The transcription/DNA repair factor IIH (TFIIH) is made of two distinct sub-complexes, the core-TFIIH and the CDK-activating kinase (CAK), bridged together by the XPD subunit. The CDK7 kinase subunit of the CAK sub-complex targets different substrates according to the protein complex it belongs to: as free-CAK it phosphorylates specific cyclin-dependent kinases (Cdks) and promotes cell cycle progression; as subunit of the entire TFIIH (holo-TFIIH), CDK7 phosphorylates and activates RNA polymerase II and specific transcription factors. In this context, the bridging factor XPD plays a key role in modulating the association/dissociation between the CAK and the core-TFIIH, thus linking transcription to cell cycle control and DNA repair. Mutations in the ERCC2/XPD gene are responsible for distinct clinical entities, including the cancer-prone xeroderma pigmentosum (XP) and the multisystemic cancer-free trichothiodystrophy (TTD). To understand how mutations in the same gene give rise to hereditary disorders with opposite cancer proneness, we investigated whether XPD mutations affect the cellular composition of TFIIH complex and, in turn, the CAK-mediated signaling pathways. Therefore, by native chromatin immunoprecipitation studies, we investigated the association/dissociation dynamic of the two TFIIH sub-complexes throughout the cell cycle and found that in XPD mutated fibroblasts, isolated from either XP or TTD patients, most of the CAK sub-complex dissociates from the chromatin and from the core-TFIIH. Considering that the CAK substrate specificity depends on it free or core-bound state, we looked at the protein interaction profile of CDK7 kinase subunit both in physiological and pathological conditions. The CDK7 immunoprecipitation followed by mass spectrometry analysis allowed the identification of new CDK7 interactors either associated or detached from the chromatin. Some of the identified proteins display a different interaction profile in TTD and/or XP cells, compared to control fibroblasts. We focused our attention on DDX1, an ATP-dependent RNA helicase that is involved in the resolution of RNA/DNA hybrids accumulated at DNA double strand breaks. We found that the interaction between DDX1 and the chromatin-bound CDK7 is stronger in TTD primary and immortalized fibroblasts than in XP or control fibroblasts. To gain knowledge on the functionality of the identified interaction we performed various in vitro assays. Upon purification of DDX1 from E. coli and XPD, CAK and TFIIH complex from Baculovirus and in vitro co-immunoprecipitation experiments, we established that DDX1 binds not only the CAK sub-complex but also the XPD subunit and the core-TFIIH. Moreover, the presence of XPD stabilizes the DDX1 interaction with the CAK but not with the core-TFIIH. We found that the C-terminal region of XPD is essential for DDX1 binding and that TTD- and XP-specific point mutations mapping in the C-terminus of XPD do not affect their interaction in vitro. Finally, by in vitro kinase assay we assessed that DDX1 is a substrate of the free-CAK but not of the holo-TFIIH complex. Future studies will clarify the functional relevance of the CAK-XPD interaction with DDX1. Overall, our results point to an altered association of the CAK to the chromatin as consequence of XPD mutations and identify DDX1 as a novel interactor of CDK7, showing a different binding profile in TTD compared to control and XP fibroblasts.

Identification of DDX1 as a novel interactor of CDK-Activating Kinase and its implication in Trichothiodystrophy disorder

FERRI, DEBORA
2020-01-10

Abstract

The transcription/DNA repair factor IIH (TFIIH) is made of two distinct sub-complexes, the core-TFIIH and the CDK-activating kinase (CAK), bridged together by the XPD subunit. The CDK7 kinase subunit of the CAK sub-complex targets different substrates according to the protein complex it belongs to: as free-CAK it phosphorylates specific cyclin-dependent kinases (Cdks) and promotes cell cycle progression; as subunit of the entire TFIIH (holo-TFIIH), CDK7 phosphorylates and activates RNA polymerase II and specific transcription factors. In this context, the bridging factor XPD plays a key role in modulating the association/dissociation between the CAK and the core-TFIIH, thus linking transcription to cell cycle control and DNA repair. Mutations in the ERCC2/XPD gene are responsible for distinct clinical entities, including the cancer-prone xeroderma pigmentosum (XP) and the multisystemic cancer-free trichothiodystrophy (TTD). To understand how mutations in the same gene give rise to hereditary disorders with opposite cancer proneness, we investigated whether XPD mutations affect the cellular composition of TFIIH complex and, in turn, the CAK-mediated signaling pathways. Therefore, by native chromatin immunoprecipitation studies, we investigated the association/dissociation dynamic of the two TFIIH sub-complexes throughout the cell cycle and found that in XPD mutated fibroblasts, isolated from either XP or TTD patients, most of the CAK sub-complex dissociates from the chromatin and from the core-TFIIH. Considering that the CAK substrate specificity depends on it free or core-bound state, we looked at the protein interaction profile of CDK7 kinase subunit both in physiological and pathological conditions. The CDK7 immunoprecipitation followed by mass spectrometry analysis allowed the identification of new CDK7 interactors either associated or detached from the chromatin. Some of the identified proteins display a different interaction profile in TTD and/or XP cells, compared to control fibroblasts. We focused our attention on DDX1, an ATP-dependent RNA helicase that is involved in the resolution of RNA/DNA hybrids accumulated at DNA double strand breaks. We found that the interaction between DDX1 and the chromatin-bound CDK7 is stronger in TTD primary and immortalized fibroblasts than in XP or control fibroblasts. To gain knowledge on the functionality of the identified interaction we performed various in vitro assays. Upon purification of DDX1 from E. coli and XPD, CAK and TFIIH complex from Baculovirus and in vitro co-immunoprecipitation experiments, we established that DDX1 binds not only the CAK sub-complex but also the XPD subunit and the core-TFIIH. Moreover, the presence of XPD stabilizes the DDX1 interaction with the CAK but not with the core-TFIIH. We found that the C-terminal region of XPD is essential for DDX1 binding and that TTD- and XP-specific point mutations mapping in the C-terminus of XPD do not affect their interaction in vitro. Finally, by in vitro kinase assay we assessed that DDX1 is a substrate of the free-CAK but not of the holo-TFIIH complex. Future studies will clarify the functional relevance of the CAK-XPD interaction with DDX1. Overall, our results point to an altered association of the CAK to the chromatin as consequence of XPD mutations and identify DDX1 as a novel interactor of CDK7, showing a different binding profile in TTD compared to control and XP fibroblasts.
10-gen-2020
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/1301311
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