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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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Trichothiodystrophy: A Disorder Highlighting the Crosstalk between DNA Repair and Transcription


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Trichothiodystrophy (TTD) is a rare autosomal recessive multisystem disorder characterized by sulfur-deficient brittle hair, mental and physical retardation, ichthyosis, and, in many patients, cutaneous photosensitivity but no cancer incidence. All sun-sensitive TTD cases appear to be defective in nucleotide excision repair (NER) as a consequence of alterations in one of three genes, namely XPB, XPD and TTDA. Intriguingly, in view of the very marked differences in the clinical phenotypes, defects in two of the genes altered in TTD (XPB and XPD) can also cause the cancer-prone disorder xeroderma pigmentosum (XP) or, in rare cases, the combined symptoms of XP and Cockayne syndrome (XP/CS). A breakthrough in understanding the perplexing features of this complex triad of hereditary disorders came from the discovery that the genes mutated in TTD are all related to TFIIH, a multiprotein complex involved in both transcription and NER. XPB and XPD encode two subunits of TFIIH, and also TTDA has been recently identified as a new component of the TFIIH complex. The discovery of this unexpected link between DNA repair and transcription was crucial to rationalize the TTD pathological phenotype as well as the puzzling genotype-phenotype relationships related to defects in XPB and XPD. The last few years have witnessed significant progress in this field. It was shown that the mutations associated with the three disorders are located at different sites in the XPD gene and that all the genetic and molecular alterations responsible for the NER-defective form of TTD cause a decrease by up to 70% in the cellular concentration of TFIIH. This implies that a limited availability of TFIIH interferes with NER but is compatible with life and therefore its impact on transcription must be selective only under certain conditions or in selective cellular compartments. We are also beginning to understand how different mutations in the XPD gene result in different clinical entities: all the XPD mutations found in patients, independent of the associated phenotype, are detrimental for the XPD helicase activity, thus explaining the NER defect, but only those responsible for the TTD phenotype affect basal transcription. Emerging evidence indicates that the involvement of TFIIH in transcription is multifaceted, ranging from transcription by RNA polymerase I and II to regulation of gene expression. Although the multiple roles of TFIIH in transcription remain to be fully explored, these discoveries represent major advances in our understanding of fundamental cellular processes. They have important impacts on clinical medicine with implications for cancer prevention, aging, differentiation and development.

Trichothiodystrophy (TTD), variously known as Tay syndrome, Pollit syndrome, Amish hair-brain syndrome and Sabinas syndrome, is a rare autosomal recessive disorder that was first described as a distinct clinical entity in 1980 to characterize the condition of patients with sulfur-deficient brittle hair and other neuroectodermal symptoms and signs.1 The identification of DNA repair defects in a consistent number of TTD cases has stimulated further investigations aimed at clarifying the pathogenesis of this disorder whose clinical symptoms are more easily attributable to transcription anomalies than DNA repair alterations.

Clinical Features>

The main diagnostic criteria of TTD are: brittle hair, mental and growth retardation, face characterized by receding chin, small nose, large ears and microcephaly, nail dysplasia and ichthyosis (fig. 1). The acronyms PIBIDS, IBIDS, BIDS, have been used on the basis of the presence or absence of the following symptoms: Photosensitivity, Ichthyosis, Brittle hair, Impaired intelligence, Decreased fertility and Short stature. At birth, children often present with ichthyosiform erythroderma, and they may be encased in a collodion-like membrane. In addition, numerous patients suffer from repeated and severe infectious illnesses, mainly of the gastrointestinal and respiratory tract. Forty to 50% of patients exhibit marked photosensitivity but there are no reports of cancer (for a recent review see ref. 2).

Figure 1. The four Italian patients affected by trichothiodystrophy, firstly described as NER-defective and classified into the XP-D group.

Figure 1

The four Italian patients affected by trichothiodystrophy, firstly described as NER-defective and classified into the XP-D group. From reference .

TTD is most strikingly characterized by hair abnormalities which are considered as the key factors in the recognition of these patients (fig. 2). Scalp hair, eyebrows, and eyelashes are short, thin, brittle and dry. Light microscopy reveals irregular hair surface and diameter, trichoschisis, a decreased cuticular layer with twisting, and a nodal appearance that mimicks trichorrhexis nodosa. Polarisation microscopy of the hair typically shows alternating light and dark bands that confer a “tiger tail” pattern. Scanning electron microscopy usually reveals absent or severely damaged cuticle scales, an irregular hair surface, trichoschisis and trichorrhexis nodosa, and torsions in the flattened hair shaft. The hair, and often the nails, of affected individuals are characterized by a reduction in cystine, cysteic acid and sulphur content, a reduction that derives from decreases in sulphur-rich matrix proteins. Sulphur proteins are altered not only quantitatively but also qualitatively and there is an abnormal distribution of the sulphur-rich proteins in the cortex and in hair cuticles.

Figure 2. Hair features in TTD (courtesy of Dr.

Figure 2

Hair features in TTD (courtesy of Dr. Stefano Marinoni, Trieste, Italy). A) Short, sparse and broken scalp hair. B) Polarizing microscopy of hair shafts showing tiger-tail pattern.

Clinical Photosensitivity Is Usually Associated with an Altered Cellular Response to UV

In 1986, we first reported the results of DNA repair investigations on four Italian patients who showed clinical symptoms diagnostic of TTD together with acute photosensitivity. Upon UV irradiation, cells from these patients showed a notable reduction in levels of survival and UV-induced DNA repair synthesis (UDS), a failure to recover normal DNA and RNA synthesis rate, and an increased mutability.3 The occurrence of these abnormalities in cellular response to UV light indicates the presence of a defect in nucleotide excision repair (NER), pathway that removes of a wide spectrum of DNA lesions, including UV-induced damage. Defects in NER had previously been identified in two other hereditary disorders, namely xeroderma pigmentosum (XP) and Cockayne syndrome (CS), which are characterized by certain unique clinical symptoms. In particular, XP displays various manifestations of cutaneous UV-genotoxicity, notably photosensitivity, pigmentation abnormalities, early onset of precancerous lesions and a greatly increased incidence of cancer in the photoexposed area of the skin. In contrast, cancer-proneness has never been reported in CS patients, in whom skin symptoms are confined to photosensitivity, and the major diagnostic criteria for CS are postnatal growth failure and progressive neurological dysfunction (reviewed in ref. 4).

Although the respective clinical symptoms are distinct, the cellular phenotype that we observed in the four TTD patients was closely similar to that of XP. This finding prompted us to investigate genetic homology between XP and TTD. The investigation consisted of a classical complementation test that was based on the analysis of the capacity of somatic cell hybrids to perform the UDS (fig. 3). TTD cells were fused with XP cells representative of the seven NER-deficient complementation groups hitherto identified (designated XP-A to XP-G), and the UDS level was analyzed in the heterodikaryons. Since parental cells in each cross were labelled with latex beads of two different sizes, heterodikaryons were unambiguously identified as binuclear cells containing beads of both sizes. Restoration of normal UDS levels was observed in all cases except in the crosses between TTD and XP-D cell strains. These results indicated that UV hypersensitivity in the four Italian TTD patients was due to the presence of a defect in XPD.3

Figure 3. Classical complementation assay by cell fusion for nucleotide excision repair defects.

Figure 3

Classical complementation assay by cell fusion for nucleotide excision repair defects. The fibroblast strains used as partners in the fusion are grown for three days in medium containing latex beads of different sizes that are incorporated into the cytoplasm (more...)

Since then, DNA repair investigations have been extended to other TTD cases from different countries. The cellular response to UV appeared to be normal in patients with normal cutaneous photosensitivity, and defective in patients who showed photosensitivity, together with other classic diagnostic symptoms for TTD.5-8

Heterogeneity of the Repair Defect in TTD

Genetic analysis of the DNA repair defect, performed so far in about forty patients, has led to the identification of three genetically different DNA repair defects associated with TTD. The majority of the repair-deficient TTD patients have the same genetic defect as that present in the XP-D group. This group comprises many patients with XP and two patients who showed clinical symptoms of XP in association with those of CS (for recent reviews see refs. 9, 10). In TTD patients mutated in XPD, the severity of the pathological phenotype varies from moderate (short stature, delayed puberty, mental development at preschool or primary school level, axial hypotonia, reduced motor coordination and survival beyond early childhood) to severe (very poor mental and motor performances and speech, failure to thrive and death during early childhood). It is of interest that in these patients heterogeneity has been observed also in the degree of severity of the repair defect, with UDS levels ranging between 50% and less than 10% of normal. The Italian patients and some of the non-Italian cases show a drastic reduction in the UDS levels with a cellular sensitivity to UV similar to or even greater than that in XP patients classified in the XP-D group. Other non-Italian TTD patients are characterized by a lower reduction in UDS level with survival significantly affected only at high UV doses.8 No correlation was observed between the degree of the repair deficiency and the severity of the clinical symptoms.

A different defect was found in one French family with two afflicted children showing a mildly affected phenotype with hair abnormalities but without physical and mental impairment, and partially reduced UDS levels (40% of normal). In these patients the repair defect appeared to be the same as that found in the XP-B group.11,12 XP-B is a very rare defect and to date has only been identified in two families with the XP/CS phenotype.13-15

A new NER defect that has been designated as TTD-A was found in one English patient16 and, more recently, in three additional cases.17 TTD-A patients show consistently reduced UDS levels (15-25% of normal) and clinical features of moderate severity. In conclusion, genetic characterization of the repair defect in TTD patients led to the identification of a new gene that is involved in NER (the TTDA gene), and to the demonstration that two defects (the XP-B and XP-D defects), which have already been described as responsible for XP and XP/CS pathological phenotypes, are associated with TTD. Therefore, two unexpected findings have emerged from DNA repair investigations on TTD, namely: i) the lack of cutaneous skin abnormalities and skin cancer even in those patients whose DNA repair defect is the same as that of XP-D, and ii) mutations in either XPB or XPD gene are associated with at least two clinically distinct entities (TTD, XP and/or XP/CS), sharing only cutaneous photosensitivity.

The Genes Mutated in TTD Are All Related to the Transcription Factor TFIIH

These intriguing aspects have been rationalized by the discovery that the genes mutated in TTD encode three distinct subunits of TFIIH, a multiprotein complex that has a dual role in NER and general transcription.17-20 TTDA is involved in the stabilization of the TFIIH complex,18 whereas XPB and XPD encode two subunits of TFIIH.19,20

The transcriptionally active form of TFIIH (holo-TFIIH) is made up of a total of ten subunits (fig. 4). Five of these (XPB, p62, p52, p44 and p34) are tightly associated in a subcomplex called coreTFIIH. XPD is less tightly associated with the core and mediates the binding of the CDK-activating kinase (CAK) subcomplex, which includes the three subunits, cdk7, cyclin H and MAT1 (for reviews see refs. 21-23). The recently identified tenth subunit of TFIIH (TTDA or TFB5) is a 8 KDa protein involved in the stabilization of the complex.17 XPB and XPD are ATP-dependent helicases with opposite polarity. TFIIH participates in local DNA unwinding, either around the promoter, to allow the synthesis of the RNA transcript by RNA polymerase II, or around a damaged site, to permit damage-specific nucleases to cleave the DNA on either side of the damage. The 3'-5' helicase activity of XPB is essential for both transcription and repair, whereas the XPD 5'-3' helicase activity is necessary for repair but dispensable for in vitro basal transcription, although XPD substantially stimulates the transcription activity in vitro. This probably accounts for the rarity of XP-B patients compared with the relatively high frequency and variety of pathological phenotypes associated with the XP-D defect. The TFIIH complex also shows ATPase activity associated with the XPD and XPB helicases and kinase activity from the cdk7 subunit, which is able to phosphorylate numerous substrates, including the C-terminal domain (CTD) of the large subunit of RNA polymerase II, converting it from the initiating IIa form to the elongating IIo form. This kinase activity is involved in the regulation of transcription and cell cycle (reviewed in refs. 24, 25, respectively), and in the negative control of repair.26 The enzymatic activities of TFIIH are tightly controlled by interactions within the TFIIH complex or through interactions with many general and regulatory transcription factors. For example, p44 interacts with XPD to stimulate its helicase activity, p52 regulates the function of XPB through pair-wise interactions, MAT1 and cyclinH regulate cdk7 kinase activity, and phosphorylation of cyclinH by cdk8/cyclinC represses both the activation of transcription by TFIIH and the ability of TFIIH to phosphorylate RNA polymerase II. Recently, it has been shown that in Drosophila Xpd negatively regulates the CAK activity of Cdk7 and it is down-regulated at the beginning of mitosis, thus contributing to the upregulation of mitotic CAK activity, to the positive regulation of mitotic progression and also, likely, to the mechanism of mitotic silencing of basal transcription.27 Thus, the structural function of XPD in TFIIH assembly appears to be used by cells in a dynamic way to regulate and coordinate the diverse cellular functions of the different sub-complexes in transcription, DNA repair and cell cycle progression.25

Figure 4. TFIIH composition and enzymatic functions of its subunits.

Figure 4

TFIIH composition and enzymatic functions of its subunits. The five subunits composing the coreTFIIH subcomplex are in red, the three subunits of the cdk-activating kinase (CAK) subcomplex are in light blue, the XPD subunit that bridge the two TFIIH subcomplexes (more...)

The identity of TTDA, the third gene responsible for the photosensitive form of TTD, has been for years a key unanswered question. It has been suggested that the function defective in TTD-A is involved in the stabilization of TFIIH or in its protection against degradation.18 The gene has been recently cloned and its product was shown to have a role in regulating the level of TFIIH.17

Mutational Analysis in Repair-Defective TTD Patients

The dual function of TFIIH in repair and transcription led to the notion that clinical features diagnostic for XP result from mutations that interfere only with the DNA repair function of TFIIH, while those typical of TTD and CS are due to a subtle impairment of its transcriptional role.11,28 This hypothesis requires that the mutations associated with the three disorders are located at different sites in the gene. This was explored by determining the sites of mutations in many patients. Different mutations in the XPB gene were found in association with the TTD and the XP/CS phenotype, although the study was limited to five cases from three families, due to the rarity of the XP-B defect (Table 1).12,15,29 Mutation analysis in XP-D patients has been performed in a significant number of cases, namely twenty nine with XP,30-35 twenty eight with TTD32,34,36-39 and two with XP/CS.31,40 Each mutated site was indeed found in either XP or TTD or XP/CS individuals (fig. 5). The only exceptions are two alleles, resulting in either substitution of arg616 or a leu461val change associated with deletion of amino acid region 716-730. However, these alleles are completely inactive, at least in S. pombe orthologue, suggesting that the phenotype is determined by the second XPD allele that was found always different in XP and TTD cases.32 Most of the mutations are clustered in the C-terminal third of the protein whereas a few are close to the N-terminus and the region between amino acids 260 and 460 is devoid of mutations.10 The XP phenotype often results from mutations in the helicase motifs of XPD. Although the mutational pattern delineates neither a “TTD domain” nor an “XP domain”, a limited number of mutations has consistently been found in association with a given pathological phenotype. Most of the mutations in TTD are localized at three sites (arg112, arg658, and arg722) whereas 70% of mutations in XP patients are localized at a single site, arg683. These may be “TTD-specific” and “XP-specific” mutations. Accordingly, a mouse generated with the TTD-specific arg722trp mutation had many of the features of TTD.41,42

Table 1. Mutations in the XPB gene found in TTD and XP/CS patients.

Table 1

Mutations in the XPB gene found in TTD and XP/CS patients.

Figure 5. Mutations in the XPD protein in TTD, XP and XP/CS patients.

Figure 5

Mutations in the XPD protein in TTD, XP and XP/CS patients. The diagram shows the XPD protein with the helicase domains (black boxes). The amino acid changes resulting from the mutations found in the different pathological phenotypes are shown boxed. (more...)

Besides providing data supporting the hypothesis that the site of the mutation in the XPD gene determines the clinical phenotype, the mutational analysis of the Italian TTD patients has shed new light on the basis of the heterogeneity observed among TTD patients in the degree of severity of the repair defect and of the clinical symptoms. The different degrees of impairment in the cellular responses to UV in TTD appeared to be related to specific mutations.39 Both in the homozygous patients and in the functionally hemizygous patients, substantial UV sensitivity was associated with the arg112his substitution whereas a mild UV sensitivity was associated with mutations resulting either in the change of arg658 or in the loss of the final portion of the XPD protein. An intermediate UV sensitivity, similar to that found in XP patients defective in XPD, was associated with the arg722trp change. In contrast, the severity of the clinical symptoms did not correlate with the magnitude of the DNA-repair defect but it appears to be influenced by the dosage of the mutated allele. The most severe clinical features were found in patients who were functionally hemizygous, suggesting that TFIIH in these patients not only contains a mutated XPD subunit, but it also could be present at only half of the normal amount. This may well result in a more severe impairment of the transcriptional activity of TFIIH. This hypothesis implies that, in tissues critically affected in TTD, the level of XPD and, by implication, of TFIIH is rate limiting factor for transcription. Further investigations have demonstrated, however, that a more complex situation underlies the TTD pathological phenotype as well as the clinical outcome of mutations in the XPD gene.

The Cellular Amount of TFIIH Is Reduced in All Repair-Defective TTD Patients

The analysis of the steady-state level of TFIIH in fibroblasts from patients representative of distinct clinical, cellular and molecular alterations demonstrated that mutations in any of the three genes (XPD, XPB and TTDA) responsible for the photosensitive form of TTD cause a decrease by up to 70% in the cellular concentration of TFIIH.43 The reduction in the amount of TFIIH, however, did not correlate either with the residual repair capacity or with the severity of clinical symptoms (fig. 6). Substantial reductions in TFIIH levels have been detected in patients showing relatively moderate psychomotor retardation but no increased proneness to infections whereas less marked reductions by 35-45% of normal levels were found in patients who had drastically compromised pathological phenotypes. These observations strikingly indicate that the severity of the TTD pathological phenotype cannot be related solely to the effects of mutations on TFIIH levels.43

Figure 6. Levels of TFIIH, UV-induced DNA repair synthesis and clinical severity in TTD patients mutated in the TTDA, XPD and XPB gene.

Figure 6

Levels of TFIIH, UV-induced DNA repair synthesis and clinical severity in TTD patients mutated in the TTDA, XPD and XPB gene. The level of TFIIH was analyzed by Western Blot of cell lysates using antibodies against the subunits cdk7, p44 and p62. The (more...)

What is the basis of the reduced amount of TFIIH in TTD cells? In vivo, the ectopic expression of the XPD wild-type protein in XPD mutated TTD cells appeared to be sufficient to restore normal levels of the other TFIIH subunits. This increase was paralleled by the restoration of normal repair activity (fig. 7). These findings indicated that a normal XPD protein provides stability to the TFIIH complex, probably by restoring proper protein-protein interactions, and ensures its correct functioning in NER.43 In vitro evidence further indicates that a mutated TFIIH subunit may affect the stability of the entire complex. It has been shown that mutations in XPB and p52 prevent the XPB anchoring within the core TFIIH,44 and mutations in p44 prevent incorporation of the p62 subunit within the core TFIIH.45 Mutations in XPD or in p44 that modify the XPD-p44 interaction may affect the composition of TFIIH by decreasing the amount of XPD and CAK subunits associated with the core and/or weakening the anchoring of CAK to the core TFIIH.46,47 Furthermore, mutations in the C-terminal region of XPD have been shown to play an important role in maintaining TFIIH architecture whereas mutations in the N-terminal region of XPD do not affect either the composition of recombinant TFIIH complexes or the contact with MAT1, the interacting partner of XPD within the CAK complex.48

Figure 7. The expression of the XPD wild-type protein in TTD8PV cells restores normal levels of the other TFIIH subunits and normal UV-induced DNA repair synthesis capacity.

Figure 7

The expression of the XPD wild-type protein in TTD8PV cells restores normal levels of the other TFIIH subunits and normal UV-induced DNA repair synthesis capacity. TTD8PV fibroblasts and stably transfected TTD8PV fibroblasts ectopically expressing the (more...)

Mutations in the XPB and XPD genes may render either the transcript or the protein unstable, or interfere with correct folding or proper associations of the protein with the other components of TFIIH, leading to rapid degradation of uncomplexed proteins. Alternatively, a mutated subunit may induce slight conformational changes in the architecture of the TFIIH complex that, in turn, may favor its degradation. This hypothesis may hold true also for the TTD-A defect. Alterations in a factor with a role in the stabilization of the complex might prevent the conformational changes required for optimal functioning of THIIH. The findings in TTD-A cells suggest that a 3-4 fold reduced amount of TFIIH complexes with normal composition consistently reduce NER efficiency (to 25% of normal) and confers subtle defects in transcription resulting in a TTD phenotype with a physical and mental impairment of moderate severity.18 Less severe reductions may be necessary but they are not sufficient to generate the TTD phenotype. Reduced levels of TFIIH were indeed found also in some XP-D cell strains from XP and XP/CS patients, although the levels were in general not as low as in the TTD cell strains.43 This finding already suggested that the clinical outcome of XPD mutations is the combined result of the reduction in TFIIH content and the effects of the specific mutations on the interactions of TFIIH with other components of the transcription machinery that may differentially compromise transcription activity. Interestingly, it has been reported that in Drosophila having mutations in haywire, the XPB homolog, the penetrance of diverse phenotypes also does not correlate with the type of mutation.49

Consequences of XPD Mutations on the Activities of TFIIH

Explanations for the nature of the various clinical features associated with the XP-D defect have been provided by recent in vitro studies with recombinant TFIIH complexes in which the XPD subunit carries amino acid changes found in patients. All the analyzed mutations, independent of the associated pathological phenotype, appeared to affect the helicase activity of XPD but only those responsible for TTD diminish the basal transcription activity of TFIIH.48 Inhibition of the XPD helicase activity caused by every mutation found in patients explains the NER deficiency present in all the patients mutated in XPD. In particular, the mutations resulting in amino acid changes in the first two-thirds of the protein, including residue 602, abolish the XPD intrinsic helicase activity whereas the mutations in the last C-terminal third of XPD prevent its interaction with p44 and, by consequence, stimulation of the helicase activity of XPD. Remarkably, while mutations associated with TTD confer also a significant in vitro basal transcription defect, mutations associated with the XP or the XP/CS phenotypes, even those that completely disrupt XPD helicase activity, still allow RNA synthesis. Two alleles, resulting in either the substitution of arg616 or the leu461val change associated with the deletion of amino acid region 716-730, which were found to be null alleles in S. pombe,32 completely abolished basal transcription. A total defect in transcription is obviously incompatible with life. This explains why these two alleles, despite being relatively frequent among patients, have never been found in the homozygous state but always in association with other mutated XPD alleles, all of which interfere with basal transcription less drastically.

TTD Symptoms Reflect Subtle Defects in Transcription

Therefore, two specific defects affect the TFIIH complex in TTD cells: a reduced steady-state level and an impaired in vitro transcription activity. A general implication of the reduced amount of TFIIH found in TTD is that a limited availability of TFIIH interferes with NER but is compatible with life. This implies that the TFIIH level albeit reduced in TTD cells does not seriously impede transcription of most genes but it might impair transcription only under certain conditions or in specific cellular compartments affecting, for example, only a limited set of genes that critically demand optimal TFIIH function. Several lines of evidence support the hypothesis that the reduced amount of TFIIH in TTD patients may become limiting in terminally differentiated tissues, in which the mutated TFIIH might get exhausted before the transcriptional program has been completed. The link between TFIIH instability and the hair and skin features that are the diagnostic hallmark of TTD is supported by the peculiar situation described in four TTD patients with fever-dependent reversible deterioration of TTD features.50 The XPD mutation responsible for the pathological phenotype in these patients (resulting in the arg658cys change) confers a temperature-sensitive defect in transcription and repair due to thermo-instability of TFIIH. During the terminal differentiation of hair, nails and skin, the gene family of cysteine-rich matrix proteins is the last to be transcribed at a very high rate: the encoded proteins constitute a major fraction of hair and ensure keratin-crosslinking before cell death. In the case of destabilizing TTD mutations, TFIIH may be depleted before terminal differentiation is complete. As a consequence, crosslinking of keratin filaments is not finished, leading to incomplete (brittle) hair. This phenomenon becomes more pronounced in TTD patients carrying the arg658cys mutation, when high fever further destabilizes TFIIH.50

Further evidence supports the hypothesis that many of the clinical features of TTD result from inadequate expression of a diverse set of highly expressed genes. A systematic study of eleven TTD patients demonstrated that all of them showed wholesome hallmarks of β-thalassemia trait: β-globin mRNA levels were reduced in the reticulocytes, and β-globin synthesis was also reduced relative to α-globin. Besides offering an easily measurable diagnostic test for TTD, these data provide a direct evidence for a transcriptional deficiency in TTD.34 A marked reduction in T cell proliferation in response to mitogens was detected in TTD lymphocytes from TTD patients3 and alterations in T cells and dendritic cells (DC) suggestive of a subtle transcriptional defect of a set of genes involved in DC maturation and function, have been reported in a TTD child with a severe immunodeficiency.51 These observations are paralleled by the finding of reduced transcription of the skin-specific, differentiation-related gene SPRR2 in the TTD mouse expressing the arg722trp mutated XPD protein.41 The SPRR2 gene is a member of the small proline-rich protein (SPRR) family expressed in epidermis. It encodes a structural component of the cornified envelope and is expressed in the final stage of terminal differentiation. Reduced SPRR2 expression in TTD mouse skin reflects defective gene transcription in late stages of terminally differentiating epidermal keratinocytes.

The observation that XPD mutations associated with TTD interfere with transcription may help to explain the lack of cancer proneness in TTD, despite the reduced efficiency of NER. The defect in transcription together with limiting amounts of TFIIH could prevent tumoral transformation and/or progression. However, also the involvement of TFIIH in cell-cycle regulation could play a role in the lack of cancer susceptibility in TTD. All the studies performed so far, have not identified any cellular and biochemical parameter whose alteration unequivocally correlates with the different cancer susceptibility in XP and TTD patients defective in the same repair pathway, and even in the same gene (reviewed in ref. 9).

It has been suggested that the other symptoms of TTD, namely those that confer the aging phenotype, are caused by unrepaired oxidative damage that compromises transcription and leads to functional inactivation of critical genes and enhanced apoptosis, resulting ultimately in functional decline and depletion of cell renewal capacity.42 In this context, it is worthwhile mentioning that defects in RNA pol I transcription have been recently suggested as an alternative explanation for the pathological phenotype of CS, a disorder that shares many features of aging and developmental anomalies with TTD.52 The involvement of TFIIH in transcription by RNA polymerase I has been demonstrated both in vitro53 and in vivo.54 Therefore, it cannot be ruled out that a mutated XPB or XPD subunit might interfere with the role of TFIIH in RNA pol I transcription, thus accounting for the overlapping symptoms in CS and TTD.

XPD Mutations May Interfere with the Regulation of Gene Expression by TFIIH

An extra layer of complexity in the interpretations of mutational effects of TFIIH has been added by recent discoveries that extend the roles of TFIIH in transcription (reviewed in ref. 24). Apart from its role in basal transcription by RNA polymerase I and II, TFIIH maintains complicated cross-talk with different types of factors involved in RNA Pol II-mediated transcription and has a regulatory role during transcription.

It has been shown that TFIIH interacts with some nuclear hormone receptors, including the estrogen (ERα), androgen (AR) and retinoic acid (RARα and RARγreceptors (reviewed in ref. 55). As a consequence of their interaction with TFIIH, ERα and RARs are phosphorylated in their N-terminal A/B region by cdk7, a component of the TFIIH CAK subcomplex, which is anchored to the coreTFIIH by XPD. This phosphorylation process plays a critical role in transcription mediated by the liganded receptors, allowing ligand-dependent control of the activation of the hormone-responsive genes. A reduced ligand-dependent activation of transcription by the three receptors RARα, ERα and AR was observed in XP-D cells with the mutation arg683trp, i.e., the change most frequently found in XP patients.56 Conversely, the arg683trp mutation had no effect on the transactivation mediated by the Vitamin D receptor, which lacks the typical A/B domain targeted by cdk7.48 Transcriptional activation by RARα was significantly reduced also in one TTD cell line with the arg722trp change whereas almost optimal transactivation was found in a TTD cell line with the arg112his substitution and in TTD-A cells.48,56 These findings indicate that mutations located in the C-terminal end of XPD, independent of the associated pathological phenotype, prevent the action of nuclear receptors having an A/B domain. This might account for the developmental and neurological defects encountered in both XP and TTD patients having mutations at position arg658 (TTD), arg683trp (XP), and arg722trp (TTD).48

TFIIH interacts also with some tissue specific transactivators. It has been demonstrated that the transcriptional activator FUSE Binding protein (FBP), a regulator of c-myc expression, binds specifically to TFIIH.57 In two cell lines defective in either the XPB or XPD gene (derived from the XP/CS patient XP11BE and from the XP patient XP6BE, respectively), this interaction was either abolished or attenuated, resulting in impaired regulation of c-myc expression.58 Mutations altering TFIIH conformation may therefore affect to different degrees its stereo-specific interactions with tissue-specific transcription regulators (activators and repressors). We can speculate that while XP-type mutations could interfere with a specific class of regulators involved in carcinogenesis, TTD-type mutations may affect the interaction of TFIIH with transcription factors involved in other regulated pathways such as differentiation, development and neurogenesis. The possibility that TTD mutations differentially affect activated transcription mediated by the interaction of TFIIH with different transcription regulators may be an alternative explanation of the lack of cancer proneness in TTD, despite the reduced efficiency of NER.

TFIIH Defects and Clinical Outcome

It is apparent from recent studies that the activity of TFIIH in transcription is multifaceted, ranging from transcription by RNA pol I and RNA pol II to regulation of gene expression. It has also become clear that the role of TFIIH in fundamental cellular processes, transcription, DNA repair and cell-cycle, is more complex than initially thought, which has important implications for the interpretation of phenotypes caused by alterations in TFIIH. The effect of mutations in the XPB or XPD genes may each be subtly different, affecting in slightly different ways the stability and the conformation of TFIIH and, consequently, its functional activities. The phenotypic consequences of a mutated allele will depend on the precise balance and inter-relationships between these effects and the clinical outcome in patients will reflect the combined effects of each allele on TFIIH activity and the gene dosage. This might explain the puzzling variety of pathological phenotypes, ranging from Cerebro-Oculo-Facio-Skeletal syndrome (COFS)59 to combined XP/TTD features,60 that have been recently identified in association with defects in XPD. A complex phenotype of moderate severity with some features of both XP and TTD has been found in two patients, coded XP38BR and XP189MA.60 In both patients, polarized light microscopy revealed a tiger-tail appearance of the hair, and amino acid analysis of the hairshafts showed levels of sulfur-containing proteins between those of normal and TTD individuals. The patient XP189MA, showing undetectable levels of UDS but mild features of XP, was compound heterozygote for two novel mutations, one resulting in a protein truncation likely to be totally nonfunctional (fig. 5). It might well be that the mutation present in the less severely affected allele confers an extremely mild defect in transcription that does not completely prevent the phenotypic consequences of the repair defect, as usually found for the mutations associated with TTD. Alternatively, a peculiar situation in the genetic background of this patient may mitigate the defects in both transcription and NER, resulting in mild TTD and XP features.

In the patient XP38BR one of the XPD alleles contained a novel mutation resulting in the leu485pro change, which appeared to be lethal in S. pombe (fig. 5). The second allele contained the mutation G413A, causing the arg112his substitution, that has been found in several patients, all with the clinical features of TTD, yet the major features of XP38BR were of XP. The finding of a single mutation resulting in more than one phenotype is unprecedented for NER-defective syndrome, with only one exception. The same set of mutated CSB alleles has been observed in two brothers with a severe form of XP and in one patient with the classical form of CS.61 A further unexpected finding in XP38BR was that the UDS level was substantially higher than that in patients with the same mutation but the TTD phenotype (30% compared to 10% of normal). The most likely explanation for these perplexing findings is that this patient contains an as yet unidentified modifying mutation in another gene that partially suppresses the defects in both transcription and NER that are usually associated with arg112his alteration. As well as resulting in only partial TTD features, this milder putative transcription defect might permit the development of the skin abnormalities characteristic of XP, albeit again with a mild phenotype.

It is worthwhile mentioning that we have found the arg112his change in the state of functional hemizygosity in an Italian patient that shows a severe physical and mental impairment without the hair abnormalities typical of TTD. No cellular and biochemical alteration has been so far identified that may account for the unexpected genotype-phenotype relation in this patient (our unpublished observations). Thus, unusual phenotypes related to defects in the XPD gene are emerging and perhaps this is not so surprising in view of the complexity of the role of the TFIIH complex and of its multifaceted activities. During transcription, TFIIH interacts with a variety of factors, including tissue-specific transcription factors, nuclear receptors, chromatin remodeling complexes and RNA, suggesting that in some cases the genetic background may also play a role in the clinical outcome. In this context, the variety of clinical features associated with XP-D defects provides a unique tool to dissect the complex interplay between repair and transcription and the phenotypic consequences of mutations that affect the stability and/or the activity in repair and transcription of the TFIIH complex.

The finding that TFIIH is central to the onset of the photosensitive form of TTD, suggests that the search for the genes implicated in the nonphotosensitive form should be addressed to genes encoding factors of transcriptional regulation. This is still an unexplored research field.

In conclusion, although it is evident that there is still much more to learn about TTD, the last few years have witnessed significant progress in our understanding of the genetic and molecular bases of this disorder. We are also beginning to understand the links between molecular defects and clinical symptoms in TTD: the pathological phenotype that was difficult to explain on the basis of a repair defect appeared to be correlated to subtle defects in transcription. We have still to fully understand why a defect in the same gene causes either a cancer-prone phenotype as in XP, or multi-system abnormalities, as in TTD and how the transcriptional deficiency results in a cancer-free phenotype, even when NER is severely reduced. In this perspective, it is likely that future research will help in elucidating not only the biological roles of the functions defective in TTD, but also the crucial aspects of tumor formation, aging, differentiation and development.


I am very grateful to Alan Lehmann for our long-lasting and fruitful collaboration characterised by stimulating discussions and pleasant interactions. I acknowledge Tania Pedrini and Jean-Marc Egly for lively and helpful discussions. I thank Elena Botta, Tiziana Nardo and all the other members of my group at the IGM CNR, Pavia for their contribution to the work over the years. Our studies mentioned in the text have been supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), the EC (grants SC1-232, CHRX-CT94-0443 and QLG1-1999-00181) and the MIUR (Functional Genomics Project and FIRB grant RBNE01RNN7).


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