Molecular Mechanisms of Biallelic Effects
We next turned to UV-based cellular assays including unscheduled DNA synthesis after UV irradiation (UV-UDS), recovery of RNA synthesis after UV irradiation (UV-RRS), and UV survival, which report on the NER subpathways (global genome NER and transcription-coupled NER) and total NER, respectively. In none of these assays was the response to UV improved in compound heterozygotes relative to TTD homozygotes (Figure 4A–4C). However, unlike the in vivo TTD phenotypes described above, in which XpdTTD/TTD and XpdTTD/KO animals were indistinguishable, XpdTTD dosage effects were observed in UV survival, UV-UDS, and UV-RRS, indicating that cellular parameters as measured in fibroblasts here do not always correlate with the phenotype at the level of the intact organism. XpdTTD/KO hemizygous cells were thus used as the baseline on which to compare the activity of compound heterozygous cells. Relative to XpdTTD/KO hemizygote cells, UV survival was improved by the homozygous lethal Xpd†XPCS allele in XpdTTD/†XPCS compound heterozygous cells and to a lesser degree by the Xpd†XP allele (Figure 4A). Because of embryonic and cellular lethality, we were unable to test UV survival associated exclusively with the Xpd†XPCS or Xpd†XP alleles. However, homozygous XPDXP (XPDR683W) and hemizygous XPDXPCS (XPDG602D) human cells are known to be highly sensitive to UV [19,25], as are cells from a homozygous viable XpdXPCS/XPCS (XPDG602D/G602D) mouse model (Figure 4A, dotted line) [23]. Thus, the survival of XpdTTD/†XPCS (and XpdTTD/†XP) cells likely represents a level of UV resistance that neither mutant allele can impart on its own (Table 2). Significant effects of compound heterozygosity on NER subpathways relative to XpdTTD/KO cells were observed in XpdTTD/†XP cells but only for UV-UDS activity. Finally, none of the mutant TFIIH combinations (carrying alterations associated with TTD [XPDR722W], XPCS [XPDG602D], or XP [XPDR683W]) exhibited synergism in an in vitro NER reaction reconstituted with different mutant TFIIH complexes (Figure 4D). Taken together, these data are consistent with interallelic complementation of UV sensitivity in cells but underscore the lack of any correlation between UV-related repair characteristics and TTD progeroid phenotypes in animal models.
Figure 4  TFIIH Functions and Mechanisms of XPD-Associated Disease Pleiotropy
(A) Cellular survival after UV irradiation. Rescue of hemizygous XpdTTD/KO survival by Xpd†XPCS and Xpd†XP alleles is illustrated by arrows marked A and B, respectively. UV survival of homozygous XpdXPCS/XPCS cells (asterisk) from the normally expressed viable allele (XpdXPCS) is depicted by a dotted line. Survival curves represent an average of four independent experiments; 1–2 cell lines per genotype were included in each experiment. Error bars indicate SEM between experiments.
(B) UV-UDS, a measure of global genome repair. Number of experiments: n = 15 (XpdTTD/TTD), n = 6 (XpdTTD/KO), n = 4 (XpdTTD/†XPCS ), n = 2 (XpdTTD/†XP ); 1–2 cell lines per genotype were included in each experiment. The asterisk indicates significant difference with XpdTTD/TTD; crosses indicate significant differences with XpdTTD/KO.
(C) UV-RRS, a measure of transcription-coupled repair of UV-induced lesions. Number of experiments: n = 7 (XpdTTD/TTD), n = 2 (XpdTTD/KO), n = 4 (XpdTTD/†XPCS ), n = 2 (XpdTTD/†XP ); 1–2 cell lines per genotype were included in each experiment.
(D) Incision/excision activity of combinations of altered TFIIH complexes in a reconstituted NER reaction. Equal amounts of single or mixed populations of recombinant TFIIHs (containing XPD, XPB, p62, p52, His-p44, Flag-p34, cdk7, cyclin H, Mat1, and p8) were mixed with recombinant XPG, XPF/ERCC1, XPC/hHR23B, RPA, and a radiolabelled synthetic NER substrate. The excision products (26–34 nucleotides in length) were visualised at nucleotide resolution on a denaturing polyacrylamide gel as indicated . Note the weak activity corresponding to each single and combined TFIIH complex (lanes 3–8) relative to the wt (lane 1) and negative controls (lane 2).
(E) Xpd dose-dependent reduction of TFIIH in homozygous XpdTTD/TTD, hemizygous XpdTTD/KO, and compound heterozygous XpdTTD/†XPCS and XpdTTD/†XP cells by comparative immunofluorescence of the p62 subunit of TFIIH. Roman numerals represent different microscopic slides and Arabic numerals different cell lines labelled as follows: (I) wt cells (1) labelled with 2-μm beads, XpdTTD/TTD cells (2) with 0.79-μm beads, and XpdTTD/KO cells (3) with no beads; (II) wt cells (1) labelled with 0.79-μm beads and XpdTTD/†XPCS cells (4) with no beads; and (III) wt cells (1) labelled with 0.79-μm beads and XpdTTD/†XP cells (5) with no beads.
(F) Quantification of immunofluorescent signal from at least 50 nuclei per cell line and 2–6 experiments per genotype. Bars representing cells analysed on the same microscopic slide are depicted side by side, with wt set at 100%. The p-value indicates minimum significant difference between wt and the indicated cell lines analysed on the same microscopic slide within one experiment. Next we asked whether the Xpd†XPCS and Xpd†XP alleles, despite decreased mRNA levels, ameliorated TTD symptoms by increasing overall TFIIH levels in compound heterozygous XpdTTD/ †XPCS and XpdTTD/ †XP cells. Previously, using comparative immunohistochemistry, we and others have shown an up to 70% reduction of TFIIH levels in primary fibroblasts from patients with TTD compared with wt controls due to reduced stability [16,17]. Despite overexpression of mRNA from the XpdTTD allele relative to the wt allele (Figure 1E), TFIIH protein levels were reduced by 50% in primary mouse XpdTTD/TTD fibroblasts (Figure 4E and 4F), thereby mimicking the situation in human patients with TTD. In accordance with the gene dosage, a further reduction of up to 70% of the wt level was observed in hemizygous XpdTTD/KO cells. Consistent with low mRNA expression levels, neither the Xpd†XPCS nor the Xpd†XP allele was able to restore TFIIH abundance to wt levels in XpdTTD compound heterozygote cells (Figure 4E and 4F). Thus, the improved UV survival observed in compound heterozygote cells (Figure 4A) and likely the rescue of TTD progeroid symptoms (Figure 3) were not due to normalisation of TFIIH levels, suggesting a qualitative rather than a quantitative effect on these phenotypes in vivo.
In contrast, the level of XPCS mRNA expression did affect the ability of the encoded protein (XPDG602D) to restore the TTD hair phenotype to normal. Notably, XpdTTD/ †XPCS animals had a partial TTD hair phenotype, correlating with low levels of Xpd†XPCS expression, whereas XpdTTD/XPCS animals had wt hair, correlating with normal expression levels from the viable XpdXPCS allele (Table 2 and unpublished data). Thus, the range of expression levels from these two mutant alleles affected their ability to complement some phenotypes (hair). An overview of the functional relationships between Xpd alleles, phenotypes, and the presumed underlying TFIIH function in mice and cells is presented in Table 2.