (crucial NER-factor) by depletion of the SWI/SNF-chromatin-remodeler renders cells hypersensitive to Platinum-based chemotherapeutics (PMID: 30287812). Mutations in SWI-subunits are frequently found in different cancer types, illustrating the importance of this finding for stratifying tumors (biomarker) and that SWI/SNF may serve as a potential drugable target (PMID:30897376).
12) We developed a unique imaging platform to determine NER in living C. elegans (worm). NER in this, easy accessible (genetically/microscopically), model organism is highly similar to mammalian, making it perfectly suited to quantify cell-specific repair in vivo. Since this platform is more economical and with less ethical issues than mouse-models, future studies on the complex genotype-phenotype relationship of NER-deficiency will be performed with this system.
1) Transcription-coupled NER (TC-NER), removes DNA damage that block gene transcription. TC-NER-deficiency causes the premature aging Cockayne syndrome (CS). We developed a sensitive TC-NER assay (>20 x more sensitive than previous), allowing TC-NER quantification at physiological low doses of DNA damage (PMID:28088761).
2) We developed a new research-approach by combining live-cell imaging with targeted isolation of cellular proteins; interacting proteins can be discerned by Mass-Spectrometry (MS). Cells were engineered to express fluorescently-marked (GFP) NER-proteins. This GFP-mark allows NER tracking in living cells, and to specifically isolate NER-interacting proteins. The function of newly identified interactors was directly tested in living cells, using the same GFP-NER protein expressing cells. With this procedure we identified novel interactors of the TC-NER factor CSA, that are required to properly shape CSA (PMID:29531219), others are under investigation.
3) We found a new role for the TC-NER factor CSB in removing oxidative DNA lesions (PMID:29955842), resolving a long-standing controversy in the field on its involvement in removing these lesions.
5) We found that specific XPF mutations that cause XP and CS, induced persistent DNA binding of NER that disturbs all DNA-associated processes. This provided, for the first time, direct insight into the genotype-phenotype relationship of XPF mutations (PMID:30165384).
Together these findings, disclosed important new insight into the vital TC-NER process.
6) We identified a novel DNA damage signaling pathway, induced by lesion-blocked transcription which triggered non-canonical ATM (well-known DDR signaling kinase) activation (PMID:26106861). This observation is highly relevant to understand the thus far unexplained neurologic features in ATM-deficient patients.
7) DNA is compacted by wrapping around histone proteins, designated chromatin. To allow DNA repair, chromatin needs to be remodeled. We identified a specific chromatin-remodeler that facilitates TC-NER (PMID:24990377).
8) We found a specific DNA damage-induced chemical modification of histones (acetylation) (PMID:30104204) and identified the HUWE1 enzyme that modifies histones with the ubiquitin protein (PMID:29127375). Both processes control DNA damage signaling and revealed that chromatin-modification is involved in this response, one of the main project’s research goals.
10) We found that TC-NER and base excision repair (next to previously known other repair pathways) are required to remove DNA lesions induced by Platinum-based chemotherapeutics (PMID:30137419). This finding is important for stratifying tumors for their vulnerability to chemotherapeutic drugs.
11) We showed that affecting the stability of TFIIH (crucial NER-factor) by depletion of the SWI/SNF-chromatin-remodeler renders cells hypersensitive to Platinum-based chemotherapeutics (PMID: 30287812). Mutations in SWI-subunits are frequently found in different cancer types, illustrating the importance of this finding for stratifying tumors (biomarker) and that SWI/SNF may serve as a potential drugable target (PMID:30897376).
12) We developed a unique imaging platform to determine NER in living C. elegans (worm). NER in this, easy accessible (genetically/microscopically), model organism is highly similar to mammalian, making it perfectly suited to quantify cell-specific repair in vivo. Since this platform is more economical and with less ethical issues than mouse-models, future studies on the complex genotype-phenotype relationship of NER-deficiency will be performed with this system.