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Genomics 90 (2007) 703–711 www.elsevier.com/locate/ygeno

Mutations in the helix termination motif of mouse type I IRS genes impair the assembly of keratin

Shigekazu Tanaka a, Ikuo Miura b, Atsushi Yoshiki c, Yoriko Kato a, Haruka Yokoyama b, ⁎ Akiko Shinogi b, Hiroshi Masuya b, Shigeharu Wakana b, Masaru Tamura a, Toshihiko Shiroishi a,b,

a Mammalian Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8450, Japan b Mouse Functional Genomics Research Group, RIKEN Genomic Sciences Center, 3-1-1 Koyadai, Tsubaka, Ibaraki 305-0075, Japan c Experimental Animal Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan Received 26 April 2007; accepted 20 July 2007 Available online 24 October 2007

Abstract

Two classical mouse hair coat mutations, Rex (Re) and Rex wavy coat (Rewc), are linked to the type I inner root sheath (IRS) keratin genes of chromosome 11. An N-ethyl-N-nitrosourea-induced mutation, M100573, also maps close to the type I IRS keratin genes. In this study, we demonstrate that Re and M100573 mice bear mutations in the type I IRS gene Krt25; Rewc mice bear an additional mutation in the type I IRS gene Krt27. These three mutations are located in the helix termination motif of the 2B α-helical rod domain of a type I IRS keratin . Immunohistological analysis revealed abnormal foam-like immunoreactivity with an antibody raised to type II IRS keratin K71 in the IRS of Re/+ mice. These results suggest that the helix termination motif is essential for the proper assembly of types I and II IRS keratin protein complexes and the formation of keratin intermediate filaments. © 2007 Elsevier Inc. All rights reserved.

Keywords: Rex; Wavy coat; ; Inner root sheath; Krt25; Krt27; Proline; Deletion; Helix termination motif; ENU mutagenesis

Keratins, a family of intermediate filament (IF) , are To date, a wide variety of keratin and keratin-related genes have primarily involved in the mechanical and structural functions of been reported [2–4]. Multiple mouse mutants with similar de- epithelial tissue. Keratin proteins are composed of four α- fects in hair have been reported; the mutations in these animals helical rod domains, 1A, 1B, 2A, and 2B, which are interrupted mapped to chromosome 11 or 15 [5]. Identification of the cor- by a nonhelical linker and flanked by nonhelical head and tail responding genes in these mutant mice has provided new insight domains. The N- and C-termini of the α-helical rod domain are into the functions of these proteins. more highly conserved across all IF proteins than the α-helical Caracul (Ca) mice, which bear a dominant mutation mapped region of other α-helical rod domains. These termini are to mouse chromosome 15, possess curly hair and vibrissae [6]. referred to as the helix initiation motif and helix termination Ca mice are reported to have a mutation in keratin 71 (Krt71; motif, respectively. Type I (acidic) and type II (basic) keratin formerly designated mK6irs1/Krt2-6g) [7–9], a member of the proteins form obligate heterodimers with their α-helical rod type II inner root sheath (IRS) keratin genes. The Krt71 protein domains [1]. The genes encoding these type I and type II kera- is expressed specifically in the IRS of hair follicles [10–12]; Ca tins are located on mouse chromosomes 11 and 15, respectively. mice exhibit morphological abnormalities of the IRS [7–9]. The gene responsible for another classical hair coat mutation, Rex ☆ Sequence data from this article have been deposited with the EMBL/ (Re), maps to an interval between Hoxb5 and Wnt3 that con- GenBank/DDBJ Data Libraries under Accession No. AB288231. tains the type I keratin gene cluster [13–15]. Re, a semidomi- ⁎ Corresponding author. Mammalian Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka nant mutation, confers a similar phenotype to Ca mice, with 411-8450, Japan. Fax: +81 55 981 6817. curly hair and vibrissae [13,16,17]. Vibrissae of the homozygote E-mail address: [email protected] (T. Shiroishi). are more strongly curled than those in the heterozygote [16].

0888-7543/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2007.07.013 704 S. Tanaka et al. / Genomics 90 (2007) 703–711

The diameter of the hair shaft in Re mice is irregular due to irregular and fragile (Fig. 1C). This phenotype becomes ap- morphological abnormalities in all three layers of the IRS, parent at 1 month of age (Figs. 1B and 1C);thewaveofpelage Henle's layer, Huxley's layer, and the cuticle [17,18]. Although becomes weaker thereafter [13,16–18]. Our histological ana- the Re mutant was first described in 1939, the causative gene lysis of 1-month-old Re/Re mice revealed bent hair follicles has not yet been identified. Re mice are derived from a com- and fragile hair shafts (Fig. 1E). Re/Re mice demonstrated mercial breeder in Essex in the United Kingdom [13]; the enlargement of sebaceous glands compared to the sizes seen in genetic background in which Re originally arose is unknown. wild-type mice (Fig. 1E). At this stage of development, the The recent discovery of four human type I IRS keratin genes, average length of hair follicles in Re/Re mice was shorter keratin 25 (KRT25), keratin 26 (KRT26), keratin 27 (KRT27), than that seen for wild-type mice (Figs. 1D and 1E). The and keratin 28 (KRT28), and identification of many type I and dermal papillae of Re/Re mice, however, were morphologi- type II prompted refinement of the previous nomen- cally normal (Figs. 1D and 1E). To identify the hair follicle clature of the keratin gene family [2,19–24]. We previously defect, we performed toluidine blue staining. Henle's layer of reported the isolation of a mouse skin-specific keratin cDNA, the IRS in Re/+ mice was nonuniform and discontinuous in Krt1-c29 [25]. This gene is now classified as a mouse homo- the hair follicles of all hair types (white arrowheads in Figs. logue of the type I IRS keratin genes and has been renamed 1G,1I,1K,1M,and1O), a histological phenotype similar to Krt27. Porter et al. [26] also reported three mouse type I IRS that of Ca mice [7–9]. keratin gene homologues, keratin 25 (Krt25, mIRSa1), keratin 27 (Krt27, mIRSa3.1, Krt1-c29), and keratin 28 (Krt28, Linkage analysis of Re mIRSa2). The expression patterns of the type I IRS keratin proteins are similar to those of type II IRS keratin proteins in the The spontaneous mutation responsible for the Re phenotype IRS of both human and mice [12,22,24]. As the Re mutation is was first reported in 1939; the genetic background upon which linked to the mouse type I IRS keratin gene cluster and the the mutation arose is unknown. This uncertainty of genetic phenotype of Re mice is similar to that of Ca mice, we hypo- background hampered genetic analysis of the Re mutation. In thesized that the gene product responsible for the Re phenotype our laboratory, we have maintained the Re mutant stock by is a keratin interacting with the type II keratin Krt71, the protein repeated backcrosses of phenotypic carriers onto C57BL/6J mutated in Ca mice. (B6), which reached 50 generations in September 2004. Using In this study, we confirmed the tight linkage of the Re mu- backcrossed progeny at 56 and 57 generations, we performed tation with the type I IRS keratin gene cluster. Subsequently, our linkage analysis of 224 progeny. Backcrossed progeny analysis of the type I IRS keratin genes Krt25, Krt26, Krt27, were phenotyped and genotyped for microsatellite markers to and Krt28 in Re mutants discovered a single nonsynonymous distinguish the original Re background from that of B6. In the base substitution in the helix termination motif of the 2B α- progeny, four markers, D11Mit264, D11Mit124, D11Mit330, helical rod domain of the type I IRS gene Krt25. This mutation and D11Mit198, were polymorphic, indicating persistence of results in an amino acid change of leucine 381 to proline. the original Re genome region in the B6 background. Three Immunohistological analysis of Re/+ skin with an anti-K71 additional markers, D11Mit99, D11Mit123,andD11Mit329, antibody demonstrated a rough, foam-like pattern of the keratin were monomorphic (Fig. 2). The phenotype in all of the pro- filament, suggesting that the mutation in Krt25 is responsible geny was completely concordant with the genotype of for the Re phenotype. D11Mit124, D11Mit330,andD11Mit198. On the other hand, We also performed a mutational analysis of two independent 7 of the 244 progeny exhibited a discordant phenotype with the hair coat mutants, the classical Rex wavy coat (Rewc) mouse genotype of D11Mit264 mice (Fig. 2). Thus, while the gene [27] and a new N-ethyl-N-nitrosourea (ENU)-induced mutant causing the Re phenotype is segregated from D11Mit264,itis M100573 mouse, both of which exhibit a phenotype resembling tightly linked to the type I IRS keratin gene cluster residing in Re. The results revealed a missense mutation in the K25 helix the interval between D11Mit124 and D11Mit198 (Fig. 2). This termination motif in M100573 and a deletion of exon 6, which result also suggested that the region distal to D11Mit99 may encodes the helix termination motif of K27, in Rewc. This is the have been replaced by the B6 genetic background in the Re first report to identify disease-causing mutations in the type I mutant line, although we cannot exclude the possibility that we IRS keratin genes Krt25 and Krt27, and these results suggest could not discriminate between Re and B6 genotypes by that the helix termination motif of the 2B α-helical rod domain microsatellite markers because the original Re genetic back- is essential for proper assembly of type I and type II keratin ground was not polymorphic for B6. Previously, linkage protein complexes and formation of keratin IFs. analysis suggested that Re is an allele of Rex denuded (Reden) [28]. Reden has a mutation in the nonkeratin gene Gasdermin Results A3 (Gsdma3, this gene symbol is not officially approved by the nomenclature committee, currently designated Gasdermin 3, Characterization of Re phenotype Gsdm3) [29],whileRexwavycoat(Rewc) [27],which had been thought to be an allele of Re, had no mutation in The gross anatomy of the hair coat of Re/Re mice exhibits Gsdma3 [30]. An additional skin and hair coat mutant, more waved pelage and smaller body size compared to Re/+ Bareskin (Bsk), which is genetically linked to Re,bearsa mice (Fig. 1A) [13,16–18]. Vibrissae of Re/+ mice seem to be mutant allele of Gsdma3 (now designated Gsdma3Bsk) [30,31]. S. Tanaka et al. / Genomics 90 (2007) 703–711 705

Fig. 1. Phenotype of Re mice. (A) Re/Re mice exhibit more strongly curled pelage and smaller body size compared to Re/+ mice at 1 month after birth. Compared with (B) the vibrissae of wild-type animals, Re/+ mice display (C) ragged and fragile vibrissae. HE staining of dorsal skin sections from (D) wild-type and (E) Re/Re mice at 1 month of age demonstrated that all hair follicles were bent; the hair shafts were extremely fragile in Re/Re mice. Sebaceous glands were enlarged in Re/Re mice (E) in comparison to wild-type mice (D). Longitudinal sections of (F, G) vibrissae and both (H, I) large and (J, K) small hair follicles from wild-type (F, H, J) and Re/+ mice (G, I, K) at 1 month of age are compared with cross sections of (L, M) large and (N, O) small hair follicles from 1-month-old wild-type (L, N) and Re/+ mice (M, O) using toluidine blue staining. Arrowheads indicate the break points in Henle's layer of the IRS. The scale bars indicate (D, E) 100 and (F–O) 25 μm.

This study excluded Gsdma3 as the causative gene of the Re genes are the most likely candidates for the causative gene of phenotype. Re (Fig. 2). Using a mouse genome database, we searched for mouse homologues of type I IRS keratin genes found in the Exploration of Re candidate gene homologous region of the human genome. We identified three putative IRS keratin genes, Krt25, Krt26, and Krt27, and one A search of several public genome databases revealed that partial sequence, Krt28, in this region. To determine if this the genomic region between D11Mit264 and D11Mit99 con- partial sequence encodes the mouse Krt28 homologue, we tains 19 nonkeratin genes, 6 epithelium keratin genes, 4 IRS isolated the predicted cDNA that contains an open reading keratin genes, 2 genes, and 13 keratin-associated frame using reverse transcriptase–polymerase chain reaction protein genes. The phenotype of Re and previous insights from (RT-PCR). Phylogenetic analysis with other type I keratin genetic analysis of Ca suggested that the type I IRS keratin genes indicated that the newly identified cDNA is a mouse 706 S. Tanaka et al. / Genomics 90 (2007) 703–711

Fig. 2. Schematic representation of the Re congenic region. A total of 224 backcrossed progeny (wild-type n=117, Re/+ n=107) were used for linkage analysis. Black bar indicates the Re congenic region. The physical positions (bp) are shown in parentheses beneath the marker names. The order of markers was referenced by the NCBI Mouse Genome Database build 36.1. The broken line indicates the positions of the type I IRS keratin genes. orthologue of the human KRT28 gene and that the putative Project, http://www.gsc.riken.go.jp/Mouse/). These facts sug- Krt25, Krt26, and Krt27 sequences are mouse orthologues of gested that M100573 is an allele of Re. We analyzed genomic the respective human type I IRS keratin genes (Fig. 3A). We sequences of the four type I IRS keratin genes of these two deposited the newly isolated cDNA sequence as mouse Krt28 mutants. M100573 was determined to carry a single nonsynon- in the DDBJ/GenBank/EMBL database (Accession No. ymous base substitution, 1135 T to A, in Krt25, which results in AB288231). an amino acid change of tyrosine 379 to asparagine. Tyrosine Then, we systematically compared the genomic sequences of 379 is also highly conserved throughout mammalian species. In these Re-derived genes with those from four inbred strains, addition, this position is very close to leucine 381, the BLG2, HMI, NJL, and KJR, derived from genetically divergent responsible mutation in Re mice (Fig. 3C). Sequencing of the wild-type mice of different subspecies. Krt25 from Re mice has Krt26, Krt27, and Krt28 genes of M100573 revealed no a single-base nonsynonymous substitution, 1142 T to C, which alteration. Next, genome sequencing of Rewc mice detected a results in an amino acid change of leucine 381 to proline (Fig. 1004-bp genomic deletion that includes the whole of exon 6 of 3B). This single-base substitution was not observed in any of Krt27 (Fig. 3D). We confirmed no mutation in Krt25, Krt26,or the four inbred strains or in B6. Our search for reported Krt28 in Rewc mice. single-nucleotide polymorphisms (SNPs) in this gene revealed that none of 11 standard laboratory inbred strains or 4 additional Immunohistological analysis of Re mutant with anti-K71 wild-derived inbred strains had this substitution (NCBI SNP protein antibody database, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD= search&DB=snp), indicating that the proline 381 substitution is The type II keratin protein K71 is a putative partner of the highly specific to Re mice. type I K25 protein for heterodimerization; in situ hybridization Cross-species comparison of amino acid identity is useful to and immunohistological analyses demonstrate coexpression judge the biological significance of amino acid residues at par- and colocalization of these proteins [10–12,22,24]. In addition, ticular positions in a functional motif or domain. Phylogenetic transient expression of both keratin cDNAs in cultured cells analysis of the K25 helix termination motif from opossums induces formation of keratin filaments [26]. Immunohistologi- (Marsupialia) to humans (Primates) indicated that leucine 381 is cal analysis of Ca mice revealed that K71 is ectopically completely conserved in the K25 protein across multiple species expressed in the hair shaft, with both K71 and K25 aggregating (Fig. 3C). in Henle's and Huxley's layers of the IRS [7–9].We hypothesized that if Re bears a K25 mutation, the K71 protein Detection of mutations in Krt25 of M100573 and in Krt27 of Rewc should also exhibit abnormal localization in the IRS of Re mice. We performed immunohistological analysis of the The Rewc strain was generated by chronic neutron irradiation vibrissae of wild-type and Re/+ mice at 5 weeks of age using of an unspecified strain in 1968 [27]. The similarity of the Rewc an anti-K71 polyclonal antibody (Figs. 4A and 4B). In both phenotype to that of Re mice suggested that the mutated gene wild-type and Re/+ mice, the K71 protein was specifically might be an allele of Re, although the defects are more severe in detected in Huxley's layer of the IRS in the lower portion of Rewc than in Re mice [17,27]. M100573 is a dominant mouse hair follicles (Figs. 4A and 4B) and in both Henle's and mutation generated by the RIKEN ENU-mutagenesis project. Huxley's layers of the IRS in the upper portion of the hair bulb M100573 is mapped to mouse chromosome 11 by rough (Figs. 4C and 4D). Re/+ mice, however, exhibited irregular linkage analysis. M100573 mutant mice exhibit curly vibrissae Henle's and Huxley's layers, with abundant foam-like and pelage; the pelage phenotype tends to disappear with immunoreactive signals for K71 throughout these layers of increasing age, as in Re mice (RIKEN Mouse Mutagenesis the IRS (arrowheads in Figs. 4B–4D). The keratin filament S. Tanaka et al. / Genomics 90 (2007) 703–711 707

Fig. 3. Phylogenetic and sequence analyses of type I IRS keratin genes. (A) We generated a phylogenetic tree of mouse (m) and human (h) type I IRS keratin proteins using the amino acid sequences of mouse and human (K10) and keratin 39 (K39), two type I epithelial and hair keratin proteins, as an outgroup. The numbers on the branches indicate the bootstrap probabilities (%), while the scale is the number of amino acid substitutions per site. (B) Sequence analysis of Krt25 revealed that Re mice bear a single base substitution, T 1142 C, which leads to a single amino acid substitution of leucine (Leu) 381 to proline (Pro) in keratin 25 (K25). (C) Alignment of the predicted K25 orthologues among mammals revealed that tyrosine (Y) 379 and leucine (L) 381 in mouse are conserved from opossums (Marsupialia) to humans (Primates) (indicated by an arrowhead). Amino acid residues that are identical among all members are marked with an asterisk below the alignment. A colon and a period indicate substitutions with highly conserved and less conserved residues, respectively. Chimp, chimpanzee; Tenrec, lesser hedgehog tenrec. # indicates the amino acid sequences of the cloned cDNA. (D) In this schematic diagram of the genomic structure of mouse Krt27, the noncoding and coding exons are represented by white and black boxes, respectively. The exons are numbered. Rewc has a genomic deletion of 1004 bp, denoted by a box that corresponds to the region from intron 5 to intron 6 of Krt27. 708 S. Tanaka et al. / Genomics 90 (2007) 703–711

all types of IF proteins (Fig. 5B). Helix initiation and term- ination motifs are required for both the formation and the stability of two-chain coiled-coil keratin IF heterodimers [32]. The amino acid residues mutated in the Krt71 type II keratin gene of eight Ca mice are also highly conserved among mam- malian species. This position is located in the helix initiat- ion motif of the 1A α-helical rod domain and in the helix termination motif in the 2B α-helical rod domain [7–9] (Fig. 5A). Many mutations in the human keratin genes have been described in hereditary skin disorders (Human Intermediate Filament Database, http://www.interfil.org/index.php). Epider- molysis bullosa simplex Dowling–Meara has K14/K5 (type I/ type II) mutations, and bullous congenital ichthyosiform ery- throderma/epidermolytic hyperkeratosis contains K10/K1 (type I/type II) mutations. These mutations in the human genes are also located predominantly in the helix initiation or termination motifs, rather than in other parts of the α-helical rod domain or the nonhelical regions (Fig. 5B). The helix termination motif within the 2B α-helical rod domain is structurally important for all types of IF proteins. In the helix termination motif, leucine 381 of K25, which is mutated in Re, is not conserved in other type I IRS keratins. Only in K25, it is completely conserved in multiple species (data not shown), suggesting that the leucine residue at this position is important only for the K25 orthologues. In the Re mutation, leucine 381 is substituted by proline. While proline is normally found in the nonhelical linker and head and tail domains, it is less frequently present in the α-helical rod domains of normal keratin proteins. Proline residues destabilize α helices [33,34], and those in such helical domains are thought to be deleterious for the formation of the coiled-coils and for the assembly of individual subunits into IFs [35,36]. Letai et al. [35] demonstrated that a point mutation in the human K14 protein that gives rise to an amino acid change, L419P, in the helix termination motif has a more deleterious effect on keratin Fig. 4. Immunohistological analysis using an anti-K71 antibody. Longitudinal assembly than a simple deletion mutation (del 418-LLEGE- sections of vibrissae at (A, B) the lower hair follicle and (C, D) the upper hair bulb 422). In many mutations responsible for human inherited from wild-type (A, C) and Re/+ (B, D) mice at 1 month of age were stained with diseases, proline substitutions are often located in the helix an anti-K71 antibody (green). Counterstaining utilized DAPI (blue). The arrow- heads indicate a region of numerous foam-like signals. Cu, cortex; Hu, Huxley's termination motifs, regardless of heptad position (underlined layer; He, Henle's layer; ORS, outer root sheath; Ma, matrix. Scale bar indicates residues, Fig. 5B). We showed that Re mice revealed abnormal 10 μm. distribution of the type II K71, the putative heterodimeric partner of the type I K25, and disruption of the keratin filament network was abnormal and rough in Huxley's layers of the IRS network in IRS. All results support the supposition that the Re in Re/+ mice (Fig. 4B). mutation impairs Krt25 function and results in the mutant phenotype similar to that of Ca mice. Discussion The Rewc mutant has a deletion in exon 6 of Krt27, which encodes the helix termination motif of the 2B α-helical rod In this study, we analyzed three independent wavy coat domain (Fig. 5A). Thus, mutations in the helix termination mouse mutations, Re, M100573, and Rewc. We demonstrated motif of the 2B α-helical rod domain of type I IRS keratins are that Re and M100573 have nonsynonymous base substitutions common to all three mouse mutants (Fig. 5A). Again, this result in the type I IRS keratin gene Krt25, while Rewc has a deletion suggests that the helix termination motif is essential for keratin in the type I IRS keratin gene Krt27. filament assembly in the IRS. The substituted amino acid residues in both Re and M100573 All the results in this study suggest that the mutations in are located in a highly conserved region, the helix termination Krt25 and Krt27 are the direct cause of the wavy coat phenotype motif, in the 2B α-helical rod domain of K25 (Fig. 5A). Tyro- of Re, M100573, and Rewc. Therefore, now we propose that Re sine 379, which was mutated in M100573, is conserved across and M100573 are two mutant alleles of Krt25, and should be S. Tanaka et al. / Genomics 90 (2007) 703–711 709

Fig. 5. Mutation sites in the IRS keratins of wavy coat mutants. (A) Schematic of the secondary structure of the K25, K27, and K71 proteins. Keratin proteins are composed of an α-helical rod domain (white boxes, 1A, 1B, 2A, and 2B), which is interrupted by a nonhelical linker (black bars, L1, L12, and L2) and flanked by nonhelical head and tail domains. The N- and C-termini of the α-helical rod domain are the helix initiation and termination motifs, respectively (gray boxes). The black arrowheads denote the positions of the point mutation in K25 seen in M100573 mice and the eight mutations in K71 seen in Ca mice. The open arrowhead denotes the position of the amino acid substitutions in Re mice. The region deleted in Rewc is indicated by a black line. (B) We aligned the helix termination motifs of the 2B α-helical rod domains of multiple types of mouse and human IFs. The amino acid residues denoted by boldface specify the mutations identified in Re, M100573, and Ca mice and in human IF diseases, such as EBS-DM, BCIE/EHK, myofibrillar myopathy (MFM), and Emery–Dreifuss muscular dystrophy (EDMD) (Human Intermediate Filament Database, http://www.interfil.org/index.php). Amino acid substitutions of the proline residue are underlined. MFM contains mutations in human (DES), type III IF. EDMD has mutations in human A/C (LMNA), type V IF. (NEFL) and beaded filament structural protein 2 (BFSP2) are type IV and type VI IFs, respectively. The heptad position is denoted as a-b-c-d-e-f-g, in which positions a and d are primarily occupied by hydrophobic residues. Amino acid residues that are identical in all members are marked with an asterisk below the alignment. A colon and a period indicate substitutions with highly conserved and less conserved residues, respectively. Two patterns of conservation among keratin IFs and all types of IF, respectively, are shown. referred to as Krt25Re and Krt25Rgsc573, and that Rewc is an contributions of each protein to the IRS may explain the differ- allele of Krt27 and should be referred to as Krt27Re-wc. ence in number of mutant alleles identified in the type I and the Of the type II IRS keratin genes, eight mutant alleles have type II IRS keratin genes. been detected in Krt71 alone (Fig. 5A) [7–9]. In humans, the Histological analysis revealed that the phenotype of Re mice expression patterns of four type I and four type II IRS keratin is very similar to that of Ca mice. It is puzzling that the sebo- genes in three layers of the IRS are not completely comple- cytes are enlarged in both Re and Ca mice, despite no expres- mentary. Krt71 is expressed in all three layers of the IRS, while sion of Krt25 and Krt71 in the sebaceous glands. Krt10-null the other three type II IRS keratin genes, Krt72, Krt73, and mutant mice also show hyperproliferation of basal cells and Krt74, are expressed only in one layer of the IRS [12,22].In sebocytes, though Krt10 is not expressed in basal cells and contrast, the three type I IRS keratin genes Krt25, Krt27, and sebocytes [37,38]. It is interpreted that in Krt10 mutant activ- Krt28, although not Krt26, are expressed in all three layers of ated MAP kinase signaling in the suprabasal cells and the the IRS [24]. Thus, Krt71 stands out as a ubiquitously ex- interfollicular epidermis results in proliferation of the basal cells pressed, critical gene among the type II IRS keratin genes and as and the sebocytes [37,38]. In the same way, it is possible that the a heterodimeric partner of the type I IRS keratins, whereas the MAP kinase signaling is activated in Re and Ca mice. type I IRS keratin genes may contribute equally to the formation As another explanation, the Re phenotype of the sebaceous of heterodimerization with type II IRS keratins. The relative glands may be associated with hair growth. One of the functions 710 S. Tanaka et al. / Genomics 90 (2007) 703–711 of the sebaceous gland is to support separation of the hair shaft Cloning and sequence analysis of mouse type I IRS keratin genes from the IRS by sebum secretion [38]. The enlargement of the sebaceous glands of Re mice may be due to decreased sebum We homogenized dorsal skin samples in Isogen (Nippon Gene, Tokyo, Japan) and extracted total RNA according to the manufacturer's recommendations. secretion by rupture of the sebocytes, which is accompanied cDNAs were synthesized from total RNA using Superscript III RNase H− reverse with delayed hair growth in Re mice. transcriptase (Invitrogen). To isolate cDNAs encoding type I IRS keratin genes, Additional morphological and immunohistological analysis we performed PCR amplification using gene-specific primers based on the of these mutants, Re, M100573, and Rewc, would provide new sequences of Krt25 (4631426H08Rik; GenBank Accession No. NM_133730), insight into the role of type I IRS keratin proteins and further Krt26 (4732407F15Rik; GenBank Accession No. NM_001033397), and Krt27 (Krt1-29c; GenBank Accession No. NM_010666). As the previously cloned understanding of the functional differences between type I and Krt28 sequence (4733401L19Rik; GenBank Accession No. XM_203344) was type II IRS keratin proteins in hair formation. only a partial cDNA, we designed PCR primers based on the putative Krt28 gene in the genome database (mouse build 36.1). To attempt to identify a mutation in Material and methods these candidate genes, we sequenced the PCR products either directly or after subcloning into the pCR-Blunt II-TOPO vector (Invitrogen) using an ABI Prism Mice 3700 genetic analyzer (Applied Biosystems, Foster City, CA, USA) and a BigDye Terminator Cycle Sequencing Ready Reaction Kit, version. 3.1 (Applied The B6 strain, originally purchased from The Jackson Laboratory (Bar Biosystems). All RT-PCR amplification procedures utilized the high-fidelity – Harbor, ME, USA), was maintained at the Genetic Strains Research Center, DNA polymerase KOD Plus according to the manufacturer's instructions National Institute of Genetics (NIG; Mishima, Japan). The B6-TrJ Re/+ mutant (Toyobo, Osaka, Japan). Direct sequencing of PCR products from genomic DNA strain was purchased from The Jackson Laboratory in 1991. Mice carrying the was performed by the Hitachi High-Tech Science Systems Corp. (Ibaraki, Japan) Re mutation have been maintained at the NIG by repeated backcrosses of using the primers listed in the supplementary material. phenotypic carriers to B6 mice. Rewc was also purchased from The Jackson Laboratory. Genomic DNA was extracted from Rewc/Rewc frozen liver samples Bioinformatics and phylogenetic analysis stored at the RIKEN BioResource Center (BRC) (Tsukuba Japan). The M100573 mutant was generated from B6 mice by ENU mutagenesis at the Using BLAST, we conducted a homology search of the GenBank (NCBI; RIKEN Genomic Sciences Center (Tsukuba, Japan) and stocked at the RIKEN http://www.ncbi.nlm.nih.gov) EST database and the mouse M. musculus build BRC [39]. Four inbred strains derived from wild-type mice of different sub- 36.1 and human Homo sapiens build 35.1 genomic databases. A phylogenetic species, BLG2 (Mus musculus musculus; origin: General Toshevo, Bulgaria), tree was constructed using the neighbor-joining method [42] in the ClustalW HMI (M. m. castaneus; origin: Heimei, Taiwan), NJL (M. m. musculus; origin: multiple alignment package. The predicted amino acid sequences of the K25 Northern Jutland, Denmark), and KJR (M. m. yamasinai; origin: Kojuri, Korea), homologue proteins used for multiple alignments were derived from the Ensembl have been described previously [40,41]. All animal experiments were approved database (http://www.ensembl.org/index.html) for the following organisms: by the Animal Care and Use Committee of the National Institute of Genetics and chimpanzee (Ensembl Transcript ID GENSCAN00000093133), macaque RIKEN Tsukuba Institute. (Ensembl Translation ID ENSMMUP00000001987), cow (Ensembl Translation ID ENSBTAP00000009540), dog (Ensembl Translation ID ENSCAFP0000 Histological analysis 0032235), rat (Ensembl Transcript ID ENSRNOT00000015150), lesser hedge- hog tenrec (Ensembl Transcript ID GENSCAN00000074943), elephant (En- Back skin isolated from wild-type and Re littermates was initially fixed with sembl Transcript ID GENSCAN00000032495), and opossum (Ensembl Trans- 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C overnight. lation ID ENSMODP00000016809). Goat (GenBank Accession No. AY510111) Skin samples were placed in 70% ethanol and minced into rectangular pieces and sheep (GenBank Accession No. AF227759) are reported [17]. Classification (5×10 mm) and then dehydrated in an ethanol series, embedded in paraffin, and of these mammalian K25 proteins was confirmed by phylogenetic analysis using α sectioned at 5 μm. Sections were stained with hematoxylin and eosin or the -helical regions of the amino acid sequences (data not shown). Alignment of α toluidine blue. the 2B -helical rod domains of the mouse and human keratin proteins for all types of IFs utilized the following cDNA sequences: human KRT25 (GenBank Accession No. NM_181534), mouse Krt71 (GenBank Accession No. NM_ Immunohistological analysis 019956), human KRT14 (GenBank Accession No. NM_000526), human KRT5 (GenBank Accession No. NM_000424), human KRT10 (GenBank Accession Paraffin sections for immunohistological analysis were prepared as men- No. J04029), human KRT1 (GenBank Accession No. NM_006121), human tioned above. After removing the paraffin, sections were microwaved in 10 mm desmin (DES) (GenBank Accession No. NM_001927), human neurofilament sodium citrate buffer (ph 6.0) for 5 min. After being washed in water, sections (NEFL) (GenBank Accession No. NM_006158), human lamin A/C (LMNA) were blocked with PBS containing 5% bovine serum albumin for 1 h at 4°C. (GenBank Accession No. NM_005572), and human beaded filament structural After being washed in PBS three times for 5 min each, sections were incubated protein 2 (BFSP2) (GenBank Accession No. NM_003571). with primary antibodies at 4°C overnight. A rabbit polyclonal antibody against mK71 (mK6IRS/KRT2-6G) (1:3200) [10] was used as the primary antibody. After being washed in PBS, sections were incubated with secondary anti- bodies for 1 h at room temperature. For immunofluorescence, we utilized an Acknowledgments AlexaFluor 488-conjugated anti-rabbit IgG (1:500) (Invitrogen, Carlsbad, CA, USA) as a secondary antibody and 4,6-diamino 2-phenylindole (DAPI) for nuclear staining. Sections were analyzed and photographed using a BX51 We thank K. Hiratsuka and A. Sugiyama for maintaining the fluorescence microscope (Olympus, Tokyo, Japan). mouse mutant lines; Y. Mizushina, H. Kobayashi, N. Sakurai- Yamatani, H. Yamamoto, and A. Okagaki for their excellent Genotyping of microsatellite markers technical assistance and valuable advice; and C. Yamanaka for the sequence analysis of M100573. We thank Y. Shimomura of We performed PCR base genotyping using seven microsatellite markers, Niigata University for kindly providing the anti-K71 antibodies. D11Mit99, D11Mit123, D11Mit124, D11Mit198, D11Mit264, D11Mit329, and D11Mit330. Using genomic DNA extracted from tails, PCR was performed We also thank T. Takada for valuable advice. This work was for 35 cycles of 95°C for 1 min, 55–58°C for 1 min, and 72°C for 1 min in a supported in part by a grant-in-aid from the Ministry of BioMetra T1 thermal cycler (Westburg, Leusden, The Netherlands). Education, Culture, Sports, Science, and Technology of Japan. S. Tanaka et al. / Genomics 90 (2007) 703–711 711

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