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Available online at Molecular Phylogenetics and Evolution 47 (2008) 637–649 The human progesterone receptor shows evidence of adaptive evolution associated with its ability to act as a transcription factor Caoyi Chen a,1, Juan C. Opazo a,2, Offer Erez b, Monica Uddin a, Joaquin Santolaya-Forgas b,c,Morris Goodman a,d, Lawrence I. Grossman a, Roberto Romero a,b,*, Derek E. Wildman a,b,e,* a Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA b Perinatology Research Branch, NICHD, NIH, DHHS, Bethesda, MD 20892, USA c Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA d Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI 48201, USAe Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI 48201, USA Received 1 August 2007; revised 6 December 2007; accepted 11 December 2007 Available online 1 February 2008 The gene encoding the progesterone receptor (PGR) acts as a transcription factor, and participates in the regulation of reproductive processes including menstruation, implantation, pregnancy maintenance, parturition, mammary development, and lactation. Unlike othermammals, primates do not exhibit progesterone withdrawal at the time of parturition. Because progesterone-mediated reproductive fea-tures vary among mammals, PGR is an attractive candidate gene for studies of adaptive evolution. Thus, we sequenced the progesteronereceptor coding regions in a diverse range of species including apes, Old World monkeys, New World monkeys, prosimian primates, andother mammals. Adaptive evolution occurred on the human and chimpanzee lineages as evidenced by statistically significant increases innonsynonymous substitution rates compared to synonymous substitution rates. Positive selection was rarely observed in other lineages. Inhumans, amino acid replacements occurred mostly in a region of the gene that has been shown to have an inhibitory function (IF) on theability of the progesterone receptor to act as a transcription factor. Moreover, many of the nonsynonymous substitutions in primatesoccurred in the N-terminus. This suggests that cofactor interaction surfaces might have been altered, resulting in altered progesterone-reg-ulated gene transcriptional effects. Further evidence that the changes conferred an adaptive advantage comes from SNP analysis indicatingonly one of the IF changes is polymorphic in humans. In chimpanzees, amino acid changes occurred in both the inhibitory and transac-tivation domains. Positive selection provides the basis for the hypothesis that changes in structure and function of the progesterone recep-tor during evolution contribute to the diversity of primate reproductive biology, especially in parturition.
Ó 2007 Elsevier Inc. All rights reserved.
Keywords: Primates; Positive selection; Hormone activity; Parturition; Chimpanzee The steroid hormone progesterone is a vital regulator of * Corresponding authors. Address: Perinatology Research Branch, reproduction in mammals NICHD, NIH, DHHS, Wayne State University/Hutzel Women's Hospi- tal, Center for Molecular Medicine and Genetics, 3990 John R, Box 4,Detroit, MI 48201, USA. Fax: +1 313 993 2694.
The role of progesterone in mammalian E-mail addresses: (R. Romero), pregnancy maintenance is well established ( (D.E. Wildman).
1 Present address: Nantong University, 9 Seyuan Road, Nantong, JS 226019, PR China.
). In primates, progesterone partici- Present address: Instituto de Ecologı´a y Evolucio´n, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile.
pates in the regulation of normal menstrual cycles, ovula- 1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2007.12.026 C. Chen et al. / Molecular Phylogenetics and Evolution 47 (2008) 637–649 tion (and embryo implantation 1.1. Structure of progesterone receptor ). Many ofthe biological actions of progesterone are mediated The gene encoding human PR has eight exons and is through the progesterone receptor (PR; HUGO gene sym- located on chromosome 11q22.1. PR is characterized by bol = PGR), a member of the nuclear receptor superfamily alternatively spliced isoforms (), as of ligand-activated transcription factors shown in . The two well-studied major isoforms of PR are PR-B and PR-A. Another isoform, PR-C, has also gene encoding this receptor arose early in vertebrate evolu- been described ().
tion via a series of duplications of an ancestral estrogen Exons 1 and 2 can be considered alternatively spliced receptor The progesterone receptor has and/or translated exons because all of exon 1 and most been implicated in the initiation of human parturition as of exon 2 are absent from PR-C and part of exon 1 is the relative abundance of PR isoforms changes near the absent from PR-A. The remaining exons are considered end of gestation ( constitutively spliced since they are included in all major The major isoforms vary in length but not in amino acid Humans and their closest relatives, the chimpanzees, dif- sequence (i.e., there is no frame-shift). PR-B is 933 amino fer from each other in numerous ways that could be related acids in length, while PR-A lacks 164 amino acids at the to progesterone and its receptor. The duration of parturi- amino terminus. In vitro, PR-B is a stronger transactivator tion in humans is, on average, longer than the 40-minute than PR-A, whereas PR-A acts as a transrepressor of PR- to eight hour range observed in chimpanzees B and some other steroid receptors ( ). Chimpanzee mammary glands, like ). The domain architecture of PR those of other mammals, are enlarged during lactation is shown in C. Structurally, both isoforms consist of whereas human mammaries are consistently evident an amino-terminal region, a centrally located DNA-binding beyond puberty. Conversely, unlike in humans, cyclic domain (DBD), and a carboxy-terminal hinge region con- swelling of the skin surrounding the anogenital region of taining nuclear localization signals, as well as a ligand-bind- chimpanzees is obvious.
ing domain (LBD, sometimes called HBD). There are three In spite of these differences between humans and chim- transcription activation function (AF) domains. AF-1 is panzees, the hormonal profiles and length of the menstrual located upstream of the DBD domain whereas AF-2 is cycles of humans and chimpanzees are mostly similar. The located in the LBD (The other activation mean length of gestation in chimpanzees is 227 days com- function domain (AF-3) is unique to the PR-B isoform, and pared to 280 in humans is located within the N-terminal region This difference is actually less than it seems.
). There is also an inhibitory function Human gestational length is measured from the first day of (IF) region located between AF-3 and AF-1, which has been the last menses about two weeks before conception. In con- proposed as the region responsible for autoinhibition and trast, chimpanzee gestations are measured to begin from the transrepression of PR last day of maximal sex skin tumescence during a cycle in ). The function of PR-C is still which copulation was observed. Therefore, the actual differ- ence in gestation length between human and chimpanzees is It is translated from an in-frame translation start site, between 20 and 30 days. In addition, the placental morphol- Met595, at the end of the second exon of PGR. Presence of ogy and serum progesterone concentrations of humans and the PR-C isoform has been suggested in human ( chimpanzees are similar, while rhesus and baboon have lower progesterone levels ( Among all of these features, the unique role of PGR in ), and cow Since PR-C lacks human parturition is of considerable interest because of the DBD, if bound to PR-B the resulting heterodimer would the special challenges humans face during the birth process.
be unable to function as a transcription factor ( Natural selection during human evolution has resulted in Moreover, the absence of the DBD in PR-C would anatomical changes including the remodeling of the pelvis also prevent PR-C homodimers from acting as a transcrip- during the emergence of bipedalism and the expansion of tion factor.
the cranium associated with encephalization that may haveaffected parturition 1.2. Adaptive evolution in progesterone receptor ). These anatomical innovations that resultedin a relatively smaller birth canal and larger head are likely A recent genome-wide scan (of to have required adjustments underlying the labor and human and chimpanzee genes placed PGR among the 50 birth process (i.e., parturition has a longer duration in genes showing the strongest statistical evidence of positive humans than in chimpanzees). The current study examines selection as measured by calculating the ratio of nonsyn- the evolution of mammalian PGR within this broad con- onymous to synonymous nucleotide substitution rates on text of human evolution.
a per site basis. This ratio is called dN/dS or x. Generally, C. Chen et al. / Molecular Phylogenetics and Evolution 47 (2008) 637–649 Chromosome Bands Localized by FISH Mapping Clones Old UCSC Known Genes Based on UniProt, RefSeq, and GenBank mRNA PGRB (1) PGRA (495) PGRC (1785) Fig. 1. Location and structure of the human progesterone receptor. (A) Location and orientation of PGR in the human genome. The gene usually containseight exons and the full-length receptor spans 92 kb. (B) Gene structure of PGR. Translation initiation sites for PR isoforms are indicated with arrows.
Exons 1 and 2 are alternatively spliced and shown in gray (not drawn to scale). (C) Schematic illustration of previously identified functional domains in thehuman progesterone receptor. Numbers indicate the amino acid positions that delineate the beginning and end of each domain while bent arrows point tothe translation initiation site of the three isoforms (PR-A, PR-B, and PR-C). AF, activation function region; IF, inhibitory function region; DBD, DNA-binding domain; NLS, nuclear localization signal; H, hinge region; HBD, hormone/ligand binding domain.
x = 1, >1, and <1, indicate neutral evolution, positive 2. Materials and methods selection, and purifying selection, respectively Nielsen et al. identified 11 2.1. DNA samples and sequencing nonsynonymous and 0 synonymous PGR substitutionsbetween human and chimpanzee. Therefore, changes in Genomic DNA was used as a template for the polymer- structure and function of the progesterone receptor during ase chain reaction to obtain the entire coding region of pro- gesterone receptor in Homo (Homo) sapiens (Human), between humans and chimpanzees. As the Nielsen et al.
Homo (Pan) troglodytes (Common chimpanzee), Homo study included only humans and chimpanzees, the authors (Pan) paniscus (Bonobo), Gorilla gorilla (Gorilla), Pongo could not determine whether the positive selection occurred pygmaeus (Orangutan), Hylobates lar (White handed gib- on the human, the chimpanzee, or both lineages. More- bon), Macaca sylvanus (Barbary macaque), Papio anubis over, the study did not explore whether positive selection (Olive baboon), Chlorocebus aethiops (African green mon- was limited to humans and chimpanzees or whether this key), Colobus guereza (Black-and-white colobus monkey), phenomenon occurred on other mammalian lineages as Trachypithecus obscurus (Dusky leaf-eating monkey), well. Finally, the study did not describe the locations of Cebus apella (Tufted capuchin monkey), Pithecia irrorata the nucleotide substitutions in terms of domains and/or (Gray saki monkey), and Ateles paniscus (Black spider monkey). For the following species, exon 1 was obtained: Therefore, in order to determine if positive selection on Tarsius bancanus (Western tarsier), Otolemur crassicauda- PGR occurred only in humans, in chimpanzees, or among tus (Thick-tailed bushbaby), Mirza coquereli (Giant other mammals as well, we collected DNA sequence data mouse-lemur), Dasypus novemcinctus (Nine-banded arma- from all major primate and non-primate placental mammal dillo), Bradypus sp. (Three-toed sloth), Loxodonta africana lineages. With these nucleotide sequence data in hand, (African elephant), Dugong dugon (Dugong). All sequences using both probabilistic maximum likelihood and parsi- have been deposited in GenBank under accession numbers mony based approaches, we performed the following anal- DQ234979–DQ234989 and DQ485133–DQ485143. Pub- yses: (1) detection of the effects of natural selection on the lished sequences from GenBank for PGR used in analyses PGR during primate, and especially human and chimpan- are Mus musculus (Mouse) (NM_008829), Rattus norvegi- zee, evolution; (2) reconstructions of the evolutionary his- cus (Rat) (NM_022847), Oryctolagus cuniculus (Rabbit) tory of the PGR and determination of the specific (M14547) and Canis familiaris (Dog) (NM_001003074).
lineages on which amino acids were replaced; and (3) local- PCR reactions were performed in an Eppendorf (Ham- ization of putatively functionally important amino acid burg, Germany) thermal cycler under the following condi- replacements in the gene.
tions: 5 min at 95 °C followed by 30–34 cycles of 94 °C for C. Chen et al. / Molecular Phylogenetics and Evolution 47 (2008) 637–649 30 s, a variable annealing step of between 45 and 68 °C for (and visually inspected to ensure that pro- 30–40 s, and 68/72 °C for 30 s–3 min, followed by a final tein coding sequence indels preserved the correct reading extension at 72 °C for 5 min. PGR exons 2–8 were ampli- frame. The alignment files are available online as fied using Taq DNA polymerase (Qiagen Inc., Valencia, CA). The first exon was amplified with the Takara LAPCR Kit, Version 2.1 (Takara, Shiga, Japan). Amplifica-tion products were isolated by 1% agarose gel electropho- 2.3. Tests for selection resis and purified with QIAquick Gel Extraction Kit(Qiagen Inc., Valencia, CA). Purified PCR products were Aligned sequences were tested for evidence of positive either sequenced directly with both sense and antisense selection using codon-based models PGR-specific primers or cloned into the pGEM-T easy vec- as implemented in PAML tor (Promega, Madison, WI) and sequenced with SP6 and v.3.15 ). These tests are conducted from esti- T7 primers. Primer sequences are available on request. For mates of the dN/dS ratio (i.e. x) that compare the rates of nonsynonymous (amino acid changing) substitution to syn- sequencing primers were used. Sequencing was performed onymous (amino acid maintaining) substitution on a per site using the ABI BigDye terminator cycle sequencing kit basis. We conducted three conventionally applied tests of v3.1, and sequence electrophoresis was performed on an selection: neutrality, nested lineage, and branch-sites tests automated capillary ABI3700 sequencer (Applied Biosys- tems, Foster City, CA).
In order to determine whether PGR dN/dS ratios were variable among the different branches of the tree, we testedthe neutral prediction that x is the same on all lineages by 2.2. Sequence assembly and alignment comparing a fixed ratio model (model 0) to the free ratiomodel (model 1). In addition, to test lineage-based hypothe- The DNA sequence data were edited and contigs were ses four nested models as well as model 0 were examined assembled using Sequencher 4.5 (Gene Codes, Ann Arbor, using the species with full coding region sequences ().
MI). Both the full-length coding region and exon 1 A two x model A) estimated one ratio for primates sequences of all taxa were aligned using ClustalX 1.83.1 (as a total group) and another ratio for the outgroup species.
Oryctolagus cuniculus Oryctolagus cuniculus Rattus norvegicus Rattus norvegicus Oryctolagus cuniculus Oryctolagus cuniculus Rattus norvegicus Rattus norvegicus Fig. 2. Nested models of progesterone receptor evolution. In addition to the one x ratio (model 0) the maximum likelihood scores from alternativescenarios of nucleotide substitution rate variation were calculated using PAML and tested against one another using likelihood ratio tests. (A) Two x ratiomodel estimated separate ratios for non-primates and primates; (B) three x model estimated one ratio for non-primates, one ratio for the human–chimpanzee total group, and one for the other primates; (C) four x model estimated one ratio for non-primates, a ratio for the human–chimpanzee crowngroup, a ratio for the human–chimpanzee stem lineage, and a ratio for the other primates; (D) a five x model estimated a separate ratio for each of thefollowing five groups: non-primates, the human terminal branch, the chimpanzee clade, the human–chimpanzee stem, and the other primates. Hypothesistests were conducted using a nested procedure.
C. Chen et al. / Molecular Phylogenetics and Evolution 47 (2008) 637–649 A three x model (B) estimated one ratio for non-prima- (70) to identify the specific lineages and sites where nucleo- tes, and two ratios for primates, namely the human–chim- tide substitutions and amino acid replacements occurred.
panzee total group (all descendant branches in the human– With these data, we compared the alternatively spliced chimpanzee clade including their stem lineage), and all the exons (i.e. exons 1 and 2) to the exons found in all PGR iso- other primates. A four x model (C) estimated one ratio forms (i.e., the constitutively spliced exons 3–8). In partic- for non-primates and three ratios for primates––the human– ular, we assessed the number of nucleotide substitutions chimpanzee crown group (the descendent lineages from the per site in human descent from the last common ancestor last common ancestor of humans and chimpanzees), the of anthropoid primates for different exon classes: each human–chimpanzee stem lineage, and all the other primates.
alternatively spliced exon (1 and 2), exons 3–8, and the full A five x model D) estimated five different ratios as fol- gene. The exon classes were compared to one another by lows: (1) non-primates, (2) the human terminal branch, (3) test or Fisher's exact test. Previously published the chimpanzee clade, (4) the human–chimpanzee stem, phylogenetic trees of primate and mammalian relationships and (5) the other primate lineages. In every model tested, were employed to describe the pattern of nucleotide substi- the likelihood values were calculated three times, with differ- tution during evolution ent starting seed values for the x parameter (0.5, 1, and 2) to increase the probability of reaching the optimal likelihoodscore.
Branch sites tests of were conducted to detect individual PGR codons that had experienced posi- 3.1. Characterization of sequences and evolutionary changes tive selection. These tests compare a priori determined fore- in progesterone receptor ground and background branches. In our test, branches inthe human and chimpanzee crown group were the fore- The multiple sequence alignment of coding nucleotide ground lineages. The remaining branches were the back- sequences including indels spanned 2853 bp. Aligned exon ground. Model A is the alternative hypothesis and 1 sequences are 1688 bp. Exons 2–8 have an alignment assumes that on the foreground branch(es) some codons length of 1165 bp. We obtained two slightly different clones have undergone purifying selection while other codons from Tarsius, suggesting that there is heterozygosity or a are neutral or positively selected. Selection is not allowed second copy of PGR in that sample.
on background branches. Model A was compared to the All the taxa included in our analysis have alternative null model which fixed x = 1 on the foreground in the translation initiation sites (TIS) for the three major iso- two site classes that allow selection. Bayes Empirical Bayes forms, PR-B/PR-A/PR-C. PR-B/A TIS, Met1 and Met165 (BEB) posterior probabilities for site classes were examined are inside the first exon region. PR-C uses an in-frame to infer sites that likely were under positive selection.
translation start site, Met595, at the end of the second exon.
Likelihood scores for alternative models were compared The PR-C in-frame TIS not only exists in the taxa included by likelihood ratio tests, and a test was used to deter- in the full PGR gene analysis, but also in the publicly avail- mine significance. A result was considered significant if able whole genome assemblies and Ensembl gene builds for p < 0.05. All tests were conducted using presumed phyloge- armadillo (Dasypus novemcinctus), tenrec (Echinops telfair- netic trees ().
i), elephant (Loxodonta africana), opossum (Monodelphisdomestica), chicken (Gallus gallus), frog (Xenopus tropical- 2.4. Evolutionary rates is), spotted green pufferfish (Tetraodon nigrovirdis), Japa-nese pufferfish (Fugu rubripes), and zebrafish (Danio rerio) In addition to maximum likelihood tests for selection, (data not shown).
maximum parsimony ancestral sequences (ACCTRAN In primates the first exon is more variable than the other and DELTRAN) were inferred using the software PAUP* exons. Human-specific amino acid changes are mostly in Table 1Amino acid replacements in human since the divergence from chimpanzees Amino acid position Nonsynonymous SNPs Multiple species alignment AF, activation function region; IF, inhibitory function region.
C. Chen et al. / Molecular Phylogenetics and Evolution 47 (2008) 637–649 the first exon, specifically in the IF region (Accord- revealed only one nonsynonymous SNP among the codons ing to parsimony ancestral reconstructions, there were 17 changed on the human lineage There were eight unambiguous amino acid replacements in the human and amino acid replacements observed in chimpanzees (two on chimpanzee clade Eight of these replacements the chimpanzee stem, three on the bonobo terminal lineage, occurred on the human lineage and eight in the chimpanzee and three in the common chimpanzee terminal linage). Of lineages. There was one replacement on the human–chim- these eight replacements, four are in the IF region (one of panzee stem lineage. Six of the eight inferred amino acid the amino acid changes was on the chimpanzee stem and replacements on the human terminal lineage were in the three were on the terminal linage of the common chimpan- inhibitory function region. The other two amino acid zee). The remaining replacements are scattered among the replacements were in AF3 and hinge region, respectively.
transactivation AF1 and AF3 domains, and the hinge region All human replacements occurred in exon 1 with the excep- tion of one replacement in the hinge region. Searches of the Parsimony analysis inferred a range of three to five NCBI single nucleotide polymorphism database (dbSNP) amino acid replacements on the stem lineage leading to 171: G>R
535: N>S
265: A>T
184: A>V
545: A>T
267: A>V
204: S>A
691: T>S
348: A>V
213: G>E
228: P>A
359: S>C
83: D>N
444: P>T
312: V>M
679: I>V
182: G>R
Fig. 3. Amino acid replacements in PGR during human and chimpanzee evolution. A phylogram depicts the amino acid replacements that occurred inPGR during recent human and chimpanzee evolution. Numbers refer to the amino acid position in the multiple sequence alignment of the complete gene.
This alignment included all anthropoid primates sampled (see Section ), as well as mouse, rat, rabbit, and dog. Branch lengths are proportional to theamount of amino acid replacement.
C. Chen et al. / Molecular Phylogenetics and Evolution 47 (2008) 637–649 0.46 (225.2;200.1) 0.44 (8.2;7.5) Pongo 1.22 (3.1;1.0) Pan paniscus Pan troglodytes 0.24 (3.1;5.2) Gorilla 0.30 (17.7;24.1) Hylobates 0.67 (10.4;6.3) Colobus 0.35 (7.3;8.4) Trachypithecus 0.77 (5.9;3.1) Papio 0.32 (18.7;24.0) Cebus 0.12 (41.2;141.8) Oryctolagus 0.22 (61.1;111.8) Mus 0.12 (107.7;376.9) 0.26 (79.7;107.8) 0.38 (186.4;171.0) Pan paniscus Pan troglodytes 0.14 (46.4;115.0) 0.38 (241.3;222.6) 0.20 (166.1;294.8) 0.25 (104.2;144.1) 0.26 (37.0;50.0) Dugong Fig. 4. Adaptive evolution in progesterone receptor. The free ratio model (model 1) of nucleotide substitution, which estimates a separate x value for eachbranch in the tree, is shown. The numbers shown along each branch are x, and the maximum-likelihood estimates of the numbers of nonsynonymous(N*dN) and synonymous (S*dS) substitutions along that branch. (A) Complete coding region, (B) first exon. Lineages showing evidence of adaptiveevolution are depicted in red.
C. Chen et al. / Molecular Phylogenetics and Evolution 47 (2008) 637–649 Table 2Likelihood ratio scores and x values of the different nested models macaque and baboon: (1) a minimum of three changes, all lihood = 11500.53; Similar results are observed in the IF region; or (2) a maximum of five amino acid in the analysis of the first exon B). These results indi- changes, of which four are in the IF region and one is in cate that the dN/dS ratios are indeed different among lin- the AF1 domain. Additionally, there were four unambigu- ous amino acid replacements on the haplorhine (anthro- purifying selection; the background x value (i.e., one ratio poid and tarsier) stem (two in the IF region and two in or Model 0) is 0.25. In contrast to the low background x the AF3 domain).
value, a few branches do show evidence of positive selec- Functionally important conserved amino acid sites, tion. The branch with the highest ratio for the full gene is including 5K, 7K, 55LxxLL(L1), 115LxxLL(L2), 140W, the human terminal branch (dN/dS = 1.63; N*dN = 8.3, 387IKEE, and 531K (amino acid numbers are from the human S*dS = 2.1). Most of this change occurred in exon 1 protein sequence) have been identified in PR ( (In addition to the human terminal branch, accel- erations occur in the chimpanzee clade (for both the full to our data, three of these sites (7K, 387IKEE, and 531K) gene and for exon 1), the stem papionan (macaque and are uniformly conserved. The others have experienced amino baboon) lineage for the full gene, and the stem papionan acid replacements during mammalian evolution. 5K is and stem haplorhine lineages for exon 1.
replaced by Q in mouse and rat and T in dog. 140W is replaced The dN/dS ratios estimated from the various nested by 140R in dog. L1 is replaced by LxxLV in Otolemur, SxxLL models and likelihood ratio tests are shown in .
in dog and LxxLF in Bradypus. L2 is replaced by LxxLW in The best-fit model (p = 0.021; C) for PGR NWMs and PxxAL in dog.
evolution is the 4 x model that estimated one ratio fornon-primates and three rates for primates: (1) the 3.2. Tests for selection-variable dN/dS rates among lineages human–chimpanzee crown group (the descendent lineages from the last common ancestor of humans and chimpan-zees); (2) the human–chimpanzee stem lineage; and (3) The x values estimated on lineages are shown in the other primates. In this statistical model PGR has a For the full gene (the free ratio model (model 1, >7 fold higher dN/dS in the human and chimpanzee crown log likelihood = 11440.52) fits the data significantly bet- group (2.1682) compared with all other lineages, thus pro- ter (p < 0.001) than the one ratio model (model 0, log like- viding strong evidence for positive selection. The branchsites tests identified a proportion of sites as potentiallybeing positively selected (17%, x = 5.5) although the null hypothesis could not be rejected (p = 0.22).
Significance tests of the nested models 3.3. Evolutionary rates Our data demonstrate that the 50 region of PGR con- taining the sites associated with the transactivation and Table 4Parsimony inferred rates of nucleotide substitutions in PGR during human descent from the most recent common ancestor of anthropoids The significance of the comparisons between exon 1 and the other exons are labeled as follows: *p < 0.05; **p < 0.005. The comparisons between exon 2and exons 3–8 were not statistically significant. Sub = substitution.
C. Chen et al. / Molecular Phylogenetics and Evolution 47 (2008) 637–649 transrepression function is highly variable, whereas other preserved these sites, at which point no further changes coding regions are well conserved. The number of substitu- tions per site on the anthropoid lineages leading to human Among all sampled taxa, the first exon includes a CpG between alternatively and constitutively spliced exons is island. CpG dinucleotides have high mutation rates and presented in Exon 1 evolves much more rapidly increased frequency of transitions relative to transversions than exon 2, even though both are alternatively spliced.
because they are prone to be mutated by cytosine deamina- During human descent from the last common anthropoid tion (). As such, one ancestor, exon 1 had significantly more substitutions per possible interpretation of the results is that the observed site in comparison to both the other alternatively spliced amino acid replacements are due solely to CpG status exon 2 and to the constitutively spliced exons 3–8. Most rather than adaptive evolution. However, CpG islands dif- of this acceleration is due to nonsynonymous substitution.
fer from methylated CpG dinucleotides. They usually The background dN/dS value as calculated by PAML in escape methylation in the germline and remain consistently alternatively spliced exons (exons 1 and 2) is more than nonmethylated in normal tissues, with the exception of four times higher than in other constitutive exons (dN/ some imprinted genes and inactivated X chromosomes dS = 0.27 vs. 0.06). The absolute value of dN is also increased when the alternatively spliced exons are com- pared to the constitutively spliced exons (dN = 0.53 vs.
set, the ratio of transitions over transversions (ts/tv) in the CpG island region (2.88) is lower than in the non-CpGisland region (exon 2–8) (3.09). This suggests that theisland escapes the effects of rapid mutation due to CpG sta- tus. More importantly, none of the nonsynonymous substi-tutions in the human–chimpanzee clade took place at CpG This study investigated three aspects of PGR evolution: dinucleotides. Finally, progesterone receptors are unme- (1) the strength of positive selection (i.e., adaptive evolu- thylated in normal human tissues such as uterine endome- tion) in human and chimpanzee lineages; (2) the prevalence trium, breast tissues, and bone marrow ( of positive selection within primates; and (3) the specific isoforms and domains at which amino acids were replaced.
do not attribute the increase in nonsynonymous substitu- Positive selection occurred on both the human and chim- tion rate observed in human and chimpanzees to CpG panzee lineages. Exon 1 (particularly the IF region) was the region of the gene that showed the most amino acidreplacements during human evolution after the split from 4.1. Conservation in PGR the chimpanzee lineage. The chimpanzees also showedchanges in the IF region but fewer than in the human line- The pattern of selection observed among humans and age. In primates, positive selection occurred rarely and was chimpanzees is fairly rare in the context of primate and mostly limited to the human and chimpanzee clade even mammalian PGR evolution. Of 33 mammalian lin- although the papionan (macaque and baboon) and haplo- eages examined for exon 1 (only six had dN/dS rhine stem lineages also showed evidence of selection.
ratios greater than 1, and four of these fall within the Comparatively, humans and chimpanzees showed a sig- human–chimpanzee clade. Throughout the majority of pri- nificantly higher nonsynonymous than synonymous substi- mate evolution, purifying selection has acted strongly on tution rate. This result is in general accord with the finding PGR, even in the most variable region of the gene, exon of , who showed evidence for positive 1. As mentioned before, some key sites for PR autoinhibi- selection in human and chimpanzee PGR. However, with tory and transrepression function in the exon 1 IF region the addition of data from other species we demonstrate are 7K, 387IKEE, and 531K. These residues are uniformly that positive selection occurred on both the human and conserved among all the species in our study as well as in chimpanzee lineages. Furthermore, among chimpanzees the chicken (This implies that the there is evidence for positive selection in both common inhibitory function of the IF region is highly conserved.
and bonobo chimpanzees as well as on their stem lineage.
PGR is characterized by several alternatively spliced iso- An alternative interpretation to the finding of adaptive forms, and alternative splicing is a powerful and economi- evolution is that the human and chimpanzee changes have cal way to increase protein diversity from a single gene no functional consequences and have evolved neutrally locus in the course of evolution. The three major alterna- under relaxed selection. This is unlikely because in humans tive N-terminal isoforms PR-A/B/C have some overlap- there is only one reported nonsynonymous SNP in the ping and some unique functions ( codons encoding the amino acids that changed on the human lineage, which suggests that substitutions were fixed before the last common ancestor of modern humans ( Our study provides ). We propose that natural selection first spread these evidence for the existence of these three PGR isoforms in amino acid replacements through the human lineage then all species studied (as measured by conservation of the C. Chen et al. / Molecular Phylogenetics and Evolution 47 (2008) 637–649 three alternative TIS). Until the transcripts of the different ductive tract malignancies such as endometrial ( PGR isoforms of the study species are available, a definitive and ovarian tumors () as description of the effects of alternative splicing in PGR evo- well as in breast cancer (Possible clin- lution will be lacking. Finally, the DBD and LBD are ical implications of the rapid evolutionary divergence in the highly conserved among all the mammals we sequenced progesterone receptors of humans and chimpanzees should as part of this study, and we found no evidence of rapid therefore be considered.
evolution in either the DBD or LBD.
The observed amino acid replacements may represent species-specific changes in the biologic inhibitory/repressor 4.2. Functional consequences of rapid evolution in PGR activity of the PR in human and chimpanzees. The sixhuman amino acid replacements found in the IF region PGR, and particularly the inhibitory function region, combined with the lack of nonsynonymous human SNPs have evolved rapidly during recent human evolution.
at five of the six codons raise the possibility that the Indeed, 75% (6/8) of the amino acid changes on the human changes on the human lineage were fixed before the most lineage since the human–chimpanzee most recent common recent common ancestor of modern humans. This finding ancestor (MRCA) were in the first exon in the IF region strengthens the evidence for adaptive evolution in human and in chimpanzees the amino acid changes were PGR and the conclusion that observed amino acid replace- in both the IF region and transactivation domains ments have functional importance. The IF is essential for Although the exact functional consequences of the PR-A function, and researchers have suggested that one observed amino acid replacements are unclear, we can of the functions of the AF3 domain in the PR-B isoform speculate about their effects based on their location in the is to block the inhibitory effect of the IF protein. The AF3, IF, and AF1 regions provide surfaces for transcriptional activation and repression of progester- A possible implication of the evolutionary changes in the one-modulated genes, and also are involved in PGR IF region can be related to the mechanism of human labor, autoinhibitory function. Additional functional conse- many aspects of which are unique among mammals quences that could be implicated from changes in these For example, it has been proposed that primates have regions include cofactor recruitment, cofactor-binding a functional progesterone withdrawal leading to labor affinity, the conformation of the protein complexes of PR and its coregulators, and the transcriptional activity of PR-B. In this last example it is important to note that AF3 is unique to PR-B. Nonsynonymous substitutions proposed mechanisms that effectively causes functional pro- took place in AF3 domain in humans and chimpanzees, gesterone withdrawal during parturition is the change in the and this region might directly influence the intramolecular relative expression of PR-A, PR-B, and PR-C in myome- communication between the PR-B N terminus and down- trium, fetal membranes, and placenta during the onset of stream domains The two highly conserved L1 boxes and 140W that define the AF3-related It is possible that the expression and interaction among the different PR isoforms in func- amino acid replacements in New World monkeys, Otole- tional progesterone withdrawal during human and chimpan- mur, dog, and Bradypus. These observed amino acid zee parturition are uniquely modulated via changes in the IF replacements might also affect coactivator recruitment region. Human parturition is different from that of chimpan- and direct N-/C-terminal interaction. Coactivators known zees and other mammals because in the past 6 million years to act on progesterone receptor include c-AMP responsive human anatomy has been modified by the emergence of element binding protein 1 (CREB1) and members of the bipedalism (resulting in the remodeling of the pelvis) as well steroid receptor coactivator (SRC) family as encephalization (resulting in an increased cranial size rel- ). An additional interpretation is that the observed ative to body mass) ). These amino acid replacements are compensatory substitutions anatomical changes were certainly adaptive, and the mecha- for replacement in protein cofactors, which are recruited nism of human parturition may have undergone modifica- by the activated PGR to the promoters of target genes in each species.
Chimpanzees have also been subject to evolutionary pres-sures on their reproductive biology as evidenced by the pres- 4.3. Implications of this study ence of sex skin swelling and extreme differences in sexualbehavior observed between common and bonobo chimpan- PGR is an important factor in the normal development zees (). Positively and function of the reproductive tract ( selected changes in PGR, particularly in its IF region, might well have participated in the emergence of these adaptations.
glands (and even reproductive behav- However, elucidating the exact functional consequences of ior (Changes in the expression of PR-B and PR-A have been reported in repro- C. Chen et al. / Molecular Phylogenetics and Evolution 47 (2008) 637–649 Condon, J.C., Jeyasuria, P., Faust, J.M., Wilson, J.W., Mendelson, C.R., 2003. A decline in the levels of progesterone receptor coactivators inthe pregnant uterus at term may antagonize progesterone receptor This research was funded by the Intramural Research function and contribute to the initiation of parturition. Proc. Natl.
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