On the traces of XPD: cell cycle matters - untangling the genotype-phenotype relationship of XPD mutations

Abstract Mutations in the human gene coding for XPD lead to segmental progeria - the premature appearance of some of the phenotypes normally associated with aging - which may or may not be accompanied by increased cancer incidence. XPD is required for at least three different critical cellular functions: in addition to participating in the process of nucleotide excision repair (NER), which removes bulky DNA lesions, XPD also regulates transcription as part of the general transcription factor IIH (TFIIH) and controls cell cycle progression through its interaction with CAK, a pivotal activator of cyclin dependent kinases (CDKs). The study of inherited XPD disorders offers the opportunity to gain insights into the coordination of important cellular events and may shed light on the mechanisms that regulate the delicate equilibrium between cell proliferation and functional senescence, which is notably altered during physiological aging and in cancer. The phenotypic manifestations in the different XPD disorders are the sum of disturbances in the vital processes carried out by TFIIH and CAK. In addition, further TFIIH- and CAK-independent cellular activities of XPD may also play a role. This, added to the complex feedback networks that are in place to guarantee the coordination between cell cycle, DNA repair and transcription, complicates the interpretation of clinical observations. While results obtained from patient cell isolates as well as from murine models have been elementary in revealing such complexity, the Drosophila embryo has proven useful to analyze the role of XPD as a cell cycle regulator independently from its other cellular functions. Together with data from the biochemical and structural analysis of XPD and of the TFIIH complex these results combine into a new picture of the XPD activities that provides ground for a better understanding of the patophysiology of XPD diseases and for future development of diagnostic and therapeutic tools.

A Drosophila XPD model links cell cycle coordination with neuro-development and suggests links to cancer

XPD functions in transcription, DNA repair and in cell cycle control. Mutations in human XPD (also known as ERCC2) mainly cause three clinical phenotypes: xeroderma pigmentosum (XP), Cockayne syndrome (XP/CS) and trichothiodystrophy (TTD), and only XP patients have a high predisposition to developing cancer. Hence, we developed a fly model to obtain novel insights into the defects caused by individual hypomorphic alleles identified in human XP-D patients. This model revealed that the mutations that displayed the greatest in vivo UV sensitivity in Drosophila did not correlate with those that led to tumor formation in humans. Immunoprecipitations followed by targeted quantitative MS/MS analysis showed how different xpd mutations affected the formation or stability of different transcription factor IIH (TFIIH) subcomplexes. The XP mutants most clearly linked to high cancer risk, Xpd R683W and R601L, showed a reduced interaction with the core TFIIH and also an abnormal interaction with the Cdk-activating kinase (CAK) complex. Interestingly, these two XP alleles additionally displayed high levels of chromatin loss and free centrosomes during the rapid nuclear division phase of the Drosophila embryo. Finally, the xpd mutations showing defects in the coordination of cell cycle timing during the Drosophila embryonic divisions correlated with those human mutations that cause the neurodevelopmental abnormalities and developmental growth defects observed in XP/CS and TTD patients.

Cdk7 is required for full activation of Drosophila heat shock genes and RNA polymerase II phosphorylation in vivo

TFIIH has been implicated in several fundamental cellular processes, including DNA repair, cell cycle progression, and transcription. In transcription, the helicase activity of TFIIH functions to melt promoter DNA; however, the in vivo function of the Cdk7 kinase subunit of TFIIH, which has been hypothesized to be involved in RNA polymerase II (Pol II) phosphorylation, is not clearly understood. Using temperature-sensitive and null alleles of cdk7, we have examined the role of Cdk7 in the activation of Drosophila heat shock genes. Several in vivo approaches, including polytene chromosome immunofluorescence, nuclear run-on assays, and, in particular, a protein-DNA cross-linking assay customized for adults, revealed that Cdk7 kinase activity is required for full activation of heat shock genes, promoter-proximal Pol II pausing, and Pol II-dependent chromatin decondensation. The requirement for Cdk7 occurs very early in the transcription cycle. Furthermore, we provide evidence that TFIIH associates with the elongation complex much longer than previously suspected.

Xpd/Ercc2 regulates CAK activity and mitotic progression

General transcription factor IIH (TFIIH) consists of nine sub- units: cyclin-dependent kinase 7 (Cdk7), cyclin H and MAT1 (forming the Cdk-activating-kinase or CAK complex), the two helicases Xpb/Hay and Xpd, and p34, p44, p52 and p62 (refs 1–3). As the kinase subunit of TFIIH, Cdk7 participates in basal transcription by phosphorylating the carboxy-terminal domain of the largest subunit of RNA polymerase II1,4,5. As part of CAK, Cdk7 also phosphorylates other Cdks, an essential step for their activation6–9. Here we show that the Drosophila TFIIH com- ponent Xpd negatively regulates the cell cycle function of Cdk7, the CAK activity. Excess Xpd titrates CAK activity, resulting in decreased Cdk T-loop phosphorylation, mitotic defects and lethality, whereas a decrease in Xpd results in increased CAK activity and cell proliferation. Moreover, Xpd is downregulated at the beginning of mitosis when Cdk1, a cell cycle target of Cdk7, is most active. Downregulation of Xpd thus seems to contribute to the upregulation of mitotic CAK activity and to regulate mitotic progression positively. Simultaneously, the downregulation of Xpd might be a major mechanism of mitotic silencing of basal transcription.