Genomic organization of MUC4 mucin gene Towards the characterization of splice variants Fabienne Escande 1,2,3 , Laurent Lemaitre 1 , Nicolas Moniaux 4 , Surinder K. Batra 4 , Jean-Pierre Aubert 1,2 and Marie-Pierre Buisine 1,2,3 1 INSERM Unite ´ 560, Lille, France; 2 Laboratoire de Biochimie et Biologie Mole ´ culaire, Ho ˆ pital C. Huriez, Centre Hospitalier Re ´ gional et Universitaire, Lille, France; 3 Faculte ´ de Me ´ decine Henri Warembourg, Lille, France; 4 Departement of Biochemistry and Molecular Biology, the Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, USA The human MUC4 gene encodes a large membrane-associ- ated mucin, characterized by a mucin tandem repeat domain and a growth factor-like transmembrane domain. In addi- tion to the originally published sequence (sv0-MUC4), several MUC4 cDNA sequences (called sv1-MUC4 to sv21- MUC4, MUC4/X, MUC4/Y) from various tissues and cell lines have been recently described. They differ from sv0- MUC4 by deletions and/or insertions located in the 3¢ region or, for two of them, by deletion of the central repetitive domain. To establish the nature of the mechanisms responsible for the diversity of MUC4 transcripts, the genomic structure of the 3¢ region of the human MUC4 gene was determined. Our results show that it spans approxi- mately 30.8 kb of genomic DNA and is composed of 24 exons, including one alternative exon which was exclusively reported for sv1-MUC4. Moreover, we have shown that the different MUC4 transcripts are generated by several mech- anisms, including the alternative use of cassette exons, exon skipping or use of cryptic splice donor/acceptor sites. Keywords: MUC4; mucin; membrane-associated; alternative splicing. Human mucins constitute a complex family of membrane- bound or secreted O-glycoproteins produced by epithelial cells. They contain a high percentage of threonine and serine residues carrying O-linked glycan chains and distributed in tandemly repeated motifs in the central part of the protein backbone. Mucins are known to play important roles in the lubrication and protection of mucosae but more recently, the involvement of mucins in the renewal and differentiation of the epithelia, cell adhesion and cell signaling has also been proposed [1–3]. To date, 14 human mucin genes have been identified: MUC1–4, MUC5B, MUC5AC, MUC6–8, MUC11–13, MUC16 and MUC17 [4–6]. MUC4 is a member of the membrane-bound mucin family and is believed to be the homologue of the rat sialomucin complex (SMC, rat Muc4) because of their similar structural organization [7–10]. Rat Muc4 is a well-characterized heterodimeric glycoprotein complex in which the mucin subunit ascite sialo-glycoprotein (ASGP)-1 is the major detectable glycoprotein. The other subunit ASGP-2 is membrane-associated and contains epidermal growth factor (EGF)-like domains that were shown to act as a ligand for the tyrosine kinase p185 neu [11]. The full cDNA of MUC4, also called sv0-MUC4,was entirely characterized in our laboratory [10,12,13]. The deduced amino-acid sequence of the N-terminal region contains a peptide signal, followed by three imperfect repetitions of a motif, varying from 126 to 130 residues, and by a unique threonine- and serine-rich sequence. The central region is composed of a large mucin-type domain characterized by the perfect repetition of 16 amino-acid residues. Like other mucins, this mucin-type domain exhibits a variable number of tandem repeat polymorphisms with variations ranging from 145 to 395 units. The C-terminal region can be divided into 12 domains (CT1–12) with two EGF-like domains, two cysteine-rich domains, a transmem- brane domain and a short cytoplasmic tail [10,14]. In situ hybridization studies have shown that MUC4 presents a very large expression pattern. It is expressed in numerous normal tissues such as trachea, lung, stomach, colon, uterus and prostate [15,16], but it is not detected in the normal pancreas, gall bladder, liver or biliary epithelial cells [17,18]. Interest- ingly, the abnormal expression of MUC4 was demonstrated to occur in several epithelial cancers such as lung, pancreas and gall bladder carcinoma [17–20], as well as in various cancer cell lines [21,22]. No precise functions were attributed to MUC4 until now, but dysregulations of MUC4 expression in cancers, together with its homology to SMC, suggest an important role for MUC4 in human tumor biology. Recently, we have isolated 23 distinct transcripts of MUC4 that received the designation sv1- to sv21-MUC4, MUC4/X,andMUC4/Y [14,22,23]. They were isolated by RT-PCR, carried out on human testis and pancreatic adenocarcinoma cells. They differ from sv0-MUC4 by Correspondence to M P. Buisine, INSERM U-377, Place de Verdun, 59045 Lille Cedex, France. Fax: + 33 3 20 53 85 62; Tel.: + 33 3 20 29 88 59; E-mail: buisine@lille.inserm.fr Abbreviations: ASGP, ascite sialo-glycoprotein; EGF, epidermal growth factor; BAC, bacterial artificial chromosome. Note: the nucleotide sequences reported here have been submitted to EMBL Nucleotide Sequence Database under accession numbers AJ430032, AJ430033, and AJ430034. (Received 8 February 2002, revised 30 May 2002, accepted 31 May 2002) Eur. J. Biochem. 269, 3637–3644 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03032.x deletions and/or insertions located in the 3¢ region but also for two of them by deletion of the central repetitive domain. Until now, because of the lack of knowledge on the genomic organization of the 3¢ region of MUC4, the precise mech- anisms responsible for these events could not be defined. In the present paper, we described the genomic structure of the 3¢ region of the human MUC4 gene. A comparison of the nucleotide genomic and cDNA sequences allowed us to establish the nature of the mechanisms responsible for the diversity of the MUC4 transcripts. EXPERIMENTAL PROCEDURES Oligonucleotide primers Oligonucleotides used for PCR are shown in Table 1. They were synthetized by Eurogentec (Lie ` ge, Belgium) or by MWG-Biotech (Ebersberg, Germany). PCR amplification of human MUC4 introns MUC4 introns were amplified from a bacterial artificial chromosome (BAC) clone containing the human MUC4 gene [14]. Amplifications were performed in a PerkinElmer Thermal Cycler 2400 (Applied Biosystems, Courtaboeuf, France). PCR reactions were conducted in 50-lL reaction volumes, containing 1 lg of BAC DNA, 5 lLof10· buffer (100 m M Tris/HCl, 15 m M MgCl 2 ,500m M KCl, pH 8.3), 4 lLof10 m M deoxyribonucleoside triphosphates, 10 pmol of each primer and 2 U of Taq DNA polymerase (Roche diagnostics, Meylan, France). The cycle parameters were 94 °C for 4 min, followed by 30 cycles at 94 °Cfor 45 s, 58–60 °Cfor45s,and72°C for 2 min. The final elongation step was extended for an additional 10 min at 72 °C. In some cases, Expand TM Long Template PCR System (Roche diagnostics) was used. PCR reactions were conducted in 50 lL reaction volumes containing 1 lgof BAC DNA, 5 lLof10· Expand long template PCR buffer 3, 4 lLof10m M deoxyribonucleoside triphos- phates, 1.5 lLof2.25m M MgCl 2 ,10pmolofeachprimer and 2.5 U of DNA polymerase. The cycle parameters were 94 °C for 2 min, followed by 30 cycles at 94 °Cfor10s, annealing at 60 °C for 45 s, and elongation at 71 °Cfor 2min,and68°C for 10 min. The last 20 cycles had their elongation time extended by 10 s for each new cycle. The final elongation step was extended for an additional 15 min Table 1. Primers used for DNA amplification and sequencing. a Nucleotide position is defined according to the sequence of sv0-MUC4 (AJ010901). b S, sense; AS, antisense. 3638 F. Escande et al. (Eur. J. Biochem. 269) Ó FEBS 2002 at 71 °C. PCR products were analyzed by 1% agarose gel electrophoresis and cloned directly into the pCR2.1 vector with the original TA cloning TM kit (Invitrogen, Leek, the Netherlands), according to the manufacturer’s instructions. Plasmid DNA purification Plasmid DNA was purified using the QIAprep Spin Plasmid kit (Qiagen, Courtaboeuf, France). DNA sequence analysis Sequences were determined by automatic sequencing with a DNA sequencer model 4000 L LI-COR and the Sequi- Therm Excel TM II Long-read Premix DNA sequencing Kit- LC (TEBU, Le Perray en Ivelynes, France), using standard vector primers or with ABI PRISM model 377 XL automatic sequencer with the ABI PRISM dRhodamine terminator cycle ready reaction kit (Applied Biosystems) using either universal primers or specific internal oligo- nucleotides. An analysis of nucleic acid and deduced peptide sequence data was performed using PC / GENE Software (IntelliGenetics Inc.). RESULTS Genomic organization of 3¢ region of MUC4 In order to clarify the mechanisms responsible for the diversity of MUC4 transcripts, the genomic counterpart of MUC4 cDNA was identified by PCR experiments, using as a template the BAC clone reported previously [14]. This BAC was reported to contain the full human MUC4 gene. Each genomic fragment was subcloned, sequenced, and the exon–intron organization deduced. Oligonucleotide primers were chosen according to the human sv0-MUC4 cDNA sequence (AJ010901) or preliminary results obtained from the genomic organization of the mouse Muc4 gene (A. Laine & J. L. Desseyn, unpublished results). The 3¢ region of MUC4 spans approximately 30.8 kb and the nucleotide sequence is available from GenBank under accession numbers AJ430032, AJ430033, and AJ430034. A comparison with cDNA sequences of MUC4 transcripts allowed us to establish the complete exon–intron organiza- tion of the MUC4 gene. Thus, 24 exons were identified in the 3¢ region of MUC4, including one alternative exon, which was exclusively reported for the sv1-variant (Fig. 1). Exon and intron sizes, splice junction types and sequences of the exon–intron boundaries are given in Table 2. The size of the 24 introns ranged from 94 to 2.8 kb, and the size of the 24 exons ranged from 65 to 607 bp. All of the 5¢ donor and 3¢ acceptor sites were consistent with the consensus gt–ag motifs described for splice sites in eukaryotic genes [24]. Unique tandemly repeated sequences, more and less perfect, were found in some introns: approximately 90 copies of an 15-bp repeat (AGGTATGGGTGTGGA) in intron 3, approximately 60 copies of an 26–31 bp repeat in intron 4, 23 copies of a 32-bp repeat (CAGGAGTACCCCA), four4 copies of a 34-pb repeat (AGGCCTCAACACCCCCC AGCACCTTCCCCAGGCC) in intron 23. A search of the GenBank database indicated that the consensus sequences of these four repeat were not identical with any other genomic sequence. Sequence type microsatellites were also found in others introns: (GGT) 124 in intron 7 (T/CG) 22 in intron 16 (GATA) 73 in intron 18. Such repetitive intronic sequences may participate with the repetitive sequence in the central exon to interindividual polymorph- ism and may be used as a potential intragene marker of the locus 3q29. Therefore, with the previously reported first two exons, the MUC4 genomic organization is complete: MUC4 is composed of at least 26 exons, one exon coding for the 5¢ UTR and peptide leader, one exon coding for the large repetitive domain, and 24 for the 3¢ extremity. Alternative splice events The existence of cDNA variant species characterized by deletions and/or insertions in the 3¢ region of MUC4 was recently described in normal human testis and in pancreatic adenocarcinoma cell lines (HPAF) [14,22,23]. Comparative analysis of the nucleotide sequences of MUC4 transcript variants with the nucleotide sequence of the MUC4 gene allowed us to establish the nature of the mechanisms responsible for the diversity of transcripts. They were generated by the combination of either one or more events resulting in several mechanisms of alternative splicing (Table 3) [24]. Some events correspond to deletions which result from the use of cryptic splice donor sites situated 5¢ from the normally used splice site (733–762del28), or from the use of cryptic splice acceptor sites situated 3¢ from the normally used splice site (309–386del76, 2218–2587del368), or from the use of both cryptic splice donor sites situated 5¢ from the normally used splice site and acceptor sites situated 3¢ from the normally used splice site (966–1396del429, 1020– 1699del678). Some events (762–851del89, 2400–2561del160, 474–631del156, del. exon 2, del. exons 2, 3) correspond to exon skipping by an alternative use of acceptor sites. Other events correspond to insertions arising via the use of cryptic splice donor sites situated 3¢ from the normally used splice site (175–176ins14), or via the use of cassette exons (474– 475ins209). Interestingly, in the majority of cases, it appeared that the flanking sequences at the divergent sites represented consensus splice donor/acceptor sites [25] and demonstrated a remarkable similarity with the splice donor/acceptor sequences observed in sv0-MUC4. A small part of the events recently described by Chou- dhury et al. [22] could not be explained. These events should result from more complex mechanisms. DISCUSSION The human MUC4 belongs to the mucin family. Like the other members of this family, MUC4 is found in the mucus Fig. 1. Organization of the 3¢ region of human MUC4 gene. Boxes indicate exons. They are numbered consecutively above the boxes with 2 for the central exon (black box). Shaded grey box indicate 3¢-untranslated region. Horizontal lines indicate introns. They are numbered below the lines. The length of the exons and introns are showntoscale. Ó FEBS 2002 Splice variants of MUC4 (Eur. J. Biochem. 269) 3639 secretion and corresponds to a high molecular mass O-glycoprotein. It exhibits a VNTR polymorphism corre- lated with the variation of one unit of repetition that composes its central domain. In opposition with the strictly secreted mucins, several transcripts of MUC4 were previ- ously identified. This property, to be expressed under numerous RNA forms for human mucins, appears to be shared by the members of the membrane-bound mucin subfamily. Indeed, four distinct transcripts were character- ized for MUC1 as well as for MUC3 [26–30]. In both cases, the perfect knowledge of the genomic organization allowed the authors to assimilate transcripts with alternative splice forms of the MUC1 and MUC3 genes. Right now, MUC1 is the best known and characterized mucin; therefore, some functions were identified and associated with precise MUC1 splice form. For instance, MUC1 is known to participate directly in the integrity of the epithelial surface via its interaction with the b-catenin [31]. This interaction is regulated mainly by two mechanisms, via the phosphorylation status of each partner of the b-catenin pathwaycausedbytheGSK3b, or via the interaction of MUC1 through two of its splice forms MUC1/SEC and MUC1/Y [26,27]. MUC1/Y has also been referred to enhance tumor initiation and progression in vivo [32]. MUC1/Y showed expression in various epithelial tumors, such as breast and ovarian cancers but it is undetectable in adjacent normal tissues. For the MUC4 gene, 24 distinct transcripts have already been isolated from various tissue samples as well as cell lines [14,22,23]. They received the name of sv0- to sv21-MUC4, MUC4/X and MUC4/Y. The sv0-MUC4 transcript is the main variant expressed by all the tissue samples and cell lines studied right now [23]. It corres- ponds to a 26.5-kb RNA encoding an apoprotein of 930 kDa organized in the following domains: mucin-type, cysteine-rich, EGF-like, transmembrane, and cytoplasmic. The sv0-MUC4 has a structural organization similar to its rat homologue, SMC. SMC/rMuc4 is known to be a heterodimeric complex composed of two subunits ASGP-1 and 2 [7–10]. The rMuc4 is believed to play an important role in tumorigenesis and metastasis via anti-adhesive and anti-immune recognition effects of its extracellular mucin domain [33–35]. It is also shown to participate in the ErbB2/neu signaling pathway [11], pointing out an important role in cell proliferation and differentiation of epithelial cells. A secreted form was identified for rMuc4; however, it was shown not to be caused by alternative splicing of its premRNA but by proteolytic cleavage of the membrane-bound mucin [36]. Therefore at this point, only one transcript was isolated for rMuc4,aswellasfor mMuc1, mMuc3,andrMuc3. In this study, we determined, for the first time, the genomic organization of the MUC4 gene as well as the mechanisms responsible for the diversity of MUC4 tran- scripts. We have demonstrated that the events (deletions/ insertions) observed in the 24 MUC4 variants are generated by different mechanisms of alternative splicing: alternative use of exons, which is the mechanism most commonly used to generate isoforms, and the use of cryptic donor/acceptor splice sites. The identification of the same event in several MUC4 variants, isolated from different tissues or cell lines, and the molecular characterization of the splice events strongly suggest that the diversity in the 3¢ region of MUC4 variants is not due to an error in the splicing process or an artifact. Altogether, it appears that the membrane-bound mucins possess, at least from the transcriptional point of view, a level of complexity in more than their animal homologues. This complexity seems to be the highest for MUC4,asingle gene code for at least 24 distinct transcripts. As no evidence Table 2. Characteristics of the exon–intron junctions of the MUC4 gene. 3640 F. Escande et al. (Eur. J. Biochem. 269) Ó FEBS 2002 has been available to confirm the translation of the transcripts, it is difficult to study their function. If translated, the different transcripts will generate a complex family of putative, membrane-bound, secreted and/or devoid of functional domains (tandem repeat, cysteine-rich, EGF-like domains) MUC4 isoforms (Table 4). Several splice variants encode the same protein. The potential diversity of the MUC4 isoforms in a single cell type may result in modulation of the properties of the molecule. On the other hand, the alternative RNA splice forms may only function to reduce the level of expression of the main form sv0- MUC4. Indeed, sv0-MUC4 (common in human and rat) has been reported to play a key role for the epithelial development and renewal as well as in tumorigenesis. Therefore, its level of expression should be important in maintaining the epithelial integrity. By analogy with MUC1/Y splice variant, MUC4/X and MUC4/Y may have important functions in tumorigenesis and activated Table 3. Splice events detected in MUC4 cDNA variants. The constitutive exons are depicted as open boxes and alternative exons are shaded. The solid lines show the splicing events in sv0-MUC4 and broken lines show the possible alternative splicing events in MUC4-variants. The positions of the splice events were characterized according to the sequence AJ010901. The nomenclature used for the description of splicing events corresponds to the consensus nomenclature [24]. Ó FEBS 2002 Splice variants of MUC4 (Eur. J. Biochem. 269) 3641 proliferative pathway. These hypotheses remain to be verified. We are studying to find out if some MUC4 variants are very rare and occur only in specific tissues at specific times during the development, and/or under certain physiological conditions. Understanding how the complex splicing of the transcripts encoded by this gene is regulated, however, will help to elucidate how the specificity of their expression is established as well as their putative functions. Table 4. MUC4 splice variants and deduced peptides. NT1-NT3, N-terminal domains as described in [13]; TR, tandem repeat (central) domain; CT1-CT12, C-terminal domains as described in [10]. 3642 F. Escande et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ACKNOWLEDGEMENTS This work was supported by the Association de Recherche contre le Cancer and a RO1 grant from the National Institutes of Health (CA78590). We gratefully acknowledge D. Demeyer, C. Mouton, M. Cre ´ pin for performing automatic sequences and A. Bernigaud, D. Petitprez and V. Mortelec for the excellent technical assistance. 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(1997) Reversible disruption of cell–matrix and cell–cell interac- tions by overexpression of sialomucin complex. J. Biol. Chem. 272, 33245–33254. 36. Rossi, E.A., McNeer, R.R., Price-Schiavi, S.A., Van den Brande, J.M., Komatsu, M., Thompson, J.F., Carraway, C.A., Fregien, N.L. & Carraway, K.L. (1996) Expression as a soluble, secretable form in lactating mammary gland and colon. J. Biol. Chem. 271, 33476–33485. 3644 F. Escande et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . establish the nature of the mechanisms responsible for the diversity of MUC4 transcripts, the genomic structure of the 3¢ region of the human MUC4 gene was. mechanisms. DISCUSSION The human MUC4 belongs to the mucin family. Like the other members of this family, MUC4 is found in the mucus Fig. 1. Organization of the 3¢ region of