Hair Keratin Associated Proteins: Characterization of a Second High Sulfur KAP Gene Domain on Human Chromosome 2111In fond memory of Dr Peter Steinert.
2004; Elsevier BV; Volume: 122; Issue: 1 Linguagem: Inglês
10.1046/j.0022-202x.2003.22128.x
ISSN1523-1747
AutoresMichael A. Rogers, Hermelita Winter, Iris Beckmann, Jürgen Schweizer, Lutz Langbein, Silke Praetzel,
Tópico(s)RNA Research and Splicing
ResumoAnalysis of the EBI/GeneBank database using nonhuman hair keratin associated protein (KAP) gene sequences as a query resulted in the identification of two human KAP gene domains on chromosome 21, one of which, located at 21q22.1, has recently been characterized. The second domain presented here, an approximately 90 kb domain on chromosome 21q23, harbored 16 KAP genes and two KAP pseudogenes. By comparison with known sheep and mouse KAP families, these genes could be assigned to two KAP families, KAP10 and KAP12, with the KAP10 family (12 members) being distinctly larger than the KAP12 family (four members). Systematic cDNA/3′ rapid amplification of cDNA ends isolation studies using human scalp mRNA led to the identification of eight KAP10 and two KAP12 cDNA sequences. In situ hybridization analyses of human anagen hair follicles using specific 3′-noncoding sequences of the various KAP10/KAP12 genes revealed mRNA expression of nearly all KAP10 and KAP12 members exclusively in a narrow region of the middle portion of the hair fiber cuticle. Bioinformatic analyses of the promoter regions of the KAP10/KAP12 genes demonstrated several enhancer elements that were present in nearly all of the KAP genes. Primary among these were binding elements for the ETS, heat shock factor, AML, and HOX families of transcription factors. Analysis of the EBI/GeneBank database using nonhuman hair keratin associated protein (KAP) gene sequences as a query resulted in the identification of two human KAP gene domains on chromosome 21, one of which, located at 21q22.1, has recently been characterized. The second domain presented here, an approximately 90 kb domain on chromosome 21q23, harbored 16 KAP genes and two KAP pseudogenes. By comparison with known sheep and mouse KAP families, these genes could be assigned to two KAP families, KAP10 and KAP12, with the KAP10 family (12 members) being distinctly larger than the KAP12 family (four members). Systematic cDNA/3′ rapid amplification of cDNA ends isolation studies using human scalp mRNA led to the identification of eight KAP10 and two KAP12 cDNA sequences. In situ hybridization analyses of human anagen hair follicles using specific 3′-noncoding sequences of the various KAP10/KAP12 genes revealed mRNA expression of nearly all KAP10 and KAP12 members exclusively in a narrow region of the middle portion of the hair fiber cuticle. Bioinformatic analyses of the promoter regions of the KAP10/KAP12 genes demonstrated several enhancer elements that were present in nearly all of the KAP genes. Primary among these were binding elements for the ETS, heat shock factor, AML, and HOX families of transcription factors. in situ hybridization hair keratin associated protein rapid amplification of cDNA ends In the past decade, significant advances have been made toward the characterization of human hair keratins and their associated proteins. These proteins make up the main structural components of the hair fiber. The human hair keratins consist of 15 members, which can be subdivided into two families, comprising the type I (acidic) and type II (basic/neutral) hair keratins (Rogers et al., 1998Rogers M.A. Winter H. Wolf C. Heck M. Schweizer J. Characterization of a 190-kilobase pair domain of human type I hair keratin genes.J Biol Chem. 1998; 273: 26683-26691Crossref PubMed Scopus (67) Google Scholar). Copolymers of individual type I and type II hair keratin members, which are differentially expressed in the hair fiber, make up the structural backbone of the 8–10 nm hair keratin intermediate filaments of trichocytes. The gene structures of all human hair keratins have been identified and characterized, the nine members of the type I hair keratin genes being found on human chromosome 17q21.2 and the six members of the type II hair keratin genes on chromosome 12q13.3 (Rogers et al., 1998Rogers M.A. Winter H. Wolf C. Heck M. Schweizer J. Characterization of a 190-kilobase pair domain of human type I hair keratin genes.J Biol Chem. 1998; 273: 26683-26691Crossref PubMed Scopus (67) Google Scholar,Rogers et al., 2000Rogers M.A. Winter H. Langbein L. Wolf C. Schweizer J. Characterization of a 300 kbp region of human DNA containing the type II hair keratin gene domain.J Invest Dermatol. 2000; 114: 464-472Crossref PubMed Scopus (64) Google Scholar). In addition, the expression patterns of all human hair keratins have also been characterized, with specific type I and type II hair keratin proteins being found in the hair matrix as well as the cortex and the cuticle of the hair fiber (Langbein et al., 1999Langbein L. Rogers M.A. Winter H. Praetzel S. Beckhaus U. Rackwitz H.-R. Schweizer J. The catalog of human hair keratins: I. Expression of the nine type I members in the hair follicle.J Biol Chem. 1999; 274: 19874-19884Crossref PubMed Scopus (203) Google Scholar,Langbein et al., 2001Langbein L. Rogers M.A. Winter H. Praetzel S. Schweizer J. The catalog of human hair keratins. II. Expression of the six type II members in the hair follicle and the combined catalog of human type I and II keratins.J Biol Chem. 2001; 276: 35123-35132Crossref PubMed Scopus (226) Google Scholar). Based on gene sequence characteristics, differential expression patterns, and proximity to each other, the type I and type II hair keratin gene domains could be divided into three gene groups (A-C, type I hair keratins; A and C, type II hair keratins;Langbein et al., 1999Langbein L. Rogers M.A. Winter H. Praetzel S. Beckhaus U. Rackwitz H.-R. Schweizer J. The catalog of human hair keratins: I. Expression of the nine type I members in the hair follicle.J Biol Chem. 1999; 274: 19874-19884Crossref PubMed Scopus (203) Google Scholar,Langbein et al., 2001Langbein L. Rogers M.A. Winter H. Praetzel S. Schweizer J. The catalog of human hair keratins. II. Expression of the six type II members in the hair follicle and the combined catalog of human type I and II keratins.J Biol Chem. 2001; 276: 35123-35132Crossref PubMed Scopus (226) Google Scholar). Located between the keratin intermediate filament bundles in the hair fiber is a largely amorphous mass containing the hair keratin associated proteins (KAP), which are assumed to be responsible for the lateral linkage of keratin intermediate filament bundles in trichocytes (Powell et al., 1997Powell B.C. Rogers G.E. The role of keratin proteins and their genes in the growth, structure and properties of hair.in: Jolles P. Zahn H. Höcker H. Formation and Structure of Human Hair. Birkhäuser, Basel1997: 59-148Crossref Google Scholar). In humans, the number of individual KAPs appears to be very large (>80, unpublished data). KAPs are currently divided into 23 families, the majority of which have been recently characterized in humans (Powell et al., 1997Powell B.C. Rogers G.E. The role of keratin proteins and their genes in the growth, structure and properties of hair.in: Jolles P. Zahn H. Höcker H. Formation and Structure of Human Hair. Birkhäuser, Basel1997: 59-148Crossref Google Scholar;Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Korn B.S.J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12-21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar,Rogers et al., 2002Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a first domain of human high glycine-tyrosine and high sulfur keratin-associated protein (KAP) genes on chromosome 21q22.1.J Biol Chem. 2002; 277: 48993-49002Crossref PubMed Scopus (94) Google Scholar). KAPs can be subdivided into three groups based on their chemical properties: (1) The high sulfur KAPs ( 30 mol % cysteine content) comprising the KAP4, KAP5, KAP9, and KAP17 families; and (3) the high glycine tyrosine KAP consisting of the KAP6-KAP8 and KAP18-KAP22 families. Recent studies have characterized two domains of human high/ultrahigh sulfur and high glycine tyrosine KAP genes (Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Korn B.S.J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12-21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar,Rogers et al., 2002Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a first domain of human high glycine-tyrosine and high sulfur keratin-associated protein (KAP) genes on chromosome 21q22.1.J Biol Chem. 2002; 277: 48993-49002Crossref PubMed Scopus (94) Google Scholar). The first domain encompasses approximately 600 kb on chromosome 17q21.2 and harbors the members of the KAP1-KAP3, KAP4, KAP9, KAP16, and KAP17 families, which are embedded inside the type I keratin gene cluster. The second domain, approximately 535 kb in size, is found on human chromosome 21q22.1 and contains the members of the KAP6-KAP8 and KAP19-KAP22 high glycine tyrosine KAP families, as well as the KAP11, KAP13, KAP15, and KAP23 high sulfur KAP families. The mRNA expression of many individual human KAP has been described, the majority being found in the hair fiber cortex and to a lesser extent in the cuticle (Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Korn B.S.J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12-21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar,Rogers et al., 2002Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a first domain of human high glycine-tyrosine and high sulfur keratin-associated protein (KAP) genes on chromosome 21q22.1.J Biol Chem. 2002; 277: 48993-49002Crossref PubMed Scopus (94) Google Scholar;Shimomura et al., 2002aShimomura Y. Aoki N. Rogers M.A. Langbein L. Schweizer J. Ito M. hKAP1.6 and hKAP1.7, two novel human high sulfur keratin-associated proteins are expressed in the hair follicle cortex.J Invest Dermatol. 2002; 118: 226-231Crossref PubMed Scopus (17) Google Scholar,Shimomura et al., 2002bShimomura Y. Aoki N. Schweizer J. Langbein L. Rogers M.A. Winter H. Ito M. Polymorphisms in the human high sulfur hair keratin associated protein (KAP) 1 gene family.J Biol Chem. 2002; 277: 45493-45501Crossref PubMed Scopus (40) Google Scholar). In addition, an antibody recognizing the members of the human KAP1 family has successfully been used to demonstrate KAP expression at the protein level (Shimomura et al., 2002bShimomura Y. Aoki N. Schweizer J. Langbein L. Rogers M.A. Winter H. Ito M. Polymorphisms in the human high sulfur hair keratin associated protein (KAP) 1 gene family.J Biol Chem. 2002; 277: 45493-45501Crossref PubMed Scopus (40) Google Scholar). Moreover, several KAP family specific polymorphisms have been recently identified (Shimomura et al., 2002aShimomura Y. Aoki N. Rogers M.A. Langbein L. Schweizer J. Ito M. hKAP1.6 and hKAP1.7, two novel human high sulfur keratin-associated proteins are expressed in the hair follicle cortex.J Invest Dermatol. 2002; 118: 226-231Crossref PubMed Scopus (17) Google Scholar,Shimomura et al., 2002bShimomura Y. Aoki N. Schweizer J. Langbein L. Rogers M.A. Winter H. Ito M. Polymorphisms in the human high sulfur hair keratin associated protein (KAP) 1 gene family.J Biol Chem. 2002; 277: 45493-45501Crossref PubMed Scopus (40) Google Scholar). This report, a continuation of our efforts to elucidate human KAP genes and to characterize their expression in the human hair follicle, identifies 16 novel high sulfur KAP genes as well as two KAP pseudogenes present on a second locus of human chromosome 21. We show them to be the orthologs of the single KAP10 and KAP12 family members found previously in other species, and d emonstrate the mRNA expression of all of these KAP members in a narrow region of the hair fiber cuticle. The recent characterization of a domain of high/ultrahigh sulfur KAP genes on human chromosome 17q21.2, as well as a further domain of high sulfur and high glycine tyrosine KAP genes on chromosome 21q22.1, led us to continue our search for further human orthologs of KAP genes previously described in other species. One family of interest was the KAP12 family, whose single gene had been previously isolated during the initial sequencing of the mouse genome (Cole and Reeves, 1998Cole S.E. Reeves R.H. A cluster of keratin-associated proteins on mouse chromosome 10 in the region of conserved linkage with human chromosome 21.Genomics. 1998; 54: 437-442Crossref PubMed Scopus (13) Google Scholar). Using this sequence as a query, we analyzed the human GeneBank/EBI database. Several sequences of high homology were found on a 90 kb region of human chromosome 21q23, a region that had been initially bioinformatically identified as a KAP-gene-containing domain during the sequencing of chromosome 21 (see Figure 1 and Table II;Hattori et al., 2000Hattori M. Fujiyama A. Taylor T.D. et al.The DNA sequence of human chromosome 21. The chromosome 21 mapping and sequencing consortium.Nature. 2000; 405: 311-319Crossref PubMed Scopus (881) Google Scholar). Eighteen putative KAP genes were found on the contiguous human genomic sequences AP001754 and AL163300 (previously AP001755), which covered the region in question. Sixteen of these sequences were determined to be intact genes based on the completeness of their ORF. Nearly all of the gene sequences possessed an upstream tataaa box-like structure within 100 bp of the initiation codon. In addition, putative polyadenylation signals were found within a region of 600 bp downstream of the end of the ORF. The putative genes/pseudogenes were small in size (approximately 1 kb) and consisted of a single exon. The genes were separated by 3.5–21 kb of intervening DNA sequence, and possessed no unified direction of transcription. Amino acid homology comparisons of ORF translations of each putative KAP gene with known KAP proteins from other species and their graphical representation via evolutionary tree analysis identified 12 proteins that were orthologs to sheep KAP10.1 (Powell et al., 1997Powell B.C. Rogers G.E. The role of keratin proteins and their genes in the growth, structure and properties of hair.in: Jolles P. Zahn H. Höcker H. Formation and Structure of Human Hair. Birkhäuser, Basel1997: 59-148Crossref Google Scholar) and four that were orthologs to mouse KAP12.1 (Cole and Reeves, 1998Cole S.E. Reeves R.H. A cluster of keratin-associated proteins on mouse chromosome 10 in the region of conserved linkage with human chromosome 21.Genomics. 1998; 54: 437-442Crossref PubMed Scopus (13) Google Scholar; see Figure 2). Direct amino acid comparisons of the human KAP10 and KAP12 family members with their orthologs from sheep and mouse (Figure 3, 4) further confirmed the family classification of these KAP. The KAP10 family members appear to be unique due to the relatively large size of their proteins (25.1–40.4 kDa, see Table II). The cysteine content of the KAP10 family members (24.3–27.4 mol %) places them in the category of high sulfur KAP. Similar to all KAP families described to date, the human KAP10 family possesses largely conserved, family-specific, amino- and carboxy-terminal amino acid sequences (MA(A/T)(S/C)TMS(V/I)(C/R)SS, amino-terminus; S(G/T)(Q/K)KSSC, carboxy-terminus; see Figure 3). Several exceptions occur. For example, KAP10.4 and KAP10.12 possess shorter amino-terminal and carboxy-terminal sequences; KAP10.8 shows a larger amino-terminal and a shorter carboxy-terminal sequence; and KAP10.1 and KAP10.5 exhibit shorter carboxy-terminal sequences. The center of the KAP10 protein members consists largely of varying amounts of double cysteine containing pentameric repeats (boxed in Figure 3). Interspersed between these repeats are several regions of single cysteine-containing tetrameric/pentameric repeat structures Figure 3. The four KAP12 protein family members (KAP12.1-KAP12.4) are much smaller in size (9.7–14.7 kDa) and possess a slightly larger cysteine content (21.2–23.2 mol %) compared to the KAP10 family members Table II. Like the other KAP families, the KAP12 proteins possess conserved amino- and carboxy-terminal amino acid sequences (MCHTS(C/H)S(S/p)(G/A), amino-terminus; (C/W/Y)S(T/I/N/)(p/S)(S/T)(C/G)C, carboxy-terminus; see Figure 4). The repeat structures of the KAP12 family members consist of single cysteine-containing pentameric structures.Table IIGenomic location and molecular characteristics of the KAP10/KAP12 family members: high sulfur KAP genes/pseudogenes/proteinsKAP designationAccession no. genomic sequencesORFStrandMolecular weight of protein (Da)Cysteine residues (mol %)KAP10.1AP001574254923-255771-2865926.6KAP10.2AP001574266312-267079-2561527.1KAP10.3AP001574273661-274336-2234725.8KAP10.4AP001574289381-290586+4042827.4KAP10.5AP001574295385-296200-2762526.9KAP10.6AP001574307018-308115-3679026.6KAP10.7AP001574316278-317405+3781526.7KAP10.8AP001574327774-328553+2629825.5KAP10.9AP0015753846-2724+3003625.0KAP10.0AP00157514092-14847+2557024.3KAP10.11AP00157523133-24029+3018926.1KAP10.12AP0015753874-74621+2510725.7KAP12.4AP00157530950-131288-1143223.2KAP12.3AP00157534654-34944+994622.9KAP12.2AP00157543120-43160-1468921.2KAP12.1AP00157558505-58795-973721.9ψKAP12AAP00157563033-63327+ψKAP10AAP00157578892-79440+ψ before a KAP gene designates a pseudogene. Open table in a new tab Figure 2Division of human KAP into families via CLUSTREE analysis. Multiple amino acid alignments of the KAP identified in this paper were performed together with representative members of the human KAP1-KAP5, KAP9-KAP13, and KAP15-KAP17 as well as KAP family members from other species using the CLUSTAL program. Graphical analyses of the homology data were performed using CLUSTREE. Names in black show previously described human KAP family members characterized in this laboratory; names in violet designate previously described human KAP5 family members; names in red indicate mouse KAP sequences; names in blue designate sheep KAP sequences. The CLUSTAL alignment allowed the division of KAP proteins into families, but was not statistically significant enough for determination of paralogous evolutionary relationships. *KAP23.1 did not segregate correctly. Sequence accession numbers: mouse KAP9.1 (m27685), mKAP11.1 (uo3686), hacl1 (mKAP12.1, af081797), mEMB-UHSP (mKAP13.1, af031485), mKAP14.1 (d85925), sheep KAP4.1 (x73462), sKAP5.1 (x55294), sKAP5.4 (x73434), sKAP5.5 (x73435), sKAP10.1 found inPowell et al., 1997Powell B.C. Rogers G.E. The role of keratin proteins and their genes in the growth, structure and properties of hair.in: Jolles P. Zahn H. Höcker H. Formation and Structure of Human Hair. Birkhäuser, Basel1997: 59-148Crossref Google Scholar, human KAP5 members KERA (aj006692), KERB (aj006693), and UHSP3 (x63755).View Large Image Figure ViewerDownload (PPT)Figure 3Multiple sequence alignments of KAP10 family members. The amino acid sequences shown are ORF translations of the respective gene sequences. Multialignments were constructed using the CLUSTAL program. The asterisk beside one protein name designates sheep KAP10.1 (Powell et al., 1997Powell B.C. Rogers G.E. The role of keratin proteins and their genes in the growth, structure and properties of hair.in: Jolles P. Zahn H. Höcker H. Formation and Structure of Human Hair. Birkhäuser, Basel1997: 59-148Crossref Google Scholar). Asterisks below the amino acid sequence indicate amino acid identity; dots below the sequences show amino acid homology; hyphens found within the sequences are gaps induced by the CLUSTAL software program. Sequences in bold show KAP proteins for which the respective cDNA has been isolated. Repeat structures are boxed.View Large Image Figure ViewerDownload (PPT)Figure 4Multiple sequence alignment of KAP12 family members. For details, see Figure 3. An asterisk beside one protein name indicates mouse KAP12.1 (Cole and Reeves, 1998Cole S.E. Reeves R.H. A cluster of keratin-associated proteins on mouse chromosome 10 in the region of conserved linkage with human chromosome 21.Genomics. 1998; 54: 437-442Crossref PubMed Scopus (13) Google Scholar).View Large Image Figure ViewerDownload (PPT) ψ before a KAP gene designates a pseudogene. 3′-noncoding region PCR probes derived by amplification of human genomic DNA were used to screen an arrayed human scalp cDNA library according to previously described methods (Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Korn B.S.J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12-21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar). This allowed the isolation of a total of 10 partial KAP cDNA clones encompassing eight members of the KAP10 family (KAP10.1-KAP10.5, KAP10.7, KAP10.9, KAP10.10) and two members of the KAP12 family (KAP12.1, KAP12.2, see Table I). As all of these cDNA sequences were incomplete, we tried to obtain full-length sequences by means of 3′ RACE procedures using an upstream primer located in front of the initiation of translation codon of genes KAP10.7 and KAP12.2 Table II. The 3′ RACE products were subcloned into the PCR cloning vector CR4.1 and a large number of individual clones were analyzed. The majority of these PCR clones gave incorrect products, most of them being incomplete KAP cDNA sequences. Three full-length 3′ RACE products were also isolated, however: KAP12.1 and KAP12.2, found using the KAP12.2 upstream oligonucleotide, as well as KAP10.10, obtained as a cross-hybridization product of the KAP10.7 oligonucleotide. All full-length sequences contained an intact ORF, a methionine initiation codon, and a polyadenylation signal followed by a poly A tail.Table IPCR primers and isolated cDNA clonesKAP PCR ProductsPCR primer sequenceSize of PCR productAnnealing temperaturecDNAs foundcDNA accession numbersbpC°KAP10.1ccccgtgtctcccctgtgct16062DKFZp636AJ566380gtcgagaagtgacccagagK1765Q4KAP10.2cross-hybridisationsee KAP10.9DKFZp636AJ566381KAP10.9G1021Q4KAP10.3ttccagggccatctctgacttc18659DKFZp636AJ566383ggggcaggacacagtgaccB1012Q4KAP10.4atttcctggtgtctgctgttga20658DKFZp636AJ556382gaaggctctgcggctctgK2422Q4KAP10.5acctggtcctgactcgggttag19060DKFZp636AJ566384agtgtcctggggcagagaacK2052Q4KAP10.6agatggggcaggctctttgtct143?59NFcross-hybridization–tgcccagggtggaaccagKAP12.1KAP10.7ttctttacccttgacggctctc23259DKFZp636AJ566385cctcgtttctccccacaccM2324Q4KAP10.8ggtaagtggctgcccctacctg19060NFcross-hybridization–cgaggccaagtgacccagagKAP10.1KAP10.9agcccacccagcctcagcaca18763DKFZp636AJ566386cgtccccaaccagcgaccagG0427Q4KAP10.10gcaggctctttgtcttgg3-RACE–ttagaactagagggggtgctgKAP10.11gagcgcgtcacactttc13254DSP–cagttcttctttgtcacattggKAP10.12ctgctcaggccagaagtccag25661DSP–caaggaggcgagaccacaggKAP12.1crosshybridization––3-RACE–KAP12.2KAP12.2acctgtacactccctggatg201553-RACE–gctggtttatttggctctcatKAP12.3acacctgtatccctccgtgaa17056DSP–KAP12.4ggcctcctatgaccctcgta19158DSP–tgaacccaggagaaactgtgtψKAP10Atggctgacctcataccctaca22658DSP–aaggccagtttattcattgtgψKAP12Atgacagatccacctcccactg16154DSP–ttggtcaggaaggcaaaggac KAP 3-RACE products5′primer sequencesize of productcDNA foundcDNA accession numberKAP 10.7ttcactcactcacccactcact1120scl459AJ566387KAP12.1/cactgccctctgcctcgacctt604scl460AJ566388KAP12.2761scl458AJ566389 Open table in a new tab Previous studies had shown that weakly expressed KAP genes were not always detected in our arrayed scalp cDNA library. Therefore, in order to obtain information concerning the region of expression of all the KAP10 and KAP12 family members, we subcloned all of the 3′-noncoding region PCR products generated for screening into the CR4.1 vector and used these plasmid constructs to obtain antisense cRNA probes for in situ hybridization (see Table I). All of the in situ hybridizations performed localized the mRNA of the respective KAP10 or KAP12 family members to a narrow region of the hair fiber cuticle, lying approximately 20 cell layers above the apex of the dermal papilla (Figure 5, 6). As the 3′-noncoding regions of KAP10.2 and KAP10.9 as well as KAP12.1 and KAP12.2 were almost identical, the expression of these KAP genes could only be analyzed collectively. No expression of any KAP member was seen either in the matrix, lower cuticle, cortex, and medulla, or the inner or outer root sheaths of the hair follicle. Furthermore, KAP expression in the interfollicular epidermis was also not found. Minor variations in the onset of KAP expression were not discernible using the methods described above, and therefore cannot be excluded.Figure 6Regions of mRNA expression of KAP12 family members. For details see Figure 5. (A), (B) Enlargements of KAP12.2 and KAP12.4, showing the absence of staining in the inner root sheath cuticle. Bar: (A) 150 μm; (B) 50 μm.View Large Image Figure ViewerDownload (PPT) Because all of the KAP10 and KAP12 family members showed mRNA expression in a very limited region of the hair cuticle, a bioinformatic analysis of the region 1 kb upstream of the initiation of translation codon was performed in order to identify enhancer elements common to all of the KAP genes described here Figure 7. Upon analysis, a large number of infrequently found canonical enhancer elements were detected (presented as black boxes in Figure 7) as well as eight elements that were present in nearly all of the promoter regions (colored boxes in Figure 7; see the legend for details). These were, based on their frequency of occurrence, the ETS-1 enhancer element and the heat shock factor enhancer, which were present in all KAP promoter regions, as well as the binding elements for CTCF, Yy1, AML-1, MZF1, HOXC12/HOXC13, and TBP (see below). The recent studies of human KAP genes, initiating out of nearly three decades of work by many laboratories on KAP genes and proteins derived from sheep, mouse, rat, and rabbit (for an overview, seePowell et al., 1997Powell B.C. Rogers G.E. The role of keratin proteins and their genes in the growth, structure and properties of hair.in: Jolles P. Zahn H. Höcker H. Formation and Structure of Human Hair. Birkhäuser, Basel1997: 59-148Crossref Google Scholar), have resulted in the identification of a number of human KAP genes orthologous to those found in these species (MacKinnon et al., 1990MacKinnon P.J. Powell B.C. Rogers G.E. Structure and expression of genes for a class of cysteine-rich proteins of the cuticle layers of differentiating wool and hair follicles.J Cell Biol. 1990; 111: 2587-2600Crossref PubMed Scopus (70) Google Scholar;Emonet et al., 1997Emonet N. Michaille J.J. Dhouailly D. Isolation and characterization of genomic clones of human sequences presumably coding for hair cysteine-rich proteins.J Dermatol Sci. 1997; 14: 1-11Abstract Full Text PDF PubMed Scopus (12) Google Scholar;Perez et al., 1999Perez C. Auriol J. Gerst C. Bernard B.A. Egly J.M. Genomic organization and promoter characterization of two human UHS keratin genes.Gene. 1999; 227: 137-148Crossref PubMed Scopus (11) Google Scholar;Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Korn B.S.J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12-21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar,Rogers et al., 2002Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a first domain of human high glycine-tyrosine and high sulfur keratin-associated protein (KAP) genes on chromosome 21q22.1.J Biol Chem. 2002; 277: 48993-49002Crossref PubMed Scopus (94) Google Scholar; unpublished data). To date, complete gene sequence data are available for the human KAP1-KAP4, KAP6-KAP9, KAP11, KAP13, and KAP15-KAP23 families. In addition, partial sequence data exist for the human KAP5 family members. No data existed, however, for the human ortholog of sheep KAP10.1, which was originally presented in a review paper as an unpublished observation (Powell et al., 1997Powell B.C. Rogers G.E. The role of keratin proteins and their genes in the growth, structure and properties of hair.in: Jolles P. Zahn H. Höcker H. Formation and Structure of Human Hair. Birkhäuser, Basel1997: 59-148Crossref Google Scholar; no database entry exists for this sequence), as well as for mKAP12.1, a mouse gene sequence whose human ortholog was shown by genomic hybridization to be present on chromosome 21q23 (Cole and Reeves, 1998Cole S.E. Reeves R.H. A cluster of keratin-associated proteins on mouse chromosome 10 in the region of conserved linkage with human chromosome 21.Genomics. 1998; 54: 437-442Crossref PubMed Scopus (13) Google Scholar). Using the mKAP12.1 gene sequence as a database query, it was possible to locate 12 members of the KAP10 family and four members of the KAP12 family grouped together on two Human Genome Project chromosome 21q23 DNA sequences (Hattori et al., 2000Hattori M. Fujiyama A. Taylor T.D. et al.The DNA sequence of human chromosome 21. The chromosome 21 mapping and sequencing consortium.Nature. 2000; 405: 311-319Crossref PubMed Scopus (8
Referência(s)