- Research article
- Open Access
The genomic environment around the Aromatase gene: evolutionary insights
© Castro et al; licensee BioMed Central Ltd. 2005
Received: 08 March 2005
Accepted: 12 August 2005
Published: 12 August 2005
The cytochrome P450 aromatase (CYP19), catalyses the aromatisation of androgens to estrogens, a key mechanism in vertebrate reproductive physiology. A current evolutionary hypothesis suggests that CYP19 gene arose at the origin of vertebrates, given that it has not been found outside this clade. The human CYP19 gene is located in one of the proposed MHC-paralogon regions (HSA15q). At present it is unclear whether this genomic location is ancestral (which would suggest an invertebrate origin for CYP19) or derived (genomic location with no evolutionary meaning). The distinction between these possibilities should help to clarify the timing of the CYP19 emergence and which taxa should be investigated.
Here we determine the "genomic environment" around CYP19 in three vertebrate species Homo sapiens, Tetraodon nigroviridis and Xenopus tropicalis. Paralogy studies and phylogenetic analysis of six gene families suggests that the CYP19 gene region was structured through "en bloc" genomic duplication (as part of the MHC-paralogon formation). Four gene families have specifically duplicated in the vertebrate lineage. Moreover, the mapping location of the different paralogues is consistent with a model of "en bloc" duplication. Furthermore, we also determine that this region has retained the same gene content since the divergence of Actinopterygii and Tetrapods. A single inversion in gene order has taken place, probably in the mammalian lineage. Finally, we describe the first invertebrate CYP19 sequence, from Branchiostoma floridae.
Contrary to previous suggestions, our data indicates an invertebrate origin for the aromatase gene, given the striking conservation pattern in both gene order and gene content, and the presence of aromatase in amphioxus. We propose that CYP19 duplicated in the vertebrate lineage to yield four paralogues, followed by the subsequent loss of all but one gene in vertebrate evolution. Finally, we suggest that agnathans and lophotrocozoan protostomes should be investigated for the presence of aromatase.
The cytochrome P450 aromatase (CYP19) is a member of a large superfamily of enzymes named cytochrome P450, which are involved in many physiological functions, such as steroid biosynthesis . CYP19 is a steroidogenic enzyme which catalyses the aromatisation of androgens to estrogens. Thus, aromatase activity is essential for maintaining a physiological balance between androgens and estrogens, a critical aspect in the reproductive function of vertebrates; in humans, a P450 aromatase mutation leads to sterility . For many other vertebrate groups, it as been demonstrated that CYP19 plays a key role in sex differentiation .
While several CYP450 genes are universally distributed, CYP19 is so far restricted to the vertebrate lineage. In mammals , birds , amphibians , reptiles  and cartilaginous fishes , a single gene has been isolated. In most Actinopterygii, however, two genes, Cyp19a and Cyp19b, encode two different transcripts expressed in the ovary and brain respectively . Linkage data from zebrafish clearly suggests that these two genes are most likely the result of a genome duplication in the ray-finned bony fish lineage . Despite intensive research, the ancestry of CYP19 genes is yet to be deciphered. No orthologue has been described from fully sequenced invertebrate genomes, like Drosophila melanogaster, Ciona intestinalis or Caenorhabditis elegans . Thus, it has been suggested that the CYP19 gene arose at the origin of vertebrates [10, 11]. Nevertheless, there is now strong evidence indicating that these model invertebrate species have experienced extensive gene loss . Significantly, the estrogen receptor which was thought to have emerged in vertebrate ancestry, has now been documented in the lophotrocozoan protostome Aplysia californica .
Phylogenetics, paralogy and comparative genomics can be a particularly powerful tool to address issues of gene ancestry. Here, we analysed the evolutionary history of the genes in close physical proximity to the aromatase gene(s) in several vertebrate species (Homo sapiens, Tetraodon nigroviridis and Xenopus tropicalis). Through phylogenetic analysis we demonstrate that the CYP19 region was structured most likely by "en bloc" genomic duplication (as part of the MHC-paralogon). Most importantly, we also determine that this region has retained the same gene content and overall organisation (CYP19 included) without any gene insertion in the three lineages. A single inversion of gene order has occurred in the mammalian clade. Finally, we describe for the first time an invertebrate CYP19 partial sequence from Branchiostoma floridae. We propose that the aromatase gene family is much older than previously hinted.
Results and discussion
In this study, we sequentially addressed three questions. First, we determined the duplication pattern (pre or post vertebrate radiation) of the gene families in close proximity to the human CYP19. A further test analysed the ancestry of the human aromatase genomic location (ancestral versus derived). Finally, we investigated the presence of CYP19 in other invertebrate species (B. floridae), other than those previously explored.
Phylogeny and paralogy
We began by investigating the gene complement for each gene family in vertebrate and invertebrate species through BLAST search. Phylogenetic analysis was then performed when no previous study was available to determine duplication timings (if duplication had occurred).
The ORF identified in Ensembl as NP_997264 (FLJ41287 protein), presents significant sequence similarity to three other GenBank entries. One of those is a novel tumor necrosis factor-α inducible gene, SSC-S2 , which maps to HSA5. SSC-S2 contains a motif in the amino terminus that shows a significant similarity to death effector domain II of cell death regulatory protein, Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (FLIP) . Through phylogenetic analysis we showed these four sequences to be paralogues (figure 3B). The four genes were named as follows: SSC-S2A (HSA5), SSC-S2B (HSA1), SSC-S2C (HSA15) and SSC-S2D (HSA19). Invertebrate orthologues were found in Anopheles gambiae, D. melanogaster, C. elegans (not used in the phylogeny) and C. intestinalis (scaffold 92). The duplication events date to early vertebrate origin, as indicated by the branching pattern of the tree (figure 3B). The invertebrate sequences are basal to the vertebrate genes with a significant bootstrap support (figure 3B). No homologue of SSC-S2A is found in Actinopterygii, possibly due to gene loss. Additionally, the human genes are all located in regions of MHC paralogy – HSA1, HSA19, HSA5 and HSA15 (figure 1), as expected from two rounds of "en bloc" duplication in early vertebrate ancestry. However, the tree branching pattern is not of the (A,B)(C,D) type, but sequential which is not in agreement with the "en bloc" scenario.
The Ensembl annotation identifies this gene as Collomin. This as been renamed to Colmedin (COL1) . Colmedin is a phylogenetically conserved type II transmembrane protein with collagen repeats and a cysteine-rich olfactomedin domain, with members described in C. elegans (two genes), Drosophila and vertebrates . No orthologue was detected in C. intestinalis. Colmedin has been found to be a single-copy gene in several vertebrate species. BLAST search to Danio (not shown), Fugu and Tetraodon genomes uncovered a new Colmedin gene, which we name COL1b (figure 3C). COL1b is a specific paralogue of Actinopterygii. The genomic location of this new gene is explained most likely by an extra genome duplication (see following section).
RAB-3 is a 12 WD domain protein which binds both GDP/GTP exchange protein and GTPase-activating protein for Rab3 small G protein family . These domains are found in a variety of proteins and are likely to be involved in protein-protein interactions . It shows a domain structure similar to that of DMXL1 which has 10 WD domains, and has been renamed DMXL2 [27, 29]. Our phylogenetic analysis confirms that both genes are paralogues, with invertebrate sequences basal to vertebrate DMXL1 and DMXL2 (figure 3D). Moreover, DMXL1 maps to an expected region of MHC-paralogon in HSA5 (figure 1). Thus, the duplication event which originated DMXL2 and DMXL1 resulted most likely from two rounds of "en bloc" duplications in early vertebrate ancestry.
Secretogranin III (SCGIII) is a member of the granin protein family, that is a component of intracellular dense core vesicles. Through BLAST we found this gene family to be restricted to vertebrates (not shown).
The ORF identified in Ensembl as NM_699205 codes for the hypothetical protein MGC35274. Sequence features (Lysin domain) indicate that it might be involved in cell wall catabolism. The C. elegans orthologue has been named Predicted peptidoglycan-binding protein (PPBP). Thus, we named the human gene PPBP1. A second PPBP gene can be found in vertebrate genomes, which we designate PPBP2. The phylogenetic tree indicates that both ORFs are paralogues (figure 3E). The second PPBP gene is present in Fugu (Tetraodon also has a second PPBP gene but due to the partial sequence was kept out of the phylogeny), amphibians and mammals. The tree pattern indicates that a duplication of an ancestral PPBP gene occurred specifically in the vertebrate lineage. Moreover, the second gene maps to an expected region of MHC paralogy in the human genome – HSA1.
TMOD2 and TMOD3
Popovici et al.  proposed that the TMOD gene family duplicated in the vertebrate lineage. However, no phylogenetic analysis was performed to support this assumption. In the human genome 4 tropomodulin genes have been annotated: TMOD1 (Erythrocyte tropomodulin), TMOD4 (Skeletal muscle tropomodulin), TMOD2 (Neuronal tropomodulin) and TMOD3 (Ubiquitous tropomodulin). In invertebrates a single tropomodulin gene is observed. The phylogenetic analysis by Almenar-Queralt et al.  suggests that TMOD1, 2 and 4 duplicated in the vertebrate lineage. Nevertheless, the origin of TMOD3 is still unclear. The genomic location of both TMOD2 and 3 is highly suggestive for a tandem duplication. Our phylogenetic analysis, supports this scenario (TMOD2 and 3 are tandem duplicates from an ancestral TMOD2/3 gene). The duplication event post-dates the divergence of fish and amphibians, since a single TMOD2/3 is found in both Fugu and Tetraodon (figure 3F). On the contrary, Xenopus, chicken, mouse and human have two distinct genes (mapping side by side) (the Xenopus TMOD3 orthologue has not been used in the phylogeny). Thus, we propose that a single tropomodulin gene existed in vertebrate ancestry. It duplicated to yield three TMOD genes (1, 2/3 and 4) as a result of "en bloc" duplication (probably as part of genome duplications). Later, a tandem duplication in the ancestor of Xenopus, chicken and mammals, originated the TMOD2 and TMOD3 genes.
The phylogenetic analysis of the full set of gene families within the human aromatase DNA segment, reveals that four of those have specifically duplicated in the vertebrate lineage. Only the SSC-2S gene family shows four paralogues. The tree branching pattern is not of the (A,B)(C,D) type (expected under an "en bloc" duplication) but sequential (expected under the adaptive duplication scenario). This observation has been interpreted as evidence against an "en bloc" scenario . Thus, the phylogeny (branching patterns) per se does not support the "en bloc" duplications. In this context, the suggested duplicated regions could have resulted from a complex duplication, loss and rearrangement pattern, and not from "en bloc" duplications . However, Furlong and Holland , have recently disputed this assumption.
Our analysis confirms the previous suggestion by Popovici et al.  that genes within HSA15q are part of the MHC-paralogon. We find that the paralogues for each gene family map to expected regions of MHC paralogy (figure 1). That is the case of SSC-2S (4 genes), DMXL (2 genes), PPBP (2 genes) and TMOD (3 genes). The physical proximity between these genes is also observed in other regions of paralogy besides HSA15. For example, paralogues SSC-S2B, PPBP2 and TMOD4 map closely in chromosome 1 (200 kb), while DMXL1 and SSC-S2A are separated by just 300 kb in chromosome 5 (figure 1). Furthermore, of those genes which are found to be single copy in vertebrates, only for CYP19 and SCGIII we have not found invertebrate orthologues (either in C. intestinalis, C. elegans or D. melanogaster).
CYP19 in amphioxus
The present results imply a significant theoretical change regarding the ancestry of the CYP19 gene family. This investigation started with the observation that the human aromatase gene maps to the MHC-paralogon. Nevertheless, two opposite scenarios could be draw from the phylogenetic analysis, paralogy and comparative genomics. Either the CYP19 locus was present in the invertebrate chordate unduplicated MHC-paralogon, and the presence of a single paralogue resulted from gene loss; or CYP19 originated early on in vertebrate evolution in its present position in the MHC-paralogon (figure 4). We went on to test these hypotheses. Our results can be summarised as follows: (1) vertebrate CYP19 containing regions are indeed part of the MHC-paralogon as demonstrated by the phylogenetic analysis of the gene families in close proximity; (2) comparative genomics of the aromatase region between fish, amphibians and humans shows a striking pattern of conservation without any gene insertion; (3) following the previous analysis, we found that CYP19 is not restricted to the vertebrate clade, given the description of AmphiCYP19.
Our model determine the loss of three aromatase paralogues upon duplication of the ancestral MHC-paralogon. For the vast majority of paralogy regions it is difficult to precisely determine the amount of gene loss (due to the absence of large sets mapping data from crucial organisms). In the case of the MHC-paralogon an estimate can be calculated, given the previous work of several authors [17, 21, 35]. The sequencing and mapping data of the MHC anchor genes in amphioxus, shows a significant proportion of gene families which are single-copy in both lineages (seven out of eighteen, excluding the anchor genes and those of unknown orthology; e.g. frequenin-like) [17, 35]. Thus, the return to a single copy status following the "en bloc" duplication was not a rare event upon the duplication of the MHC-paralogon. At the moment we do not known whether AmphiCYP1 9 maps along with the MHC anchor genes in a single chromosome, but this hypothesis can be tested in the future .
Finally, we speculate that the ancestry of CYP19 genes could be more ancient than invertebrate chordate origin. Two reasons support this scenario. First, the sex steroid receptors (estrogens and androgens/progesterone/corticoids) are older than previously proposed . The estrogen receptor found in Aplysia indicates that the duplication event from a sex steroid precursor receptor pre-dates the divergence of protostomes and deuterostomes . Also, the phylogenetic analysis and paralogy studies of androgen, progesterone and corticoid receptors suggests that a single receptor was present in the ancestral Urbilateria . Thus, the receptor gene kit for sex steroid hormones was already present in the primitive Bilateria (albeit not necessarily with a similar function). The second reason comes from Lophotrocozoan molluscs. These organisms respond to steroid hormones (e.g. estradiol) during their reproductive cycle . Furthermore, biochemical analysis in mollusc tissue extracts reveals the presence of an aromatase-like activity [39, 40]. In light of these findings and observations, we argue that the presence of CYP19 should be investigated in lophotrocozoan protostomes (e.g. molluscs).
We present here a detailed study of the genomic region containing the aromatase gene in three vertebrate lineages. The gene families found in close proximity to CYP19 show a clear pattern of vertebrate specific duplication, as expected from a paralogon. A key prediction from paralogy regions is their unduplicated presence in pre-vertebrate genomes. Significantly, we have also found that the genomic organisation of the human CYP19 genomic region mimics that of Tetraodon and Xenopus. Overall our analysis suggested the existence of aromatase in invertebrates. In agreement with this hypothesis we have found a CYP19 orthologue in the invertebrate chordate amphioxus. Contrary to previous suggestions, our data implies that CYP19 was present in the primitive chordate (and probably even earlier).
Phylogenetics and paralogy studies
Accession numbers for the genes used in the phylogenetic analysis.
ci0100140958 (scaffold 92)
SINFRUT00000165316 annotated as pseudogene
TMOD ( spdo )
Putative protein sequences for each gene family were aligned using the CLUSTAL X program (version 1.8). The produced alignments were further edited by eye to maximise the homologous regions (conserved domains) (given upon request). The phylogenetic reconstruction was based on conserved domains. If no domains were identified, reconstruction's were performed using the full-length alignment (without taking into account gaps or ambiguous sites). The phylogenetic trees were constructed using neighbor-joining from the CLUSTAL X program, on an amino acid distance matrix calculated with the Dayoff PAM option. Confidence on each node was assessed by 1000 bootstrap replicates. Trees were visualised with the Treeview program (version 1.6.6).
Polymerase chain reaction (PCR)
A BLAST search to the B. floridae trace archives of the Whole Genome Sequence using the DNA sequence from the stingray (D. sabina) CYP19 was done. Clone AFSA830540 presented a significant E-value (data not shown). Clone walk 5' and 3' allowed the determination of further regions of CYP19 homology. To obtain a CYP19 sequence fragment an hemi-nested PCR approach was followed using DNA purified from an 5–24 h embryo cDNA library (J. Langeland, Kalamazoo, USA). Three oligonucleotides (2 forward and 1 reverse) were designed in conserved regions using the available genomic sequence: CYPF1 5' CTGGCTAACATCCGGGACAT 3'; CYPF2 5' CAGTGCGTGACAGAAATGCT 3'; and CYPR1 5' GACGGGCTCAGTTGGTACAT 3'. PCR was carried out in 50 μl reaction mixture consisting of 10 mM Tris-HCl, pH 8.0, 1.5 mMMgCl2, KCl 50 mM, TritonX 0.1%, 10 μM of each primer, 2 mM each of dATP, dCTP, dGTP, and dTTP, 1U DNA polymerase (Appligene Oncor). The first round of PCR (oligonucleotides CYPF1 and CYPR1) had the following profile: initial cycle of denaturation, 94°C 2 min, and forty amplification cycles with denaturation at 94°C for 45 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. An hemi-nested PCR was carried out afterwards using the first PCR product as a sample. A similar PCR profile was used with the exception of the extension time – 45 s (oligonucleotides CYPF2 and CYPR1). The PCR product was separated through 2% agarose gel and purified by using the QIAquick Gel Extraction kit (QIAGEN, Germany). The product was directly sequenced in both strands using the PCR oligonucleotides by STAB VIDA (Portugal). The sequence was deposited in Genbank DQ085624.
This study was supported by the project PDCTM/MAR/15284/99 from the Fundação para a Ciência e a Tecnologia, Portugal. LFCC is funded by the Fundação para a Ciência e a Tecnologia, Portugal (BPD/19608/2004). We acknowledge Daniel Rokhsar and the Department of Energy Joint Genome Institute for the unpublished shotgun data of the amphioxus genome project, and the Holland lab, University of Oxford, UK for the amphioxus cDNA. We acknowledge also three anonymous referees for their suggestions and comments.
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