Adaptive evolution of SCML1 in primates, a gene involved in male reproduction

Hai-hui Wu 0 1 2 Bing Su 1 2 0 Graduate School of Chinese Academy of Sciences , Beijing , PR China 1 Kunming Primate Research Center, Chinese Academy of Sciences , Kunming, Yunnan , PR China 2 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences , Kunming, Yunnan , PR China Background: Genes involved in male reproduction are often the targets of natural and/or sexual selection. SCML1 is a recently identified X-linked gene with preferential expression in testis. To test whether SCML1 is the target of selection in primates, we sequenced and compared the coding region of SCML1 in major primate lineages, and we observed the signature of positive selection in primates. Results: We analyzed the molecular evolutionary pattern of SCML1 in diverse primate species, and we observed a strong signature of adaptive evolution which is caused by Darwinian positive selection. When compared with the paralogous genes (SCML2 and SCMH1) of the same family, SCML1 evolved rapidly in primates, which is consistent with the proposed adaptive evolution, suggesting functional modification after gene duplication. Gene expression analysis in rhesus macaques shows that during male sexual maturation, there is a significant expression change in testis, implying that SCML1 likely plays a role in testis development and spermatogenesis. The immunohistochemical data indicates that SCML1 is preferentially expressed in germ stem cells of testis, therefore likely involved in spermatogenesis. Conclusion: The adaptive evolution of SCML1 in primates provides a new case in understanding the evolutionary process of genes involved in primate male reproduction. - Background Proteins involved in sexual reproduction often evolve rapidly due to positive selection [1-4]. Although the selective forces are unclear, a variety of hypotheses have been proposed including mate choice, intra-sexual competition and sexual conflict, which are different forms of sexual selection. The rapid evolution of these proteins may contribute to several important biological aspects such as reproduction and speciation. It has long been recognized that gene duplication is a major source of genomic novelties. Therefore, the newly duplicated genes involved in reproduction are likely the targets of natural and/or sexual selection. Using exon trapping, van de Vosse et al [5] identified a novel gene in human located on Xp22, named as SCMlike-1 (SCML1), which is similar with the Scm gene in Drosophila. In the human genome, SCML1 spans 18 kb and contains 8 exons. Northern blot analysis detected a major SCML1 transcript of approximately 3-kb in all human adult and fetal tissues tested [5]. SCML1 gene is a polycomb group (PcG) gene. Most of the PcG genes are expressed throughout embryonic, larval and pupal development, and are required continuously to maintain restricted homeotic expression in Drosophila. [6-10]. Most mammalian PcG genes have Drosophila homologs [11,12]. Compared to Drosophila, the mammalian PcG genes have acquired novel functions during evolution because PcG knockout mice exhibit numerous phenotypes including hematopoietic defects, neural crest defects, cardiac anomalies, and sex reversal [12,13]. SCML1 is likely a recently duplicated gene during mammalian evolution due to the absence of orthologs in Drosophila, zebrafish and chicken. In the SCM family, there are other two genes, SCML2 and SCMH1, which have orthologs in all vertebrate species and are located on chromosome Xp22 [14] and chromosome 1p34 [15] respectively. SCMH1 is a core component of polycomb repressive complex 1 (PRC1) [16-18] which is involved in the maintenance of repression and can block chromatin remodeling[17], and it plays an important role in regulation of homeotic genes in embryogenesis[19]. SCML2 is also involved in PRC1's regulation[20]. A recent study showed that SCML2 is over-expressed in acute myeloid leukaemia, suggesting its role in differentiation and cell cycle regulation[21]. As SCML2 and SCMH1 are the ancient copies in the SCM family, they would serve as the ideal reference genes when dissecting the molecular evolution of SCML1 in primates. Through a genome-wide comparison, we have identified 34 candidate genes including SCML1 that showed rapid nonsynonymous sequence divergence between human and chimpanzee [22], therefore an implication of adaptive evolution of these genes during primate evolution. To test whether SCML1 is the target of selection in primates, we sequenced and compared the coding region of SCML1 in major primate lineages, and we observed the signature of positive selection. Methods DNA samples The major lineages of primates were sampled, including three great ape species (chimpanzee-Pan troglodytes, gorilla-Gorilla gorilla and orangutan-Pongo pygmaeus), two lesser ape species (white-browed gibbon-Bunopithecus hoolock and white-cheeked gibbon-Nomascus leucogenys), two Old World monkey species (rhesus macaque-Macaca mulatta and Yunnan snub-nosed monkey-Rhinopithecus bieti) and one New World monkey species (common marmoset-Callithrix jacchus). The common ancestor of the tested primate species can be traced back to about 45 million years ago [23]. All the DNA samples were from collection in Kunming Cell Bank of CAS and Kunming Blood Center in China. PCR and sequencing All the samples were sequenced for the full length coding region of SCML1. Primers for all the primates were designed by aligning the published sequences of human (Esembl ID: ENSG00000047634) and macaque (Ensembl ID: ENSMMUG00000012899, Ensemble genome browser [24]). The primer sequences are listed [see Additional file 1]. PCRs were performed with rTaq under conditions recommended by the manufacturer (TAKARA Company). Sequencing was performed in both directions with forward and reverse primers using the BigDye terminator sequencing kit on an ABI 3130 automated sequencer. There are 8 exons in SCML1 gene, and the first exon is non-translational, therefore, not sequenced in this study. Overlapping chromatogram files retrieved from the sequencer were analyzed and edited using the SeqMan program in the Lasergene software package (DNASTAR Inc). Sequence analysis The DNA sequences were aligned with the CLUSTALW program implanted in Mega [25,26] and checked manually. There are several in-dels (do not change the reading frame) in the coding region of common marmoset, and those sites were removed in the sequence analysis. The known phylogeny of primate species was used[23,27]. The ancestral sequences were inferred by PAML 3.15 [28]. The synonymous (ds) and nonsynonymous (dN) substitution rates of each branch were calculated with the use of the maximum likelihood method under the free-ratio model [28]. Test of selection Positive selection can be inferred from a higher proportion of nonsynonymous than synonymous substitutions per site (dN/dS > 1). To detect specific amino acid sites under positive selecti (...truncated)