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Regular Article |
a Emory University School of Medicine, Department of Physiology, Atlanta, Georgia 30322
ABSTRACT
The EP2 gene codes for at least nine message variants that are all specifically expressed in the epididymis. These variants putatively encode small secretory proteins that differ in their N- and C-termini, resulting in proteins that can have little or no sequence similarity to each other. We have isolated and sequenced the human EP2 gene to determine the molecular origin of these variants. The EP2 gene has two promoters, eight exons, and seven introns. Exons 3 and 6 encode protein sequences homologous to ß-defensins, a family of antimicrobial peptides. This sequence homology and the arrangement of promoters and defensin-encoding exons suggest that the EP2 gene originated from two ancestral ß-defensin genes arranged in tandem, each contributing a promoter and two exons encoding a leader sequence and a defensin peptide. The proposed evolutionary relationship between the EP2 gene and defensin genes is supported by the observation that the EP2 gene is located on chromosome 8p23 near the defensin gene cluster and is separated by 100 kilobases or less from DEFB2, the gene for ß-defensin-2. While the EP2 gene transcribes ß-defensin-like message variants, most of the known message variants code for nondefensin proteins or proteins containing only a partial defensin peptide sequence. We suggest that, during its evolution, the EP2 gene has acquired new functions that may be important for sperm maturation and/or storage in the epididymis.
gene regulation, male reproductive tract, sperm maturation,, testosterone
INTRODUCTION
Mammalian sperm are not fully mature when they leave the testis. They undergo maturational changes in the epididymis that are necessary for sperm motility [1], sperm capacitation [2], and sperm-egg interaction [3]. Proteins that affect sperm maturation are secreted by the epididymis [4] and interact with the sperm surface [5–9]. Although many of these proteins have been identified biochemically [10, 11], their physiological functions remain unknown.
Testosterone, through its derivative dihydrotestosterone, is necessary for secretion of some epididymal proteins [12]. Among these proteins is a family of androgen-dependent, epididymis-specific secretory proteins encoded by the EP2 gene [13, 14]. The EP2 gene codes for at least five different message variants in the chimpanzee [13] and for at least six different message variants in man [14–16]. Thus far, between human and chimpanzee, the EP2 gene is reported to transcribe nine different message variants that code for eight different proteins.
Each of the proteins encoded by the message variants has a leader sequence characteristic of a secretory protein. After removal of the leader sequence, each of these proteins comprises one or two out of four possible peptide modules [13]. Two of these modules have no recognizable homology to known proteins. The other two modules have a distribution of cysteine residues characteristic of ß-defensins [13], a family of proteins with antimicrobial activity [17].
Defensins are classified as 
-defensins and ß-defensins on the basis of the disulfide cross-linking pattern of their six cysteine residues [18]. Except for the six cysteines, - and ß-defensins have little homology in their amino acid sequences, but they have very similar tertiary structures [19]. Although defensins are thought to participate in the body's defense against intruding bacteria, some defensins may also have functions that are not directly related to an antimicrobial effect [20].
In humans, thus far, two ß-defensins (DEFB1 and DEFB2) have been reported [21, 22]. All known - and ß-defensin genes are clustered on the short arm of chromosome 8 (segment 8p22–p23) [23, 24], where they are postulated to have originated by multiple rounds of gene duplication and evolution of an ancestral gene [18].
To determine how the EP2 gene gives rise to defensin-like and nondefensin-like variants, we obtained a human genomic clone to analyze the gene's structure. We compared the genomic sequence with known cDNA sequences to determine the intron-exon structure of the gene and to test whether it can account for all reported EP2 cDNA variants. In addition, we tested whether the EP2 gene is part of the cluster of defensin genes on chromosome 8 and determined its evolutionary relationship with these genes.
MATERIALS AND METHODS
General Procedures
Molecular-biological procedures followed standard protocols [25] or the manufacturers' instructions. Restriction enzymes were obtained from New England Biolabs (Beverly, MA) or Life Technologies (Rockville, MD). Taq polymerase was obtained from Life Technologies or from Sigma (St. Louis, MO). Other laboratory chemicals were obtained from Fisher Scientific (Pittsburgh, PA).
The program GeneRunner (Hastings Software) was used to manipulate DNA sequences and to design polymerase chain reaction (PCR) primers. The program MegAlign of the DNAstar suite (DNAstar, Madison, WI) was used to assemble the sequencing files into a single contig of the EP2 gene. Homology (BLAST) searches (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) were used to scan for EP2-related Genbank entries. To analyze the sequences of the two promoter regions for potential transcription factor binding sites, we used the transcription element search system (TESS) online software (http://www.cbil.upenn.edu/tess/).
Isolation of a Genomic EP2 Clone
A P1 artificial chromosome clone (PAC) containing the EP2 gene (clone address: PAC-157[10E]) was custom-isolated by PCR screening a P1 phage library (Genome Systems, St. Louis, MO). The PCR primers used were the same as the STS primers used as genetic markers and mapped to chromosome 8 (Human Genome Project STS identification number SHGC-11992; Genbank accession number G19434). The sequence of the forward primer, EP2STS1, was 5'-GAC ATT TGC TCT GAT CCC TG-3'. The sequence of the reverse primer, EP2STS2, was 5'-CCC TTG GGA TAC TTC AAC AT-3'.
DNA Sequence of the EP2 Gene
To determine the 5'-terminal region of the EP2 gene containing the proximal promoter A, a 3.2-kilobase (kb) SmaI fragment containing the promoter was cloned into pBluescript (Stratagene, La Jolla, CA). The insert was sequenced in a stepwise manner by designing a sequencing primer based on a known sequence to obtain a new sequence, and the new sequence was used to design the next primer. To provide overlap among sequence traces, both strands were sequenced in intervals of 400–500 bases.
To determine the sequences of the introns, intronic DNA fragments were amplified by PCR using primers based on flanking exonic sequences. The PCR products were cloned into the pGEM-TEasy (Promega, Madison, WI) vector and sequenced. To sequence intron 3, an 8.7-kb PCR fragment that comprises most of the intron 3 sequence was subcloned using the pCR-TOPO vector (Invitrogen, Carlsbad, CA), and the PCR insert was sequenced in its entirety using the GPS-1 Genome Priming System (New England Biolabs, Beverly, MA).
The sequences obtained were combined with known EP2 cDNA and EST sequences (Table 1). One hundred and thirty five sequences totaling 70 kb were assembled into a single contig of 19.5 kb using the MegAlign program of the DNAstar suite at Emory University's Biomedical Computation Core (BIMCORE). The average coverage of the sequence was 3.6-fold. The EP2 gene accession number at Genbank is AY005129.
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Genomic Analysis of EP2 Using Yeast Artificial Chromosome Clones
Yeast artificial chromosome (YAC) clones covering the general region of chromosome 8p22–23 were selected from the literature [23] and from the STS-based map of the human genome (contig WC-1195 at URL http://carbon.wi.mit.edu:8000/cgi-bin/contig/phys_map). Yeast strains containing the YACs (737_E_5, 773_G_4, 809_H_8, 889_D_10, 871_F_3, 880_F_6, 937_E_1, 920_D_12, 820_B_4, 764_C_7, 750_F_10, 799_B_1, and 934_C_7) were obtained from Incyte Genomics (St. Louis, MO).
Genomic DNA was isolated from these YAC-containing yeast strains [26] and used as template for PCR analysis. The EP2 gene was identified using the primers EP2PCR3 (5'-AGA CAT GAG GCA ACG ATT GCT CC-3') and EP2GEN3R (5'-GGT GCG CGG TGG TAA GAG GT-3') or EP2STS1 (5'-GAC ATT TGC TCT GAT CCC TG-3') and EP2STS2 (5'-CCC TTG GGA TAC TTC AAC AT-3'). The DEFB2 gene was identified using the primers DEFB2F1 (5'-GGC CCC AGT CAC TCA GGA GAG ATC-3') and DEFB2R1 (5'-CGC ATC AGC CAC AGC AGC TTC-3').
RESULTS
Structure of the EP2 Gene
Screening of a PAC genomic library yielded a single clone. Southern hybridization analysis of BamHI and EcoRI restriction fragments gave the same bands as a genomic Southern analysis [16] and confirmed that this PAC clone contained the EP2 genomic sequence. Based on the gel electrophoresis banding pattern of the PAC clone after digestion with BamHI, EcoRI, or HindIII, we estimate the genomic insert at 120–150 kb.
The EP2 gene is 19 kb long, including 3 kb of proximal promoter region. The arrangement of eight exons, seven introns, two promoters, and three polyadenylation sites is shown in Figure 1. Introns 1, 2, 4, 5, and 7 have consensus splicing sequences at their 3'- and 5'-ends (Fig. 2). Intron 3 is not flanked by splicing sites, as it is located between the polyadenylation site at the 3'-end of exon 3 and the transcription initiation site of the second promoter at the start of exon 4. Intron 6 is flanked by the polyadenylation site of exon 6 at its 5'-end and has a splice acceptor site at its 3'-end. The exons vary in size between 76 and 337 base pairs (bp), and the introns vary in size between 223 and 9065 bp.
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The EP2 gene contains two promoters, the proximal promoter A located at the beginning of the gene and the distal promoter B located in the large central intron 3 (Fig. 1). Promoter A drives transcription of message variants EP2A, EP2C, EP2D, and EP2F through EP2I, each starting with exon 1. Promoter B drives transcription of message variants EP2B and EP2E, each starting with exon 4. Figure 3 shows a graphical alignment of the nine known human/chimpanzee EP2 message variants with the EP2 gene. Figure 3 also shows that the EP2 gene contains an internal symmetry. Excluding the two 3'-terminal exons, it contains two segments each containing a promoter followed by three exons.
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Promoters of the EP2 Gene
As EP2 messages are androgen-dependent and epididymis-specific [14, 27], we examined the EP2A and EP2B promoters for regulatory elements that might govern this expression behavior. The sequence of the proximal 1000 bp of promoter A is shown in Figure 4. Promoter A has no TATA box. However, it does have a GC-rich region at positions -35 to -60 relative to the start codon of the open reading frame. This is consistent with observations that the 5'-termini of isolated cDNAs and 5'-rapidly amplified cDNA end products are located near position -36 [13]. However, a minor transcription initiation site upstream of position -170 may also exist [14].
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Upstream of the GC-rich region, near positions -40 and -80, there is a CACCC box and an Sp1 site, respectively, both of which might interact with elements of the core promoter region. Further upstream, there is an octamer binding factor (Oct-1) recognition site at position -360 and sites for polyoma enhancer activator (PEA3) binding around positions -205 and -600 (Fig. 4).
The EP2A promoter contains four TGTTCT (or AGAACA) sites within the proximal 600 bases and three more sites within the proximal 3 kb of promoter A. This hexameric sequence corresponds to one-half of the near-palindromic consensus sequence of the androgen response element (ARE), GG(T/A)ACANNNTGTTCT [28], the binding site of the androgen receptor through which androgen-dependent transcription is mediated. Each of these putative half-ARE sites could be part of a functional full-length ARE, whose other half-site is not readily recognized by visual inspection, but that may still bind the androgen receptor by a nonpalindromic binding paradigm or through cooperative interactions with other binding sites.
The sequence of the proximal 1200 bp of the EP2B promoter is shown in Figure 5. The EP2B promoter has no detectable sequence similarity to the EP2A promoter. The EP2B promoter possesses a TATA box at position -104 relative to the translation initiation site and a GATA box 39 bp further upstream. Four potential PEA3 sites are located in the proximal 800 bp (Fig. 5). The EP2B promoter also contains four sites with sufficient homology to a full-length ARE that they could be considered candidate AREs. All of these are located between positions -500 and -1100.
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Genomic Location of the EP2 Gene on Human Chromosome 8
Human EP2A cDNA is used as a genomic marker for the Human Genome Project (marker ID: SHGC-11992, Genbank accession no. G19434). This marker has been physically mapped to chromosome 8p22–23, between the microsatellite anchor markers D8S550 and D8S552, the region of chromosome 8 in which the other known defensins are located [23, 24]. We tested a set of YAC clones that carry sequences located between the two microsatellite markers [23] for the presence of the EP2 gene. Using EP2-specific primers, we found that the EP2 gene was present in three YACs (773_G_4, 920_D_12, and 820_B_4; Fig. 6), the same YACs that also contain DEFB2 [23]. This observation that EP2 and DEFB2 are located on overlapping YACs indicates that the two genes are located in close proximity. Therefore, we tested our EP2 gene-containing PAC clone for the presence of the DEFB2 gene and found it to be PCR positive for DEFB2. As the estimated size of the genomic insert is approximately 120–150 kb, the EP2 gene and DEFB2 are separated by at most this distance and, together with DEFB1, form a cluster of ß-defensin genes adjacent to the cluster of -defensin genes.
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DISCUSSION
We isolated and sequenced the human gene from which the epididymis-specific EP2 message variants are transcribed. By cDNA library screening and PCR analysis, nine EP2 message variants have been identified in human [14–16] and in chimpanzee [13] epididymal cDNA. The sequence of the isolated human EP2 gene accounts for all reported message variants from both species as well as for the EP2 variants (EP2A, EP2D, and EP2H) in the human EST database of random cDNA clones (Table 1). On the cDNA level, there is over 98% identity between the human and chimpanzee EP2 cDNA sequences. This is consistent with the general genomic sequence similarity reported for these species [29].
Six EP2 variants, EP2A, EP2D, and EP2F–EP2I, have been reported in the human, and five EP2 variants, EP2A–EP2E, have been reported in the chimpanzee. Based on reported cDNA clone abundances in epididymal cDNA libraries [14, 15], the major message variant in the human is EP2A, followed by EP2D and EP2H, while EP2B, EP2C, and EP2E have not yet been identified (Table 1). In the chimpanzee, the major message variant is EP2B, followed by EP2A and EP2C, with minor contributions by EP2D and EP2E [13]. Thus far, EP2F–EP2I have not been identified in the chimpanzee. Therefore, in the human, promoter A appears to be more active than promoter B, whereas in the chimpanzee promoter B appears to be more active than, or at least as active as, promoter A.
The human EP2 gene contains two promoters, eight exons, and seven introns. The proximal promoter A is followed by exons 1–3 and the distal promoter B is followed by exons 4–8. The introns are relatively short, ranging from 0.2 to 1.3 kb, with the exception of the 9-kb-long intron 3. All known EP2 message variants begin either with exon 1 or exon 4, followed by at least one and as many as five additional exons (Fig. 3). The agreement between the cDNAs and the gene's exons validates our previous suggestion that the different EP2 message variants result from the alternative use of two different promoters and from different combinations of exons of the gene [13].
Promoter A and promoter B show no sequence similarity. However, both drive androgen-dependent epididymis-specific gene expression [27]. Androgen dependence could result directly from any of the four consensus hormone response element (HRE) half-sites within the proximal 1 kb of promoter A (Fig. 4) and from any of the four consensus full-length HRE-like sites of promoter B (Fig. 5), or from additional elements located further upstream on the two promoters. Alternatively, androgen dependence could be the indirect consequence of regulatory elements on the promoters that bind androgen-dependent transcription factors or cofactors, or from a combination of these factors. For example, promoter A possesses two and promoter B possesses four binding sequences for the transcription factor PEA3, which is known to participate in the regulation of epididymal protein expression [30–32].
The mature secreted EP2 proteins, EP2A–EP2E, can be viewed as being comprised of modules [13]. Module 1 is encoded by exon 2, module 2 is encoded by exons 5 and 6, module 3 is encoded by exon 3, and module 4 is encoded by exon 6 but in a different reading frame than module 2. The two leader sequences are encoded by exons 1 and 4. Thus, EP2A (HE2) protein consists of modules 1 and 2. The EP2B protein consists of module 2, and the EP2C protein consists of modules 1 and 3. The EP2D (HE2ß1) protein consists of modules 1 and 4, and the EP2E protein consists of module 4. In this framework, the EP2F (HE22) protein consists of modules 1 and 2 and is identical to EP2A protein. The EP2G (HE2ß2) protein consists of module 1, an N-terminal fragment (26 amino acids) of module 4, and a new short module (11 amino acids) encoded by exon 8. The EP2H (HE2
1) protein is the same as the EP2G protein, except that it lacks the truncated version of module 2. The EP2I (HE22) protein contains module 1 followed by 3 amino acids encoded by exon 7. Overall, nearly all variant proteins contain module 1, with the exception of EP2B and EP2E. The latter two proteins can be considered module 1-truncated versions of EP2A and EP2D, respectively. Their lack of module 1 is derived from the fact that their cognate message is transcribed from promoter B instead of promoter A. Therefore, if there is a functional consequence of the use of promoter A versus promoter B, it necessarily resides in module 1.
Promoter A and promoter B are each followed by three exons of which the third exon codes for a defensin-like peptide sequence. Exon 3 is part of the EP2C cDNA and codes for module 3, and exon 6 is part of the EP2E cDNA and codes for module 4 [13]. Both modules exhibit a cysteine distribution pattern characteristic for ß-defensins (Fig. 7). The arrangement of promoters and defensin-encoding exons (Fig. 3) and the homology of modules 3 and 4 to ß-defensins suggest that the EP2 gene originated from two ancestral ß-defensin genes arranged in tandem, each contributing a promoter and two exons. This suggestion is supported by our observation that the EP2 gene is located close to the ß-defensin-2 gene (Fig. 6) and, therefore, is part of the cluster of - and ß-defensin genes on human chromosome 8p22–23 [24]. The ß-defensin genes DEFB1 and DEFB2 have two exons, one encoding the leader sequence and the other encoding the rest of the protein. Accordingly, we hypothesize (Fig. 8) that two ancestral ß-defensin genes were placed by gene duplication in close proximity to each other, separated by about 10 kb. Each of the two EP2 precursor defensin genes evolved an additional exon, exon 2 and exon 5. The protein sequence encoded by exon 2 (module 1) is an N-terminal addition in the defensin-like EP2C and EP2D proteins and in the nondefensin-like EP2A and EP2G proteins, and it is the major constituent in the EP2H and EP2I proteins. It is possible that exon 2 evolved as an inhibitory prosequence that is removed post-translationally to activate the defensin's antimicrobial activity [33]. In contrast to exon 2, exon 5 contains 76 nucleotides and, therefore, cannot be divided evenly into triplet codons. Its inclusion leads to a shift in the reading frame of exon 6. Consequently, the defensin-like protein sequence of EP2E becomes the unrelated protein sequence of EP2B. Additional exons (exons 7 and 8) also evolved downstream of the two precursor defensin genes, contributing 3 and 11 amino acids to the EP2I and EP2G/H proteins, respectively.
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Fusion of the two precursor defensin genes occurred when the signals for transcription termination/polyadenylation were weakened, enabling transcription through the end of the first precursor gene to the next precursor gene and even further to the C-terminal exons. This permits splicing of exons of the first defensin gene onto exons of the second defensin gene and onto the additional 3'-terminal exons. As a consequence, the new EP2 gene can generate messages that encode proteins with no homology to the hypothetical ancestral defensin proteins. Moreover, these nondefensin-encoding messages that encode proteins having no similarity to known proteins are the dominant forms of message encoded by the EP2 gene.
The EP2 gene may still produce antimicrobial peptides in the form of the EP2C and the EP2D/E proteins. In particular, the EP2C protein still has the structural features of a ß-defensin, whereas EP2D/E possess a C-terminal addition of 18 amino acids rich in negatively and positively charged residues that is not typical of defensins (Fig. 7). Although the EP2 gene may retain antimicrobial functions with EP2C and EP2D/E, it may have acquired new functions residing in variants EP2A, EP2B, and EP2F–EP2I. The physiological functions of these variant proteins are not known; however, antibodies to the C-terminal residues of EP2E protein (module 4) bind to the postacrosomal and neck region of the sperm [14], and antibodies to module 1 of EP2A protein bind to the acrosome and equatorial region of sperm [16].
In conclusion, we isolated the human EP2 gene and determined its structure. The human EP2 gene accounts for all known message variants from both human and chimpanzee. Three messages code for defensin-like proteins, whereas the other six messages code for proteins with no similarity to known proteins. The EP2 gene is located on chromosome 8 in close proximity to the ß-defensin-2 gene. We hypothesize that the human EP2 gene is derived from two ancestral ß-defensin genes and has evolved new exons that give rise to new peptide modules. Although the EP2 gene still codes for ß-defensin-like proteins, its evolution has resulted in new EP2 proteins that may be involved in sperm maturation or in sperm storage.
FOOTNOTES
First decision: 5 September 2000.
1 This work was supported in part by the National Institute of Research Resources (RR05994). 
2 Correspondence: Otto Fröhlich, Department of Physiology, Emory University School of Medicine, 1648 Pierce Drive, Atlanta, GA 30322. FAX: 404 727 2648; froehlich{at}physio.emory.edu
Accepted: November 9, 2000.
Received: July 13, 2000.
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