The mouse secreted gel-forming mucin gene cluster

Biochimica et Biophysica Acta 1676 (2004) 240 – 250 www.bba-direct.com

The mouse secreted gel-forming mucin gene cluster

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Fabienne Escande a,b,c, Nicole Porchet a,b,c, Annie Bernigaud a, Danie`le Petitprez a, Jean-Pierre Aubert a,b, Marie-Pierre Buisine a,b,c,* b

a Unite´ 560 INSERM, 59045 Lille Cedex, France Laboratoire de Biochimie et Biologie Mole´culaire, Hoˆpital C. Huriez, CHRU, 59037 Lille Cedex, France c Faculte´ de Me´decine H. Warembourg, Universite´ de Lille II, 59045 Lille Cedex, France

Received 13 August 2003; received in revised form 6 January 2004; accepted 9 January 2004

Abstract Using genomic cosmid and BAC clones and genome shotgun supercontigs available in GenBank, we determined the complete gene structure of the four mouse secreted gel-forming mucin genes Muc2, Muc5ac, Muc5b and Muc6 and the organization of the genomic locus harboring these genes. The mouse secreted gel-forming mucin gene is 215 kb on distal chromosome 7 to 69.0 cM from the centromere and organized as: Muc6 – Muc2 – Muc5ac – Muc5b with Muc2, Muc5ac and Muc5b arranged in the same orientation and Muc6 in opposite. Mouse mucin genes have highly similar genomic organization to each other and to their respective human homologues indicating that they have been well conserved through evolution. Deduced peptides showed striking sequence similarities in their N- and C-terminal regions whereas the threonine/serine/proline-rich central region is specific for each other and for species. Expression studies also showed that they have expression patterns similar to human mucin genes with Muc2 expressed in small and large intestines, Muc5ac and Muc6 in stomach, and Muc5b in laryngo-tracheal tract. These data constitute an important initial step for investigation of mucin gene regulation and mucin function through the use of animal models. D 2004 Elsevier B.V. All rights reserved. Keywords: Gene family; 11p15; Mouse homologue; von Willebrand factor; Tandem repeat

1. Introduction Epithelial mucins form a specific family of secreted and cell-surface glycoproteins expressed by glandular and ductal epithelial tissues. All members are characterized by a large central region consisting of tandemly repeated sequences rich in serine and threonine residues which support numerous O-linked oligosaccharides. At least 13 different mucins are known that are usually subdivided into secreted mucins and membrane-bound mucins (for Abbreviations: BAC, bacterial artificial chromosome; cDNA, DNA complementary to RNA; RT, reverse transcription; PCR, polymerase chain reaction; 5V-RACE, rapid amplification of cDNA ends; vWF, von Willebrand factor $ The nucleotide sequences reported in this paper have been submitted to the GenBankk/EMBL Data Bank under accession numbers AJ511872 – AJ511874 (Muc2), AJ511870,AJ511871 (Muc5ac), AJ511867 – AJ511869 (Muc6). * Corresponding author. Unite´ 560 INSERM, place de Verdun, 59045 Lille Cedex, France. Tel.: +33-3-20-29-88-50; fax: +33-3-20-53-85-62. E-mail address: [email protected] (M.-P. Buisine). 0167-4781/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbaexp.2004.01.001

review, see Refs. [1,2]). Belonging to the first class are the four large gel-forming mucins MUC2, MUC5AC, MUC5B and MUC6, which are encoded by a cluster of genes on chromosome 11p15.5. The order of the genes within the cluster was established to be MUC6 – MUC2 – MUC5AC – MUC5B from telomere to centromere [3]. These mucins show striking sequence similarities in their N- and C-terminal regions which are particularly rich in cysteine residues and are involved in intermolecular association and gel formation [4]. MUC2, MUC5AC, MUC5B and MUC6 exhibit tissue- and cell-specific expression: MUC2 is mainly expressed in intestinal and colonic goblet cells, whereas MUC5AC is primarily expressed in tracheobronchial goblet cells and in gastric surface epithelial cells; MUC5B is expressed in tracheobronchial, salivary and esophageal mucous glands, pancreatobiliary and endocervical epithelial cells; MUC6 is expressed in gastric and duodenal mucous glands and in pancreatobiliary and endocervical epithelial cells [5 –7]. Little is known regarding the regulation of specific mucins in specific tissues and their functions remain to

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be determined. Nevertheless, these mucins are widely accepted to be responsible for most of the physicochemical properties of the mucous gel covering the epithelia and thus are considered to have a prominent role in protection of the epithelia against the external environment. More recently, additional functions have been proposed for these mucins in tissue-specific ontogenesis and epithelial renewal [8 – 10]. Furthermore, qualitative and quantitative alterations in the expression of these mucins in inflammatory diseases and preneoplastic and neoplastic lesions have suggested roles for them in epithelial wound healing after mucosal injury and in neoplastic processes [11 – 13]. To help us investigate by in vivo approaches the specific functions of secreted gel forming mucins MUC2, MUC5B, MUC5AC and MUC6 as well as mechanisms of mucin gene transcriptional regulation, we aimed to obtain informations (gene structure, copy number, position and expression) on their mouse homologues. At the beginning of the study, little was known regarding the mouse homologues of human secreted gel-forming mucins MUC2, MUC5B, MUC5AC and MUC6. Only the mouse Muc5b gene had been fully sequenced [14], whereas only partial sequences have been reported for mouse Muc2 [15,16] and Muc5ac [16,17], and the existence of mouse Muc6 remained to be established. Therefore, as an initial step for investigation of mucin gene regulation and mucin functions through the use of animal models, we have isolated the mouse secreted gel-forming mucin gene cluster, determined its organization and the gene structure and expression of its different members. Our strategy was largely based on the existence of high sequence similarities and conservation of genomic structures between human and animal mucin genes.

2. Materials and methods 2.1. Isolation and characterization of the mouse mucin gene genomic clones A commercial BALB/c mouse kidney genomic library in cosmid vector pWE15 (ClonTech) was screened under standard conditions using a mixture of human cDNA (DNA complementary to RNA) probes corresponding to the highly conserved D3-domains of human MUC2 and MUC5AC. The probes were generated by reverse transcription (RT)-polymerase chain reaction (PCR) as previously described [14]. Nine positive clones were isolated we named ALI1 –ALI9. Subsequently, high-density mouse bacterial artificial chromosome (BAC) colony DNA membranes (Genome Systems) were screened according to the manufacturer’s instructions using mouse genomic PCR probes corresponding to the highly conserved D1-domains of Muc2 (GenBank accession no. AF016695) [15] and Muc5b (GenBank accession no. AJ311906) [14]. These

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experiments resulted in the isolation of three BAC clones which were purchased and named DIM1 –DIM3. Cosmid DNA was prepared using the Qiagen Plasmid kit (Qiagen) without the filtration step and BAC DNA was prepared using the Large-Construct kit (Qiagen) according to the manufacturer’s instructions. Isolated clones were further characterized by digestion of DNA with several restriction enzymes (Roche Diagnostics) and Southern blot hybridization using 32P-labeled oligonucleotide probes (MWG Biotech) deduced from human, rat or mouse MUC2, MUC5AC, and MUC5B sequences (GenBank accession nos. U07615, AF121215, AJ010437, AJ298317, AJ010792, AJ311906) [14,16,18, 19, Harder et al., unpublished]. Oligonucleotide probes were chosen in well-conserved regions allowing interspecies cross-hybridization, but specific of each mucin gene. Clones were also characterized by PCR experiments conducted directly on cosmid and BAC clones using various primer couples specific for human, rat or mouse mucin genes. PCR parameters were 94 jC for 4 min, followed by 30 cycles at 94 jC for 30– 45 s, 58 jC for 30 – 45 s, and 72 jC for 2 min, followed by a final extension at 72 jC for 15 min. Restriction fragments obtained by digesting BAC DNA by PstI were subcloned in pBluescript KS(+) vector (Stratagene) and sequenced. Fragments amplified on BAC and cosmid clones were cloned directly into pCR2.1 vector (Invitrogen) and sequenced. The extremities of the cosmid clones were sequenced directly using 1.5 Ag of DNA as template. Sequencing was performed using LI-COR automatic sequencer (ScienceTec) or ABI automatic sequencer model 377XL (Applied Biosystems). Long-range PCR experiments were conducted on BAC clones using the Expand Long Range PCR system (Roche Diagnostics). PCR parameters were 95 jC for 3 min, 10 cycles at 94 jC for 20 s, 58 jC for 20 s, 68 jC for 15 min, followed by 15 cycles under the same conditions with elongation of the extension step for 20 s each cycle, and a final extension at 68 jC for 15 min. Specificity of amplified products was verified by Southern blot analysis using internal oligonucleotide probes and by direct sequencing of the extremities. Sequence analyses and searches were performed using PC/Gene software (IntelliGenetics) and BLAST program at NCBI (http://www.ncbi.nlm.nih.gov/BLAST/). 2.2. Determination of gene structures of Muc2, Muc5ac, and Muc6 Some of the exon – intron boundaries were determined by direct comparison of genomic sequences isolated from GenBank (GenBank accession nos. NW_000335 and NW_000336) with partial cDNA sequences of Muc2 (GenBank accession nos. AF016695, AF121215, AJ010437) [15,16], Muc5ac (GenBank accession no. AJ010792) [16] and Muc6 (GenBank accession no. AJ010752) [Tomasetto et al., unpublished] available in

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GenBank. Additional exon – intron boundaries were deduced from genomic sequences by comparisons with mouse Muc5b genomic and cDNA sequences (GenBank accession no. AJ311907) [14], rat and human MUC2 cDNA sequences (GenBank accession nos. U07615, M94131,M94132) [18,20,21], human MUC5AC genomic and cDNA sequences (GenBank accession nos. AJ298317, AJ001402,AJ001403) [19,22] and human MUC6 genomic and cDNA sequences (GenBank accession no.U97698) [23]. Remaining exon – intron boundaries were determined by RT-PCR and 5V-rapid amplification of cDNA ends (RACE) experiments. Total RNA was extracted from mouse stomach and small intestine as described previously [24]. RT-PCR experiments were conducted as described previously [14] using intestinal RNA as template for Muc2 or gastric RNA as template for Muc5ac and Muc6. Oligonucleotide sequences were deduced from genomic clones and chosen by comparison with the genomic organization of the mouse and human MUC5B genes to avoid hybridization of the oligonucleotides to putative intron sites (in the hypothesis of a conserved organization) [14,25 –27]. The extreme 5V-coding region of Muc6 was obtained by 5V-RACE using the 5V/3VRACE kit (Roche Diagnostics) according to the manufacturer’s instructions using gastric total RNA (2 Ag) as template and oligonucleotides 5V-CCACATAGCTT-GCCCATGTAC-3V and 5VAGGTGGAAAAGTGGCCAGCTC-3Vas first primer and nested primer, respectively. Amplified products were cloned and sequenced as described above. Long-range PCR experiments were conducted on BAC clones and/or mouse genomic DNA as described above to determine the sizes of the large central exons of Muc2, Muc5ac and Muc6. 2.3. Expression analysis of mouse secreted gel-forming mucin genes Expression of Muc2, Muc5ac, and Muc6 genes was analyzed by RT-PCR in various mouse tissues (salivary glands (n = 2), trachea and larynx (n = 2), stomach (n = 2), liver (n = 3), small intestine (n = 3), colon (n = 6) and thymus (n = 1)) as described previously for Muc5b [14]. The sequences of the employed primers were: Muc2, 5VT G T G G C C T G T G T G G G A A C T T T- 3 V w i t h 5 VCATAGAGGGCCTGTCCTCAGG-3V (558 bp); Muc5ac, 5V-GAGGGCCCAGTGAGCATCTCC-3V with 5V-TGGGACAGCAGCAGTATTCAGT-3V (361 bp); Muc5b, 5V-AGGAAGACCAGTGTGTTTGTC-3V with 5V-GTCCTCATTGAAGAAGGGCTG-3V (319 bp); Muc6, 5VT G T G G C T T G T G T G G C A A C G C C - 3 V w i t h 5 VTGGTCGAAGTACTCATTCTGG-3V (569 bp). A 241-bp h-actin fragment (GenBank accession no. M12481) was also amplified as control using 5V-GTGGGCCGCTCTAGGCACCA-3Vwith 5V-TGGCCTTAGGGTGCAGGGGG-3Vas primers. PCR products were analyzed on a 1.8% agarose gel in Tris– borate EDTA buffer.

3. Results and discussion 3.1. Isolation and characterization of the mouse genomic clones In our aim to isolate mouse homologues of human mucin genes encoding gel-forming mucins, we used cDNA and genomic probes corresponding to highly conserved domains (D1 and D3-domains) of human and mouse gel-forming mucins MUC2, MUC5AC, MUC5B and MUC6 to screen cosmid and BAC genomic libraries. Several positive clones were isolated in both cases which were further characterized by Southern blot analysis and PCR experiments using various oligonucleotides, sequences of which were deduced from 5V and 3V sequences of human, rat or mouse mucin genes. Sequence analyses of restriction fragments and amplified fragments derived from the different clones completed the study. These experiments revealed various cosmid clones for mouse Muc5b, including ALI2, which was shown in a preceding report to contain the entire Muc5b gene, and one cosmid clone for mouse Muc5ac named ALI5, which was shown here to contain most of the Muc5ac gene (Fig. 1). Among BAC clones, DIM1 was shown to contain the 5V region of mouse Muc2 at its T7 end. Moreover, a 1.1-kb PstI –PstI restriction fragment was isolated from DIM1, sequence of which showed similarities with a Muc2 partial cDNA sequence (GenBank accession no. AF121215, corresponding to the D3-domain) but could not belong to Muc2. A sequence similarity search using BLAST program identified a partial cDNA sequence (GenBank accession no. AJ010752) [Tomasetto et al., unpublished] encoding a gastric mucin-like protein we identified as mouse Muc6 after expression analysis (see paragraph 3.4) and characterization of other genomic clones. DIM2 was shown to contain the 3Vregion of Muc2 at its SP6 end, Muc5ac, and Muc5b. DIM3 was shown to contain Muc5b only. Subsequently, sequence similarity searches using BLAST program resulted in identification of two whole genome shotgun supercontigs named NW_000335 and NW_000336. NW_000335 is 2.7 Mb and consists of 210 sequences, three of which [CAAA01115770 (28.3 kb), CAAA01221083 (2.3 kb), CAAA01124892 (3.0 kb)] encompass the 5V region of Muc2. Further sequence analysis of NW _000335 revealed that four sequences [CAAA01220361 (9.9 kb), CAAA01115781 (5.1 kb), CAAA01115778 (3.1 kb), CAAA01193663 (8.2 kb)] appeared to encompass Muc6. NW_000336 is 3.6 Mb and consists of 257 sequences, three of which [CAAA01052294 (2.9 kb), CAAA01052297 (2.4 kb), CAAA01221095 (9.6 kb)] encompass the 3V region of Muc2, two of which [CAAA01052303 (36.5 kb), CAAA01052306 (56.7 kb)] encompass Muc5ac and two of which [CAAA01052306 (56.7 kb), CAAA01052309 (80.9 kb)] encompass Muc5b. It has to be noted that the major part of the central repetitive region of Muc2 and Muc5ac was absent of the clones indicating that these regions are particularly difficult to clone

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Fig. 1. Organization of the mouse secreted gel-forming mucin gene cluster. Boxes indicate the genes. Lines with vertical bars indicate genomic clones isolated from commercial libraries and GenBank. Broken lines with black arrowheads indicate fragments generated by long-range PCR experiments. Thick lines indicate central exons containing repetitive sequences. Arrows indicate positions of initiating codons and transcriptional orientations. Asterisks indicate positions of stop codons. Lengths of the clones are shown to scale.

and underrepresented in libraries, probably due to their highly repetitive nature (see paragraph 3.2). The same problem was encountered with their respective human homologues [19,28]. In contrast, a great part of the central repetitive region of Muc6 was available, probably due to its lower repetitive nature. Similarly, the overall central region of Muc5b that is composed of 10 imperfect repeats was easily obtained [14]. 3.2. Determination of gene structures of Muc2, Muc5ac, and Muc6 Some of the exon –intron boundaries of Muc2, Muc5ac, and Muc6 were determined by comparison of the genomic sequences with partial cDNA sequences available in GenBank. Our strategy to determine missing exon –intron boundaries was based on two hypotheses: (1) the existence of high sequence similarities between the different human mucin genes and between human genes and their murine homologues on both sides of the central repetitive region, and (2) conservation of the genomic organization between human mucin genes and between human and mouse genes both in intron positions and phases as described previously for MUC5B, MUC5AC and mouse Muc5b [14,22]. Because of the lower sequence similarities in the extreme 5V regions of the mucin genes, initiating codon and first introns of Muc6 were determined by 5V-RACE assay, whereas those of Muc5ac could be determined by direct comparison with cDNA sequence of its human homologue [19]. Initiating codons for Muc5b and Muc2 have been described previously [14,21]. ATG codons we determined for the two genes Muc5ac and Muc6 were embedded in a Kozak consensus sequence (Muc5ac, ACAGCATGG; Muc6, CCACCATGC) [29]. Similarly, RT-PCR experiments were performed to confirm some of the exon –

intron junctions in regions of lower sequence similarity. Moreover, long-range PCR experiments were performed to determine the sizes of the large central exons of Muc2, Muc5ac and Muc6, sequences of which were not completely available in GenBank. Long-range PCR experiments were successful only for Muc2 and Muc6, resulting in the amplification of fragments of 7.6 and 10 kb, respectively (data not shown), allowing us to evaluate the sizes of central exons: Muc2, 8 kb; Muc6, 10 kb, and thus the sizes of transcripts: Muc2 mRNA, 15 kb; Muc6 mRNA, 15 kb. The size we determined for Muc2 is consistent with previous data obtained by Northern blot analysis [17]. For Muc5ac, the size of its central exon could be estimated to approximatively 6.5 kb based on the size of the transcripts evaluated previously by Northern blot analysis [17]. The sequences of the exon – intron boundaries, as well as exon and intron sizes, are given in Tables 1– 3. Sequences and gene structures of Muc2, Muc5ac, and Muc6 have been deposited in GenBank/ EMBL Data Bank under the following accession numbers: Muc2, AJ511872 – AJ511874; Muc5ac, AJ511870, AJ511871; Muc6, AJ511867 – AJ511869. The organizations of the different genes were very similar to each other (Fig. 2A) and to their respective human homologues indicating that they have been well conserved through evolution. Muc5ac consisted of 49 exons similarly to Muc5b [14]. This organization is consistent with that has been described for MUC5AC (3V region) [22] and MUC5B [25 –27]. Muc2 consisted of 47 exons, lacking the two first exons in comparison with Muc5ac and Muc5b but similarly to its human homologue [30]. Muc6 consisted only of 33 exons, lacking the first exon and its 3V region being composed of only two exons as already described for its human homologue [23]. On the other hand, an additional exon in comparison with Muc2, Muc5ac and

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Table 1 Genomic organization of the mouse Muc2 gene Exon no.a

Exon size (bp)

3Vsplice acceptor

b

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

76 262 115 94 110 211 126 118 139 111 70 138 162 95 127 256 53 101 155 139 108 180 240 157 132 148 44 213 >671 (f 8000c) 278 175 248 184 217 179 70 98 32 178 105 41 123 99 129 40 127 223b

tctcccagAAGCCA ttccacagGGTCAG tccaacagGTGGAG ccccctagGCATAC gtcttaagCGAGCC gcttctagCCAAGA tatcacagGGACTG ctgggcagTGTCTG tcatacagAGCGAA tcttgcagGTGGTG tcttatagCCAGCT ccctttagGTCTCT ctccccagCCAACT cctcccagAGGTGC ctctgcagACAAGG tttcatagCATTTG ctccacagCCTGTC tattccagTACCAT ctcctcagTACTTG gcccccagGACTAC ctggatagGGGACA ttctgcagGGCACT ctctctagGTGGAC tcctctagCCATAT tccaatagGTTGCT ctttgcagTACATG ttgtgcagGGAAGA ttccctagTACCTT ccttgtagGTGAAC tcctgcagGTTAAT tcccacagGCTACT gactacagGTACAG gcacacagGCACCT ctgcatagCCTATT ccccacagCTGTGA cgttctagCTCCTC ctttctagGATGTG ctttgcagTTTGGA gccttcagAGTGTG cttccaagTGCCCA cccaaaagCCCGGA gttcacagGGCTTT gtccacagCCTGGG ctcttcagGGCTCC ccccacagGCATCC gtccccagGTACTC

5Vsplice donor

Intron no.a

Intron size (bp)

Phase

AGAAAGgtaagggg GGCCATgtgagtgc CTCATGgtgggcac AGGAAGgtaagtgc GAACACgtgagcca TCTGCCgtaagtag CAGAAGgtatgagc GCAGTGgtaagtct ACCAAGgtaggcca AAGAACgtgagtgc TGGCAGgtgagtgg TGCAGGgtgagtgg TTGAGAgtggtaag TACAAGgtgagttc TCTGCAgtaagtgc GCGCTGgtgagtgt GCCACAgtaagtgg GGCTATgtgagtgc CACATGgtgagctg GTTCAGgtgcgatt ATTGGGgtgagtgc TACAAGgtaggcca AGCAAGgtacgctg TATGCCgtgagtac TGGAGGgtgagcag GTCTTGgtactgag ATGAAGgtaagctg AGCAAGgtgagggt GCCCAGgtatttat AGACAGgtcagtgg GTGACTgtaaggcc GTGGAGgtatgtgg AGTGTGgtgagtcc GGACAGgtgacctg TCTGCTgtaagtgc GCCCAGgtatgctg CATGTGgtatgctc AGAGAGgtaggttc CATGTAgtaaggcc CCTGTGgtaagtga TACCAGgtgagttc AGCTCTgtaagtgt CTGAAGgtgtgtgc GTTTCAgtgagtgg AGACATgtgagtac TGCCATgtgagtgc

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

698 1222 394 92 134 187 225 82 192 86 219 101 533 222 394 221 111 374 269 507 82 117 727 224 408 592 438 1372 607 251 471 609 297 758 195 131 125 >1262 293 205 117 267 590 194 329 130

1 2 0 1 0 1 1 2 0 0 1 1 1 0 1 2 1 0 2 0 0 0 0 1 1 1 1 1 1 0 1 0 1 2 1 2 1 0 1 1 0 0 0 0 1 2

a

Exon and intron no. assigned by comparison with mouse Muc5b gene [14]. Size corresponding to the coding sequence. c Size estimated from long-range PCR experiments and from Northern blot experiments [17]. b

Muc5b was found in Muc6 in front of its large central exon (exon 30) we named exon 29A. As expected, deduced coding sequences of Muc2, Muc5ac, Muc5b, and Muc6 showed striking sequence similarities in their 5Vand 3Vregions to each other and also to prepro-von Willebrand factor (vWF) (D, B, C and Cterminal cystine knot (CK) domains), as previously described for their human homologues [19 – 23,25,26] (Fig. 2B, Table 4). Muc6 differed from the other genes by its

shorter 3Vregion lacking D4-, B- and C-vWF-type domains, similarly to its human homologue [23]. Moreover, sequence analyses revealed that Muc2 and Muc5ac contained sequences encoding cysteine-rich domains (10 cysteines/93– 104 amino acid residues) within their central region, similarly to their human homologues and to Muc5b [14,19,20]. Available genomic sequences were found to contain only a small part of the central repetitive regions of Muc2 (147 bp) and Muc5ac (534 bp). Sequencing of the 5V extremity of the

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Table 2 Genomic organization of the mouse Muc5ac gene Exon no.a

Exon size (bp)

3Vsplice acceptor

b

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

73 75 60 262 115 91 110 214 126 115 139 111 70 138 165 95 127 256 53 101 152 139 114 180 240 157 129 154 44 186 >2730 (f 6500c) 194 175 251 184 202 179 91 101 32 163 105 38 123 99 120 61 109 283b

ttccacagGCCAGG ctccatagGTACCC gtctacagCCTTCA ttttacagGGTCCA aatttcagCTGGAG tcctgcagATGTTA ttgtccagGGTATC tcttgcagCCCAGA ttctacagGTATGG acctgtagCACATG ccctacagCCTTGT ggttatagGAGATC gcccacagCCAATG ccccccagGGCTCT atccacagAAAAGT acccccagAACTGC tcacacagCAAAGC actttcagCACCTG ttctccagTGTGTG ccctacagTATAGC ctacacagCACCTG tcctccagGACAAC ccacacagAACTAT ctctacagGGCAGG tctttcagGTGGAG ttttccagCACTGT gtccacagGATGCT cttgacagCATCTG ccccacagCCTGTG gtccacagTCATGA tcttgcagGATCCA ttctgcagAGAGGA gcccacagGTGTTT gtatccagATCATC atctgcagGCACTT actcacagTACCTT ccacgcagCATTCA tttcacagGACCCT ctctacagGGTGTC ctccacagCCGGGT acccacagCCTGCA ttgcacagGTCAGA ctttccagCCTGGT ccctgcagGGCTCT ccttgcagCCTGGT tggcacagGGCCAG cctcccagAATGTA acccacagGTACTC

5Vsplice donor

Intron no.a

Intron size (bp)

Phase

GCACAGgtaagact ATGTTGgtgagtct TACGAGgtgagtga CCACCCgtgagtct CTTCTGgtgagggg CCAACAgtaagtcc AGATCTgtgaggct TCTGCTgtaagcgc CTGAGGgtaagatg CACCAAgtggtacg AGCAAGgtgaggtc CAGACGgtgagtat CTACAGgtgagggc CCTGTGgtaagaag AGAATGgtgaattt TTCTCGgtaatgct TCTGCAgtgagtgt CATTTGgtaagacc CTCCAGgtgagcct ACCTGTgtaagtga CAACTGgtatgtgg CTACAGgtatgatt CTGGGGgtgagagg TTCAAGgtgaggct GCTCATgtacggca TCTGCCgtgagtgg TGGAAGgtagactg TTCCTGgtgagtca CTGGTGgtgagctc CGCCCGgtaaggca CCTTGGgtaagtga AGACTGgtaagact GCCAATgtgagtgg AACCAGgtagacat AGTGCGgtaagctc AAGCAAgtaagact CCTGCTgtgagtgc AAGCCTgtaagtgt GCCAATgtatgtcc GTGGAGgtacgtat GCTGTGgtgagcct GCAGCAgtaagtgc TACCAGgtaggaat CCTAAGgtgagcac TACCAGgcaagtag CCGCAGgtaaagtt AGCAGAgtaagtgg CTCCATgtatgtat

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

642 812 329 248 387 645 120 95 181 190 1169 526 85 82 1450 537 269 144 426 321 627 92 91 1199 313 817 260 627 113 1149 225 451 366 92 258 497 567 125 138 504 340 207 528 79 417 203 101 466

1 1 1 2 0 1 0 1 1 2 0 0 1 1 1 0 1 2 1 0 2 0 0 0 0 1 1 1 1 1 1 0 1 0 1 2 1 2 1 0 1 1 0 0 0 0 1 2

a

Exon and intron no. assigned by comparison with mouse Muc5b gene [14]. Size corresponding to the coding sequence. c Size estimated from Northern blot experiments [17]. b

long-range PCR product encompassing the central region of Muc2 revealed that it was composed of tandem repeats of 24 nucleotides encoding a threonine-, serine- and proline-rich consensus sequence of eight amino acid residues (Fig. 3). Recently, a new sequence isolated from mouse colon that could correspond to a part of the central region of Muc2 was added in GenBank (accession no. BC024540). Translation of this sequence revealed the

presence of one cysteine-rich domain different from that we found in our sequence and somewhat different tandem repeats of 30 nucleotides (Fig. 3). Long-range PCR experiments performed on mouse genomic DNA permited us to verify that this sequence belonged to Muc2 (data not shown) (Fig. 2b). Thus, the central repetitive region of Muc2 contains two distinct repetitive regions consisting of 8- and 10- amino acid tandem repeats, respectively, and

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Table 3 Genomic organization of the mouse Muc6 gene Exon no.a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 29A 30 31 32

Exon size (bp) 61b 54 244 127 91 110 208 123 121 139 105 73 138 162 95 133 250 53 110 152 142 111 186 240 157 144 63 36 180 216 >4967 (f 10000c) 103 178b

3Vsplice acceptor ccttccagGTGCCT tttttcagCCCCAG acccatagGGTTGT ccccacagGTCCAG gcccccagGCAAAC ctattcagGCCCAG gcctgcagCCATGA gcctttagGTACAC acctgcagCCAGTG cttcccagAGCCCA ccctccagGACAAA tcttgcagACAACA ccacacagGGCTCT ctctccagAGGTGT tcccacagAGATGC tcccacagCTGTGC tcttatagTCACTG ccctgtagCATCCT cccaccagGTACCC ctccccagCACCTG ccctgcagGATGAC ccctccagGGGCTC tctagcagGATGCC ctccacagGTATAC cctctcagCCATCT caccccagGCTGTT tcttacagCACCAC ctacacagGTTCAC ctctgcagAATACC gacatcagCCCAAC atcctcagGGACCT cttggcagTTTCAA

5Vsplice donor ATGCTGgtgagtgg AATCCTgtaaggcc CATTGGgtaagctg CTTACGgtgagatc AGGACGgtaagctg AGATATgtatgtga TCTGCCgtgagttc CCCACGgtgaggat AACCTGgtgagtat CTCCAGgtaggggg AAGAAGgtaaggct AGACACgtaagtcc CCAGAGgtgagtac TCAACAgtgagttc TACAAGgtgggtcc ACTGCAgtgagtgc CATATGgtatggag TCTTTGgtaagtgc GACTGTgtaagaag GACATGgtaggtca GCCACGgtaaccac CTGGGTgtgagtgg ACCCAGgtaacgca AGCAAGgtgggtcc TCTGCCgtgagtgt CAGAAGgtatgagg CCTGTGgtaagtcc CCACAGgtgattgc CGGGAGgtaaaaat GCCAAGgtgagtta ATCCTGgtaagtaa TGTCAGgtgagttg

Intron no.a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 29A 30 31

Intron size (bp) 2539 157 66 320 81 163 131 319 96 68 105 95 249 88 96 > 56 85 102 422 154 85 516 110 666 221 901 504 396 541 431 1740 213

Phase 1 1 2 0 1 0 1 1 2 0 0 1 1 1 0 1 2 1 0 2 0 0 0 0 1 1 1 1 1 1 1 2

a

Exon and intron no. assigned by comparison with mouse Muc5b gene [14]. Size corresponding to the coding sequence. c Size estimated from long-range PCR experiments. b

spaced by one cysteine-rich domain similarly to its human homologue. Muc5ac contains tandem repeats of 48 nucleotides encoding a threonine and serine-rich consensus sequence of 16 amino acid residues as described previously by Shekels et al. [17]. For Muc6, a larger part of the central repetitive region was available (3.8 kb). It was composed of imperfect repeats of 116 – 173 nucleotides encoding threonine, serine, and proline-rich mucin-type domains (Fig. 3). Similarly, we have recently shown that the central region of Muc5b is composed of imperfect repeats [14]. These findings still confirm the notion that although non-repetitive regions are very similar between secreted mucin genes, tandem repeats are specific for each specific-mucin and show little inter-species conservation differing significantly in sequence and size. 3.3. Organization of the mouse secreted gel-forming mucin gene cluster Sequence analysis of the genomic clones led us to hypothesize that the mouse secreted gel-forming mucin

genes were clustered and organized as follows: Muc6 – Muc2 – Muc5ac – Muc5b. To verify this map, we performed long-range PCR experiments using DNA from BAC clones DIM1 or DIM2 as template. Primer pairs were selected from the extremities of clone CAAA01193663 containing the 5V region of Muc6 and clone CAAA01115770 containing the 5V region of Muc2, and from the extremities of clone CAAA01221095 containing the 3Vregion of Muc2 and clone CAAA01052303 containing the 5Vregion of Muc5ac (Fig. 1). PCR experiments resulted in the amplification of fragments of 15 and 12 kb, respectively, confirming our hypothesis. Specificity of amplified products was assessed by hybridization with internal oligonucleotide probes (data not shown) and by sequencing of the extremities. The overall distance between Muc6 and Muc2 was thus determined to be 35 kb and the distance between Muc2 and Muc5ac to be 34 kb. Distance between Muc5ac and Muc5b was calculated from clone CAAA01052306 to be 20 kb, which is consistent with the distance previously evaluated by Chen et al. (f 25 kb) [31]. The complete characterization of the mucin gene locus indicated that the four mouse mucin genes were located

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Fig. 2. Structure of mouse secreted gel-forming mucin genes and deduced peptides. (A) Organization of genes. Boxes indicate exons and horizontal lines indicate introns; open boxes indicate coding regions and shaded gray boxes untranslated regions. Splice events are indicated by elbowed lines. The lengths of the exons and introns are shown to scale. (B) Schematic representation of the deduced peptides. Signal peptide, cysteine-rich D, B, C, and CK (cystine knot) prepro-vWF-like domains, cysteine-rich domains (Cys, 10 cysteines/93 – 104 amino acid residues), and threonine, serine, proline-rich domains composed of unique sequence (TSP) or tandem repeats (TR) are shown.

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Fig. 3. Amino acid sequences of the central repetitive regions of Muc2 and Muc6. Threonine-, serine-, and proline-rich domains were aligned to maximize sequence similarity. Gaps are indicated by dashes.

within a region of 215 kb on distal chromosome 7 to 69.0 cM from the centromere near markers D7Mit46 and D7Mit14 as we previously determined for the Muc5b gene. These results are consistent with those evaluated by computer analysis by Desseyn and Laine [32]. Moreover, our results showed that Muc6 and Muc2 were face to face and transcribed in opposite orientation, whereas Muc2, Muc5ac, and Muc5b were in a tandem array and transcribed in the same orientation (Fig. 1). Thus, mouse secreted gel-forming mucin genes are clustered and organized similarly to their human homologues [3,22]: Muc6 – Muc2 – Muc5ac – Muc5b with Muc2, Muc5ac, and Muc5b arranged in the same orientation and Muc6 in opposite orientation.

3.4. Tissue distribution of mouse secreted gel-forming mucin gene mRNAs We investigated the distribution of Muc2, Muc5ac, Muc5b, and Muc6 RNAs in various mouse tissues by RTPCR analysis (Fig. 4). Strong Muc2 expression was observed in small intestine and colon and strong Muc5ac and Muc6 expression was detected in stomach. A very slight expression of Muc5ac was also detected in laryngo-tracheal tissue. Muc5b was present in laryngo-tracheal tissue. Data expression confirms previous observations by us [14] and others [15,17,32], except for Muc5b expression in stomach and Muc6 in submaxillary glands. Indeed, in the present

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Table 4 Comparison of mouse and human mucin MUC2, MUC5AC, MUC5B and MUC6 Muc2 Muc2 Muc5ac Muc5b Muc6

N-ter: 43.6, C-ter: 34.8 N-ter: 45.3, C-ter: 35.5 N-ter: 34.1, C-ter: 3.5

Muc5ac

Muc5b

Muc6

MUC2

N-ter: 43.6, C-ter: 34.8

N-ter: 45.3, C-ter: 35.5 N-ter: 60.7, C-ter: 36.5

N-ter: 34.1, C-ter: 3.5 N-ter: 35.0, C-ter: 3.2 N-ter: 31.7, C-ter: 2.5

N-ter: 81.8, C-ter: 76

N-ter: 60.7, C-ter: 36.5 N-ter: 35.0, C-ter: 3.2

N-ter: 31.7, C-ter: 2.5

MUC5AC

MUC5B

MUC6

N-ter: 76.3, C-ter: 62.5 N-ter: 75.5, C-ter: 63.9 N-ter: NA, C-ter: 45.6

Amino acid sequences located upstream and downstream from the central repetitive domain of mouse and human mucin MUC2, MUC5AC, MUC5B and MUC6 were compared. Percent amino acid identities are shown for all pairwise combinations. NA, not available.

study, no expression of Muc5b and Muc6 was detected in stomach and submaxillary glands, respectively, contrary to what had been found previously [14,32]. This discrepancy may be due to the use of mouse strains harboring different genetic backgrounds for expression studies. Similarly, whereas Muc6 is not expressed in colon of BALB/c mice, a slight expression of this gene is detected in C57BL/6 mice (data not shown). Such variations in expression patterns will have to be taken in account in investigation of mucin gene regulation and mucin function using mouse models. Secreted mucin gene expression patterns in mouse are very similar to those described in humans [5,7] with Muc2 expressed in small and large intestines, Muc5ac and Muc6 mainly expressed in stomach, and Muc5b expressed in laryngo-tracheal tract. Nevertheless, no expression of Muc5b is detectable in salivary glands and hepatobiliary tract in mouse as discussed previously [14]. Moreover, Muc5ac expression in trachea is very weak in mouse, while

it is strong in human. This result is consistent with previous studies of Muc5ac mRNA expression in mouse respiratory tract by Northern blot analysis [17,33,34]. Differences certainly reflect histological differences in tracheal mucosa between mouse and human as MUC5AC expression is confined to goblet cells which are rare in mice. 3.5. Conclusion In conclusion, we have determined the organization of the mouse secreted gel-forming mucin gene cluster and the complete gene structure of its different members. Results showed high similarities to human homologues, indicating that the cluster of secreted gel-forming mucin gene has been well conserved through evolution. These data constitute an initial step for investigation of mucin gene regulation and mucin functions through the use of animal models.

Fig. 4. Expression analysis of secreted gel-forming mucin gene mRNAs in mouse tissues by RT-PCR. Total RNA was extracted from various tissues and RTPCR was performed for Muc2, Muc5ac, Muc5b, Muc6 and h-actin. Products were electrophoresed on 1.8% agarose gel and visualized by ethidium bromide staining.

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Acknowledgements This work was supported by grants from the ‘‘Association pour la Recherche sur le Cancer’’. We thank C. Pertuiset, M.P. Delescaut, D. Demeyer, C. Mouton, M. Cre´pin, and V. Mortelec for excellent technical assistance.

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