ORIGINAL ARTICLE

Five New CYLD Mutations in Skin Appendage Tumors and Evidence that Aspartic Acid 681 in CYLD Is Essential for Deubiquitinase Activity Ste´phanie Almeida1, Caroline Maillard1, Peter Itin2, Daniel Hohl1 and Marcel Huber1

Brooke–Spiegler syndrome, familial cylindromatosis, and familial trichoepithelioma are autosomal-dominant genetic predispositions for benign tumors of skin appendages caused by mutations in the CYLD localized on 16q12–q13. The encoded protein functions as -specific protease (UBP), which negatively regulates NF-kB and c-Jun N-terminal kinase (JNK) signaling. We investigated five families affected with these skin neoplasms and identified four premature stop codons and the novel missense mutation D681G in a family in which 11 of 12 investigated tumors were trichoepitheliomas. CYLD protein harboring this missense mutation had a significant reduced ability to inhibit TNF -associated factor (TRAF)2- and TRAF6- mediated NF-kB activation, tumor necrosis factor-a (TNFa)-induced JNK signaling, and to deubiquitinate TRAF2. CYLD-D681G was coimmunoprecipitated by TRAF2, but was unable to cleave K63-linked polyubiquitin chains. Aspartic acid 681 is highly conserved in CYLD homologues and other members of the UBP family, but does not belong to the Cys and His boxes providing the CYLD catalytic triad (Cys601, His871, and Asp889). As reported previously, the homologous residue D295 of HAUSP/USP-7 forms a hydrogen bond with the C-terminal end of ubiquitin and is important for the enzymatic activity. These results underline that D681 in CYLD is required for cleavage of K63-linked polyubiquitin chains. Journal of Investigative Dermatology (2008) 128, 587–593; doi:10.1038/sj.jid.5701045; published online 13 September 2007

INTRODUCTION at the C-terminal half of the CYLD coding sequence, lead to The Brooke–Spiegler syndrome (OMIM 605041), familial the formation of truncated proteins (Bignell et al., 2000, see cylindromatosis (OMIM 132700), and familial trichoepithe- also The Human Gene Mutation Database). Until now, only lioma (OMIM 601606) are autosomal-dominant predisposi- two missense mutations have been reported in families, tions to multiple benign adnexal skin tumors (Brooke, 1892; which were mainly affected with trichoepitheliomas (Hu Spiegler, 1899). The predominating tumors are cylindroma, et al., 2003; Zheng et al., 2004). Three trichoepithelioma trichoepithelioma, and spiradenoma that are mainly loca- families were mapped to chromosome 9p21 (Harada et al., lized on the face and scalp, but less frequently on the neck 1996) but no gene mutations at this locus have been reported and trunk, and which appear generally in early adulthood to date. (Burrows et al., 1992). These diseases are caused by germline Motif analysis revealed that the C-terminal region of CYLD mutations in a single gene, called CYLD, localized on displayed good sequence similarity to ubiquitin-specific chromosome 16q12–q13 (Biggs et al., 1995; Bignell et al., proteases (UBP, also called USP) (Nijman et al., 2005), a 2000; Fenske et al., 2000; Takahashi et al., 2000). Loss of subfamily of the large group of deubiquitinating enzymes heterozygosity at the same locus was reported in a large (Amerik and Hochstrasser, 2004). The N-terminal section has number of these tumors suggesting that CYLD may function three CAP-GLY regions, a motif found previously in a number as a tumor suppressor (Biggs et al., 1995, 1996; Takahashi of microtubule-associated proteins (Bignell et al., 2000). et al., 2000). Most of the mutations, which are mainly located CYLD interacts with NEMO, TNF receptor-associated factor (TRAF)-2, and TRAF-interacting protein and negatively 1Service of Dermatology, University Hospital Center and University of regulates the canonical NF-kB pathway Lausanne, Lausanne, Switzerland and 2Department of Dermatology, (Brummelkamp et al., 2003; Kovalenko et al., 2003; Regamey University Hospital, Basel, Switzerland et al., 2003; Trompouki et al., 2003). It does so by acting as Correspondence: Dr Marcel Huber, CHUV, Service de dermatologie, 1011 , which specifically removes K63- Lausanne, Switzerland. E-mail: [email protected] linked polyubiquitin chains from TRAF2, TRAF6, and NEMO Abbreviations: HA, hemagglutinin A; JNK, c-jun N-terminal kinase; TNF, (Brummelkamp et al., 2003; Kovalenko et al., 2003; tumor necrosis factor; TRAF, TNF receptor-associated factor; Ub, ubiquitin; UBP, ubiquitin-specific protease; WT, wild type Trompouki et al., 2003). CYLD mRNA is upregulated by Received 12 December 2006; revised 4 July 2007; accepted 5 July 2007; TNFa and bacterium nontypeable Haemophilus influenza, published online 13 September 2007 providing evidence for an inducible autoregulatory feedback

& 2007 The Society for Investigative Dermatology www.jidonline.org 587 S Almeida et al. CYLD Mutations in Brooke–Spiegler Syndrome

loop controlling CYLD expression (Jono et al., 2004). Subsequently, other signaling pathways have been identified I I 1M that are regulated by CYLD. c-Jun N-terminal kinase (JNK) II II signaling pathway activated by TNFa, anti-CD40 antibody, 3 M IL-1b, and lipopolysaccharide is repressed by CYLD by reducing the activity of MKK7/JNKK2, which is the mitogen- III ∗ III activated protein kinase that phosphorylates JNK in response IV 2M 1M 2M to these inducers (Reiley et al., 2004). Functional analysis of 1M thymocytes in CYLD knockout mice revealed that CYLD functions as a positive regulator of proximal T-cell receptor I signaling by promoting recruitment of active to its II substrate Zap70 (Reiley et al., 2006). Moreover, CYLD 3M removes polyubiquitin moieties from Lck, counteracting the III activity of c-Cbl ubiquitin (Ub) ligase, which represses Lck 1M 2M 3N 4N activity. Furthermore, CYLD acts as a negative regulator of Toll-like receptor 2 signaling by deubiquitinating TRAF6 and TRAF7 (Yoshida et al., 2005). More recently, it has been shown that 12-O-tetradecanoylphorbol-13-acetate or UVB light triggers the translocation of CYLD from the cytoplasm to the perinuclear region, where it interacts and deubiquitinates the proto- Bcl3, a member of the IkB family that can conjointly with p52 act as transcriptional activator of target such as cyclin D1. Deubiquitination by CYLD prevents the nuclear translocation of Bcl3 and therefore represses Figure 1. Genetic and clinical data of families affected with skin appendage Bcl3-dependent gene regulation (Massoumi et al., 2006a). In tumors. Pedigrees of (a) family F1, (b) family F2, and (c) family F3 which summary, the deubiquitinase CYLD regulates several signal are shown in Table 1. Arrows denote the index case in each family. M and N mark the presence or absence of the heterozygous mutations in transduction pathways in which signal propagation crucially the germline, respectively. Black symbols identify individuals with tumors. depends on the polyubiquitination level of key proteins. (d) Photograph of the index case from family F3 showing trichoepitheliomas. We have identified five new CYLD mutations in families (e) Hematoxylin and eosin stained section of tumor isolated from the affected with different combinations of skin appendage shoulder of the F3 index case. Bar ¼ 50 mm. tumors. In particular, we report here the missense mutation D681G found in a family exhibiting predominantly trichoe- pitheliomas. Biochemical evidence underlines that this codons. In family F3, whose affected members suffered mutation affects the deubiquitinating enzyme activity of primarily from trichoepitheliomas (Table 1), we found an A to CYLD by strongly reducing its enzymatic activity. G change at position 2433 (GenBank no. NM_015247) of human CYLD cDNA mutating aspartic acid 681 to a glycine RESULTS residue. We investigated all available family members for the Skin biopsies and blood samples from the index case and at presence or absence of these mutations. In each family, the least one other member from family F1 to F3 (Figure 1a–c) genotype correlated well with the occurrence of tumors were collected. Pedigree analysis in these families was except in family F1 in which individual IV-1 (Figure 1a, consistent with autosomal-dominant inheritance of the asterisk) was a carrier for the mutation but did not yet show disease. In addition, specimens of a single member from any skin lesions at the age of 23. two other families (denoted as F4 and F5 in Table 1) were Aspartic acid at position 681 of human CYLD does not obtained. However, it is presently unknown whether they belong to the Cys (amino acids 590–610) or to the His box represent familial or sporadic cases, because only one family (amino acids 870–890), which are two well-conserved motifs member could be examined and no family history was within UBPs (Nijman et al., 2005). Both of these regions were available. Hematoxylin and eosin stained sections of the proposed to be essential for deubiquitinase activity (Brum- tumors were examined by an experienced dermatopatho- melkamp et al., 2003; Kovalenko et al., 2003; Trompouki logist (Table 1). Mainly, cylindromas, trichoepitheliomas, and et al., 2003; Amerik and Hochstrasser, 2004). The region a few spiradenomas were found in families F1 and F2. In surrounding D681 of human CYLD is not very well conserved contrast, 11 of 12 tumors found at different body sites in three between members of the UBP subfamily and CYLD (Figure members of family F3 were classified as trichoepitheliomas, 2b). However, it is highly conserved in vertebrate and showing horn cysts with a fully keratinized center surrounded invertebrate CYLD homologues (Figure 2a), suggesting that by basaloid cells (Table 1 and Figure 1e). it might have a crucial function. To investigate the biological Sequence analysis of genomic DNA isolated from blood of consequences of the D681G mutation on CYLD function, we the index cases detected the heterozygous mutations in the assessed the regulation of CYLD-dependent signaling path- CYLD gene shown in Table 1. In four families, these ways by CYLD-D681G in comparison with wild-type (WT) mutations were predicted to introduce premature stop protein or CYLD-C601A (active-site mutant).

588 Journal of Investigative Dermatology (2008), Volume 128 S Almeida et al. CYLD Mutations in Brooke–Spiegler Syndrome

Table 1. CYLD mutations, tumor types, and body locations in five families Family Exon Germline mutation Codon1 Individual2 Sex Age at diagnosis Age3 Tumor4 Body site

F1 15 DelT2472 L694X III-2 F 24 49 TE Buttock SP Back CYL Scalp F2 19 G2937A W849X I-1 M 35 35 CYL Scalp 57 CYL Scalp II-3 F 38 38 TE Forehead III-1 F 5 10 CYL Scalp TE Nose III-2 M 8 8 TE Forehead F3 15 A2433G D681G II-3 M N/A 55 TE Buttock 56 TE(2) Ear 62 SP N/A 63 TE Occiput TE Neck TE Trunk III-1 M 18 18 TE Buttock III-2 M 22 22 TE(2) Back TE Forehead TE Occiput F4 10 C1518T N403X F N/A 67 CYL Scalp F5 9 G1526T E379X M 35 76 CYL Scalp CYL, cylindroma; F, female; M, male; N/A, not available; SP, spiradenoma; TE, trichoepithelioma. 1Amino-acid mutation; X denotes stop codon. 2Individuals according to pedigrees shown in Figure 1. 3Age at which tumor was removed. 4(2), number of isolated tumors from the same body site.

In contrast to the WT protein, both mutants did not reduce TRAF2 after co-transfection with CYLD mutants was similar NF-kB activity induced by TRAF2 (Figure 3a) and TRAF6 to the control without CYLD (Figure 5a, upper panel). This (Figure 3b). These findings were not caused by differences in shows that D681 of CYLD is required for TRAF2 deubiqui- protein expression (Figure 3, lower panels). To address the tination. No significant differences in the expression levels of question whether CYLD-D681G can downregulate the JNK TRAF2 and CYLD proteins were observed (Figure 5a, lower pathway activated by TNFa (20 ng/ml for 15 minutes) (Reiley panels). The WT and the two mutant CYLD proteins were et al., 2004), a nonradioactive glutathione S-transferase-c-jun coimmunoprecipitated by TRAF2 (Figure 5b), showing that pull-down kinase assay was performed with cell extracts from reduced deubiquitination of TRAF2 by CYLD-D681G is not 293T cells expressing normal and mutant CYLD proteins. caused by diminished binding to TRAF2. Although WT Although CYLD-D681G and CYLD-C601A were present at protein cleaved in vitro Lys63-linked polyubiquitin chains levels similar to WT CYLD (Figure 4, lower panel), higher partially to monomeric Ub (see right lane in Figure 5c, upper levels of phosphorylated c-Jun protein were found in the panel), cleavage by CYLD mutants was strongly reduced presence of the mutants (Figure 4, upper panel), indicating that (Figure 5c), indicating that D681 in human CYLD is important they were unable to inhibit TNFa-mediated activation of JNK. for Lys63-linked polyubiquitin chain cleavage. Furthermore, we tested how the D681G mutation affected the deubiquitinase function of CYLD. 293T cells DISCUSSION were co-transfected with vectors expressing -TRAF2, It is now well established that CYLD mutations cause familial hemagglutinin A (HA)-Ub, and the different Flag-CYLD cylindroma, familial trichoepithelioma, and Brooke–Spiegler proteins. The ubiquitination level of immunoprecipitated syndrome, suggesting that these diseases represent phenoty- TRAF2 was assessed by anti-HA immunoblot analysis. pic variations of the same disease entity. We have identified Overexpression of WT CYLD caused a substantial reduction five new mutations in the CYLD gene (four premature stop in TRAF2 ubiquitination (Figure 5a, upper panel) (Brummelk- codons and one missense mutation) in families suffering from amp et al., 2003; Kovalenko et al., 2003; Trompouki et al., these skin appendage neoplasms. In particular, we examined 2003; Reiley et al., 2005), whereas the ubiquitination level of the functional consequences of the missense mutation

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30

Human Orangutan 25 Dog Cow 25 Mouse Rat 15 Zebrafish Chicken Drosophila 10 C.elegans Luciferase activity Luciferase 5 Cons 0 Flag-CYLD UBP7_HUMAN UBP5_HUMAN UBP14_YEAST Flag-TRAF2 UBP3_YEAST FAF_DROME UBP4_YEAST Flag-TRAF2 – ++ ++ UBP10_YEAST UBP6_YEAST Flag-CYLD – – + – – CYLD_HUMAN Flag-CYLD-D681G – – – + – Cons Flag-CYLD-C601A – –– – +

60 Figure 2. Sequence alignments between CYLD and UBPs. Comparisons of amino-acid sequences between (a) CYLD homologues and (b) human CYLD 50 and different UBPs. The arrows mark the conserved aspartic acids which 40 correspond to D681 in human CYLD. The consensus symbols are as follows: 30 ‘‘*’’ means identical amino-acid residues in all sequences in the alignment; ‘‘:’’ indicates conserved substitutions; and ‘‘.’’ denotes semi-conserved 20

substitutions. activity Luciferase 10

0 Flag-CYLD D681G discovered in a family affected almost exclusively with multiple trichoepitheliomas. Similarly to active-site Flag-TRAF6 C601 CYLD mutants (Trompouki et al., 2003; this work), CYLD-D681G was unable to inhibit TRAF2- or TRAF6- Flag-TRAF6 – + + + + mediated activation of NF-kB and TNFa activation of JNK, Flag-CYLD – – + – – Flag-CYLD-D681G – – – + – and to deubiquitinate TRAF2. The latter was not caused by Flag-CYLD-C601A – – – – + reduced binding to TRAF2, as shown by coimmunoprecipita- tion but by a strongly reduced ability to cleave K63-linked Figure 3. Regulation of TRAF2- and TRAF6-mediated NF-jB activation by polyubiquitin chains. This is in agreement with results WT and mutant CYLD proteins. NF-kB reporter assays in 293T cells co-transfected with WT or mutant (D681G and C601A) Flag-CYLD and mapping the TRAF2-binding site of CYLD to amino acids either (a) Flag-TRAF2 or (b) Flag-TRAF6. Luciferase activities were measured 453–457 (Kovalenko et al., 2003). 24 hours after transfection. The presence of proteins in cell extracts was CYLD belongs to the UBP family, which is mainly analyzed by immunoblots using anti-Flag antibody (lower panels). Results are involved in removing Ub moieties from proteins larger than reported as mean7SEM from duplicates of one representative experiment. 20–30 amino acids (Amerik and Hochstrasser, 2004; Nijman These assays were carried out five (TRAF2) or three (TRAF6) times with et al., 2005). Mutagenesis experiments identified a conserved essentially the same results. triad Cys-Asp/Asn-His that has a critical role in catalysis (Huang et al., 1995; Gilchrist and Baker, 2000; Baek et al., ability. Whether this is caused by reduced binding of CYLD- 2001; Amerik and Hochstrasser, 2004). Based on these D681G to polyubiquitin or by affecting the catalytic findings and sequence alignments with UBP proteins, the mechanism remains to be determined. CYLD catalytic triad was putatively assigned to Cys601, Analyses of germline CYLD mutations in familial cylin- His871, and Asp889 (Kovalenko et al., 2003). Although the dromatosis, familial trichoepithelioma, and Brooke–Spiegler region around aspartic acid 681 does not belong to the syndrome revealed the preponderance of premature stop catalytic triad, the functional importance of this region is codon mutations in all three clinical forms. The two reported emphasized by the high conservation in CYLD homologues CYLD missense mutations, E747G and D941V, were detected (Figure 2a) and, albeit to a lesser degree, in other members of in families affected primarily with trichoepithelioma (Hu the UBP family (Figure 2b). Recent structural and molecular et al., 2003; Zheng et al., 2004). Both residues are highly data of the UBP HAUSP/USP-7 showed that Asp295, which is conserved in CYLD homologues, but are not conserved in homologous to human CYLD Asp681 (Figure 2b), hydrogen other members of the UBP family and are localized to regions bonds to the amide of Leu73 from Ub. In addition, mutating outside of the active-site cysteine and the His box. The D941 D295 disabled HAUSP/USP-7 to cleave Lys48-linked diubi- residue is situated two amino acids before two conserved quitin (Hu et al., 2002). Similarly, our data indicate that residues (YXXXY). The HAUSP/USP-7 crystal struc- alteration of D681 abolishes the deubiquitinating activity of ture identified the first of these to make van der CYLD by inhibiting the K63-linked polyubiquitin cleavage Waals contacts with the C-terminal end of bound Ub (Hu

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Phospho- c-Jun (Ser63)

Flag-CYLD IP: myc-TRAF2 TNF- – + + + + IB: HA-Ub Flag-CYLD – – – – + 66 kDa Flag-CYLD-D681G – – – + – Flag-CYLD-C601A – – + – – IP and IB: myc-TRAF2 Figure 4. Regulation of TNFa-induced JNK activity by WT and mutant CYLD IB: Flag-CYLD proteins. 293T cells were transfected with WT or mutant Flag-CYLD and treated with TNFa (15 minutes, 20 ng/ml). JNK proteins were captured by myc-TRAF2 – + + + + + pull-down with glutathione S-transferase-c-jun beads. The activity of JNK HA-Ub + – + + + + was determined by incubation with ATP and detection of the level of Flag-CYLD – – – – – + phosphorylated c-jun with a specific phospho-c-Jun (Ser-63) antibody (upper Flag-CYLD-D681G – – – – + – Flag-CYLD-C601A – – – + – – panel). CYLD proteins were detected in cell extracts by immunoblots with anti-Flag antibody (lower panel). IP: myc-TRAF2 IB: Flag-CYLD et al., 2002). The functional importance of this stretch is IP and IB: myc-TRAF2 further underlined by the finding of a premature stop codon at R936 in a cylindroma family (Bignell et al., 2000). As the IB: Flag-CYLD amino acid E747 and its neighboring sequences in CYLD are myc-TRAF2 + + + + not conserved in other UBPs, it is difficult to predict the Flag-CYLD – + – – functional role of this residue at the moment. Interestingly, Flag-CYLD-D681G – – – + we report now the third missense mutation in the CYLD gene, Flag-CYLD-C601A – – + – again in a family predominantly affected with trichoepithe- liomas (F3 in Table 1). Whether there is indeed an association Flag-CYLD between CYLD missense mutations and trichoepithelioma formation awaits further molecular analysis. Previous work Ub5 provided no evidence for a genotype/phenotype correlation Ub4 (Bowen et al., 2005). Ub3 Cylindroma and trichoepithelioma are thought to originate Ub2 Ub1 from hair follicle cells (Massoumi et al., 2006b). In CYLD knockout mice, tumors are only formed after treating animals IB: Flag-CYLD Poly-Ub with a two-stage protocol, suggesting that 2–7 + + + + Flag-CYLD – – – + mutations in other genes are required for tumor development Flag-CYLD-D681G – – + – (Massoumi et al., 2006a). In humans, these tumors develop Flag-CYLD-C601A – + – – mainly on sun-exposed body sites suggesting that additional Figure 5. Deubiquitination of TRAF2 and in vitro cleavage of K63-linked UV-induced mutations may play a crucial role in their polyubiquitin by CYLD. (a) 293T cells were co-transfected with mycTRAF2, formation. The level of signaling molecules regulating HA-Ub and WT or mutant Flag-CYLD. The level of ubiquitinated TRAF2 was follicular-sebaceous-apocrine differentiation of committed examined by anti-myc immunoprecipitations (IP) and immunoblotting (IB) progenitor cells will influence the decision regarding which with anti-HA antibody (upper panel). The amount of immunoprecipitated tumor type is formed. Additional mutations may alter these TRAF2, and of CYLD proteins in total cell extracts was detected using levels changing the differentiation phenotype of the tumor, as anti-myc antibody (middle panel) and anti-Flag antibody (lower panel), respectively. (b) 293T cells were co-transfected with mycTRAF2 and WT or shown recently for LEF1 inactivating mutations that lead to mutant Flag-CYLD. After immunoprecipitation with anti-myc antibody, the the expression of sebocyte differentiation markers and the amount of coimmunoprecipitated CYLD was examined with anti-Flag formation of sebaceous tumors (Takeda et al., 2006). antibody (upper panel). The middle and lower panels depict the quantities of Furthermore, differences in the biological activity of modifier immunoprecipitated TRAF2 and of CYLD in total cell extracts, respectively. genes caused by genetic or epigenetic alterations may (c)A2mg portion of K63-linked polyubiquitin (poly-Ub2–7) chains was determine the outcome of a cancer-promoting insult. For incubated with WT or mutant Flag-CYLD protein immunopurified from example, polymorphic variants of the Patched gene decide transfected 293T cells. Cleaved proteins were detected by silver staining after electrophoresis in a 4–20% gradient SDS-PAGE gel (upper panel). Arrows whether K5Hras transgenic mice develop early-onset squa- mark the positions of CYLD proteins and the polyubiquitin chains. The mous cell carcinomas (Wakabayashi et al., 2007). In amount of immunopurified CYLD protein was estimated by immunoblotting summary, these molecular mechanisms may explain the using anti-Flag antibody (lower panel). diversity of tumor types which are initiated by mutations in the same gene.

MATERIALS AND METHODS Ethical Committee of the Faculty of Biology and Medicine at the Clinical samples University of Lausanne (Lausanne, Switzerland), was conducted Biopsies and blood samples were obtained from the patients with according to the Declaration of Helsinki Principles. Tumors were their written informed consent. The study, approved by the Medical classified by an approved dermatopathologist.

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DNA sequence analysis in three independent assays. TRAF2/CYLD coimmunoprecipitations DNA was extracted from blood using the Nucleon BACC1 (GE were carried out as described (Regamey et al., 2003) using the Healthcare, Otelfingen, Switzerland). Sequence analysis of exons aforementioned antibodies. 9–20 of the CYLD gene were carried out as described (Bignell et al., 2000). Cleavage of K63-linked polyubiquitin chains Flag-CYLD proteins were purified from transfected 293T cells by Plasmids Flag immunoprecipitation and protein elution using Flag peptide k The pNF- B-luc and pRL-TK vectors were purchased from Stratagene (500 mg/ml) (Trompouki et al., 2003). A 10 ml portion of immuno- (Amsterdam, The Netherlands) and Promega (Wallisellen, Switzer- precipitated proteins was incubated with 2 mg of K63-linked land). To generate the pCYLD2 vector, N-terminal Flag-tagged polyubiquitin chains (mixture of 2–7 Ub units; BostonBiochem, human CYLD cDNA from the pCYLD1 vector (Regamey et al., 2003) Lausen, Switzerland) at 371C for 2 hours. After electrophoresis was cloned into the pCI expression vector (Promega, Wallisellen, through a 4–20% linear gradient SDS-PAGE gel, samples were Switzerland). Point mutations were introduced into pCYLD2 using analyzed by silver staining or immunoblotting using anti-Flag M2 the QuikChangeII kit (Stratagene) to generate pCYLD2-D681G and antibody. pCYLD2-C601A. All constructs were verified by sequencing (Synergene, Schlieren, Switzerland). Vectors for expressing HA-Ub, Sequence comparison Flag-TRAF2, and Flag-TRAF6 were provided by Drs. G. Courtois and Protein sequence alignments were calculated using the T-Coffee P. Schneider. analysis program (Notredame et al., 2000). Cell culture and NF-jB reporter assays 293T cells were cultured as described (Regamey et al., 2003). Cells CONFLICT OF INTEREST in 24-well plates (50,000 cells/well) were transfected 24 hours after The authors state no conflict of interest. seeding with 0.1 mg of the indicated expression plasmids, 0.2 mgof pNF-kB-luc, and 0.01 mg of pRL-TK using Lipofectamine 2000 ACKNOWLEDGMENTS reagent (Invitrogen, Basel, Switzerland). After 24 hours, luciferase This work was supported by grants from the Swiss National Science Foundation (3100A0-105459) and Oncosuisse (OCS 01150-09-2001) to activities were measured using the Dual-Luciferase-Reporter Assay M.H. We thank Drs. G. Courtois and P. Schneider for the kind gift of 7 System (Promega). Results are reported as mean SEM from plasmids and the patients for their cooperation in this study. duplicates of one representative experiment. These assays were carried out three (TRAF6) or five (TRAF2) times with essentially the same results as shown. 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Oncogene 12:1375–7 Protein extraction, kinase reaction, and phospho c-Jun Biggs PJ, Wooster R, Ford D, Chapman P, Mangion J, Quirk Y et al. (1995) detection were carried out according to the SAPK/JNK Assay Familial cylindromatosis (turban tumour syndrome) gene localised to chromosome 16q12–q13: evidence for its role as a tumour suppressor Kit Manual ( Technology, Allschwil, Switzerland). gene. Nat Genet 11:441–3 The experiment was repeated entirely twice with similar results Bignell GR, Warren W, Seal S, Takahashi M, Rapley E, Barfoot R et al. (2000) obtained. Identification of the familial cylindromatosis tumour-suppressor gene. Nat Genet 25:160–5 TRAF2 deubiquitination assay and coimmunoprecipitation Bowen S, Gill M, Lee DA, Fisher G, Geronemus RG, Vazquez ME et al. (2005) 293T cells were transfected as described above with 0.5 mgof Mutations in the CYLD gene in Brooke–Spiegler syndrome, familial cylindromatosis, and multiple familial trichoepithelioma: lack of m expression plasmids for HA-Ub, myc-TRAF2, and 0.7 g of plasmids genotype–phenotype correlation. J Invest Dermatol 124:919–20 expressing WT or mutant Flag-CYLD proteins. Forty-eight hours after Brooke H (1892) Epithelioma adenoides cysticum. Br J Dermatol 4:269 transfection, cells were lysed in 0.5% SDS/phosphate-buffered Brummelkamp TR, Nijman SM, Dirac AM, Bernards R (2003) Loss of the saline, heated for 2 minutes at 851C, and centrifuged. Supernatants cylindromatosis tumour suppressor inhibits apoptosis by activating NF- were diluted 1:10 with RIPA buffer and used for immunoprecipita- kappaB. Nature 424:797–801 tions (Regamey et al., 2003) with mouse anti-myc antibody (9B11; Burrows NP, Jones RR, Smith NP (1992) The clinicopathological features of Cell Signaling Technology). 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