p8/TTDA overexpression enhances UV-irradiation resistance and suppresses TFIIH mutations in a Drosophila trichothiodystrophy model

Abstract

Mutations in certain subunits of the DNA repair/transcription factor complex TFIIH are linked to the human syndromes xeroderma pigmentosum (XP), Cockayne's syndrome (CS), and trichothiodystrophy (TTD). One of these subunits, p8/TTDA, interacts with p52 and XPD and is important in maintaining TFIIH stability. Drosophila mutants in the p52 (Dmp52) subunit exhibit phenotypic defects similar to those observed in TTD patients with defects in p8/TTDA and XPD, including reduced levels of TFIIH. Here, we demonstrate that several Dmp52 phenotypes, including lethality, developmental defects, and sterility, can be suppressed by p8/TTDA overexpression. TFIIH levels were also recovered in rescued flies. In addition, p8/TTDA overexpression suppressed a lethal allele of the Drosophila XPB homolog. Furthermore, transgenic flies overexpressing p8/TTDA were more resistant to UV irradiation than were wild-type flies, apparently because of enhanced efficiency of cyclobutane-pyrimidine-dimers and 6-4 pyrimidine-pyrimidone photoproducts repair. This study is the first using an intact higher-animal model to show that one subunit mutant can trans-complement another subunit in a multi-subunit complex linked to human diseases.

Figure 1. Rescue of Dmp52 (mrn) mutants by transgenic flies overexpressing Dmp8/TTDA.

(A) Schematic diagrams of the Dmp52 mutant alleles used in this work. An EP-transposable element inserted upstream of the first Dmp52 exon is indicated as an inverted triangle. The molecular nature and positions of the previously characterized point mutations are also indicated. Black boxes represent regions conserved between human and fly p52. For more details on genotypic and phenotypic characterization, see reference . (B) Rescue of lethality of homozygous mrn alleles by Dmp8/TTDA transgenic fly lines. The percentage represents the number of homozygous individuals recovered relative to the number expected with full complementation (number in parenthesis). Note that two transgene copies increased the number of viable organisms. (C) Rescue of the sterility phenotype in heteroallelic organisms by different transgenic lines. The percentage represents the number of individual flies that were fertile and had progeny that developed at least to larval stage. Ten to 25 flies were tested for each condition. Homozygous EP3605/EP3605 and heteroallelic adults (EP3605/ mrn¹, mrn³ and mrn⁵) are 100% sterile. Dmp52-mut is a transgenic line that expresses a double point mutant (E310KR314E) . (D) Rescue of brittle-bristle and cuticle-deformation phenotypes in heteroallelic EP3605/mrn flies. 100% of adult heteroallelic flies have cuticular and bristle defects . The presence of the Dmp8/TTDA transgene rescues both defects in all heteroallelic adult flies examined. The genotype is indicated in each panel and data quantification is shown in Table 1.

Figure 2. A truncated human p52 peptide (Q202/stop) equivalent to the fly mrn³ allele can assemble with other TFIIH subunits.

The human equivalent of the mrn³ allele was co-expressed with the remaining TFIIH subunits in insect cells using the baculovirus system and affinity purified using an anti-p44 subunit antibody . (A) Immunopurified recombinant TFIIH complexes (immunoprecipitated by a p44 antibody) containing either wild-type p52 or mutant p52 analyzed by Western blotting using antibodies against XPB, p62, p52, cyclin H and cdk7. The p52[Q202/stop] truncated polypeptide is indicated in the figure. IIH9 denotes a complex containing all TFIIH components except p8; IIH6 denotes the TFIIH core complex minus p8. Note that the p52/220-stop truncated polypeptide is incorporated into the TFIIH complex and allows incorporation of the XPB subunit. (B) Analysis of NER using dual-incision assays. Reaction mixtures contained recombinant XPC-HR23b, XPA, RPA, XPG and ERCC1-XPF factors with IIH9 and IIH6 complexes in the presence or absence of p8 (+ or −); closed circular plasmid DNA containing a single Pt-GTG-DNA cross-link was as a template. The lane denoted by “−” is the DNA template without TFIIH and the lane denoted by “+” contains purified human TFIIH. (C) Reconstituted transcription assay reaction mixtures contained recombinant TFIIA, TFIIB, TFIIF, TBP, TFIIE factors and purified RNA pol II with IIH9 and IIH6 complexes in the absence or in presence of p8; the adenovirus major late promoter was used as a template. Interestingly, the presence of p8 enhanced transcription by the wild-type TFIIH complex.

Figure 3. Rescue of haywire mutants by transgenic flies overexpressing Dmp8/TTDA.

(A) Schematic diagrams of the different hay (DmXPB) mutant alleles used in this work. The molecular characterization of the mutants has been previously described . hay^nc2 and hay^nc2rv8 alleles are conditional mutations that are homozygous lethal at the non-permissive temperature (25°C). hay^nc2rv7 generates a truncated Hay protein. The black boxes represent the helicase motifs and the white box the ATPase domain. (B) The rescue of homozygous hay alleles lethality by a Dmp8/TTDA transgene is show in table format. The percentage represents the number of homozygous individuals recovered relative to that expected with full complementation (number in parenthesis).

Figure 4. Dmp8/TTDA overexpression recovers XPD and XPB levels in Dmp52 mutant flies.

Salivary glands from third instar larvae of wild-type, heteroallelic EP3605/mrn³ and homozygous mrn³ flies with two copies of the Dmp8/TTDA transgene in chromosome 2 (TTDA/+; mrn³/mrn³) were dissected and co-immunostained with H3 and XPD anti-antibodies (panel A), TBP and XPD antibodies (panel B), H3 and XPB antibodies (panel C) or XPB and TBP antibodies (panel D). XPD/H3, XPD/TBP, XPB/H3 and XPB/TBP ratios were calculated using nuclei from at least ten salivary gland cells in each condition. The error bars indicate standard errors of the means.

Figure 5. Viability, CPD and 6,4-PP repair rates, and CTD phosphorylation analysis in Dmp8/TTDA transgenic and wild-type lines after exposure to different doses of UV-B irradiation.

(A) Third instar larvae were irradiated and then allowed to develop to adults. Survival rate is indicated for each strain. The graph represents the results of at least three independent dose-response experiments for each genotype. The statistical analysis by ANOVA indicates a P value<0.001 for the more UV-resistant transgenic lines (TTDA5, TTDA1 and TTDA9) compared to the wild-type strain. The asterisks indicate significant differences compared to the wild type. Transgenic lines overexpressing Dmp8/TTDA are denoted as TTDA1, 5, 8 and 9. (B) Representative Southwestern dot-blot analysis using an anti-CPD antibody against genomic DNA extracted at different times from wild-type and Dmp8/TTDA transgenic third instar larvae after UV-B irradiation at 200 J/m². N.I. denotes non-irradiated DNA. The quantification of three independent Southwestern experiments is represented as a plot in which the average signal value for each sample at different times is shown relative to the signal value obtained 10 min after UV irradiation (normalized to one). The amount of DNA loaded on each dot blot was visualized in ethidium bromide stained agarose gels. Different times are indicated. (C) Quantification of 6-4PPs by ELISA assays using an anti-6-4PP–specific antibody. Three independent DNA samples derived from wild-type and transgenic third instar larvae irradiated at 200 J/m² were analyzed in a typical ELISA assay. The average for each sample value is shown as the signal at different times relative to the value of the signal obtained 10 min after UV irradiation (normalized to one). (D) CTD-Ser-5 phosphorylation levels in different Drosophila lines. Total protein extracts from wild type, transgenic and rescued flies were analyzed in western blot experiments using an antibody that recognize the phosphorylated Serine 5 at the CTD domain of the RNA polymerase II large subunit (indicated in the figure as CTD-Ser-5-P). As internal loading control an anti-β-tubulin antibody was used. The phenotypes are indicated in the figure panel and the ratio between the CPD-Ser-5-P and β-tubulin signals are indicated at the bottom.