Genetica 118: 193–208, 2003.© 2003Kluwer Academic Publishers. Printed in the Netherlands.

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Birth of ‘human-specific’ genes during primate evolution

Jean-Louis NahonInstitut de Pharmacologie Moleculaire et Cellulaire, CNRS UMR 6097, 660 route des Lucioles, Sophia-Antipolis,06560 Valbonne, France (Phone: +33-4-93-95-77-54; Fax: +33-4-93-95-77-08; E-mail: nahonjl@ipmc.cnrs.fr)

Key words: anthropoids, molecular evolution, primates, retroposition, segmental duplications

Abstract

Humans and other Anthropoids share very similar chromosome structure and genomic sequence as seen in the98.5% homology at the DNA level between us and Great Apes. However, anatomical and behavioral traitsdistinguishHomo sapiens from his closest relatives. I review here several recent studies that address the issueby using different approaches: large-scale sequence comparison (first release) between human and chimpanzee,characterization of recent segmental duplications in the human genome and analysis of exemplary gene families.As a major breakthrough in the field, the heretical concept of ‘human-specific’ genes has recently received somesupporting data. In addition, specific chromosomal regions have been mapped that display all the features of ‘genenurseries’ and could have played a major role in gene innovation and speciation during primate evolution. A modelis proposed that integrates all known molecular mechanisms that can create new genes in the human lineage.

Introduction

What makes us different at the genetic level from otherprimates and particularly from our closest relatives,the Great Apes? This central question in biology, withmajor consequences on our social life, ethical andphilosophical perception of ourselves, has obsessedfor decades and yet attracts many geneticists and evo-lutionary biologists. Genetic differences may lie at dif-ferent levels including gross alterations in cytogeneticarchitecture, local chromosomal rearrangements, genefamily duplication, single gene modifications (creationor loss) and differences in gene transcription and al-ternative splicing of mRNA (reviewed by Gagneux &Varki, 2001). Using the R-banding technique in the1970s, a very close organization of chromosome ban-ding with an identical euchromatin was revealed in thekaryotypes of human and other primates (reviewed byDutrillaux, 1979). Later, chromosome painting resultsnot only confirmed the high degree of conservation ofchromosomal synteny but also identified translocationand fission events that were genomic landmarks for theorigin of Anthropoids (Müller et al., 2000; reviewedby Hacia, 2001). The widespread use of cloning andmolecular biology techniques in the early 1980s gave

a direct access to genomic sequences and allowedus to trace the evolutionary history in primates ofsingle genes, or gene families such as theβ-globingene cluster (reviewed by Goodman, 1999). Fi-nally the completion of numerous whole-genome-sequencing projects culminated in February 2001 withthe publication by public and private laboratoriesof a working draft sequence of the human genome(IHGSC, 2001; Venter et al., 2001). This paved theroad for a Primate Genome Project (or Human Ge-nome Evolution Project) which was initially calledfor by McConkey and Goodman (1997) and is nowunderway with the recent release of the first gener-ation human–chimpanzee comparative genome map(Fujiyama et al., 2002).

DNA sequence comparison at many different lociamong human and African Great Apes revealed astrong similarity, close to 98.5% sequence identities(Fujiyama et al., 2002; reviewed in Hacia, 2001).Even higher percentage of amino acid sequence iden-tities was estimated in coding regions (99%). Thisled to the proposal that few novel genes are associ-ated with the ‘human-specific’ traits such as biped-ism and higher cognitive functions (Gibbons, 1998)and that marked differences should be found in the

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Figure 1. Primate phylogenetic relationships. An overview of accepted phylogenies of primates (some relevant clade names for present dayspecies are provided) based on molecular and fossil record analyses (Goodman, 1999), Mya, million years ago.

tissue-specific regulation, developmental and levelof gene expression across primate species (Normile,2001; Enard et al., 2002a). Recently, the discoveryof chimeric genes that originated at the time of emer-gence of Anthropoids, the characterization of recentsegmental duplications in the human genome and thefirst comparative analysis of the expression of humanand non-human primate gene using cDNA microarraytechnology provide some clues to evaluate which partsof our genome make us human. This review will fo-cus on the mechanisms that drive the birth of genes inprimate lineage but also address the general conceptsof ‘young’ genes, ‘gene nursery’ at segmental duplica-tion, evolving of ‘human-specific’ protein-coding (andnon-coding) genes under positive selection pressure.

Primate evolution dating

Many studies addressing human evolution have fo-cused on comparison with our closest relatives, thechimpanzees and gorillas (Gagneux & Varki, 2001;Hacia, 2001; Kaessmann & Pääbo, 2002). Recently,comparative neuroanatomy has revealed an expan-sion of both the neocortex, with a burst of size andneuronal interconnectivity during hominid evolution(Stevens, 2001; Semendeferi et al., 2002), and theright side of the human brain compared to chimpanzee(Gibbons, 2002a,b). However, other studies indicated

the progressive concerted evolution of groups of brainareas defining the so-called taxon-specific cerebro-types (Clark, Mitra & Wang, 2001), according tothe principle of ‘integrated phylogeny’ (Rapoport,1990). Indeed, it is most likely that some aspectsof human cognitive behaviors and inferred neuronaland molecular mechanisms were present to some ex-tent a long time before the emergence of modernHuman, 150,000–200,000 years ago (Gibbons,2002a,b; Kaessmann & Pääbo, 2002). Therefore, thesearch for gene and function novelties in human lin-eage should include our most distant ancestors, that is,the Anthropoid primates (Goodman, 1999).

Estimating the speciation dates is obviously thefirst task to address. Fossil record and genetic datahave provided vital, but sometimes differing conclu-sions. The scope of this review is not to discuss,and certainly not argue for or against the calibrationof the pre-mentioned approaches. I refer thereforeto the most recent statistical methods that have usedthe model of local molecular clocks (Yoder & Yang,2000) to propose an estimation of the divergence datesthat fit with fossil calibration and I consider not toocontroversial (Figure 1). Some authors (Goodman,1999; Gagneux & Varki, 2001) proposed to in-clude modern humans (Homo sapiens) and chimpan-zees (Pan paniscus andPan troglodytes) in the samegenus (Homo) with a common ancestor living about4–6 million years ago (Mya). A divergence time of

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Figure 2. Evolution of chromosomes equivalent to human chromosome 5 (HSA5). The symbols correspond to chromosomal rearrangementsas indicated. The species three-letter code is given in Dutrillaux (1979).

approximately 7–9 Mya is accepted for separation ofgorilla (Gorilla) and human/chimpanzee clades. Anusual estimate of 14 Mya for the divergence of orang-utan (Pongo) and African Apes is considered herewhile recent studies put back this time up to 20.5 Mya(Penny, Murray-McIntosh & Hendy, 1998). Gibbons(Hylobates) form parts of the Hominoidea superfam-ily together with human, Great Apes and gorilla.Gibbon-lineage divergence took place about 18 Mya.The Old World monkeys (Cercopithecoidea) includemany primate species, the most studied being baboons(Papio), mandrills (Mandrillus) and Cercopitheques(Cercopithecus), mainly found in Africa as well asmacaques (Macaca), predominant in Asia. Divergencedate for Hominoids and Cercopithecoidea was es-timated to 25 Mya, albeit a shift to 30–40 Mya wasalso proposed (Yoder & Yang, 2000). Finally, simian(Anthropoids) origin remains largely debated both interms of timing, 35 Mya versus 55 Mya, and place,Africa or Asia (Martin, 1993; Kay, Ross & Williams,1997).

Although it may seem esoteric in the context ofgenome evolution, the debate regarding the calibrationof divergence times for primate speciation is of prime

importance when considering the adaptive changessuch as climate and diatery variations that apparentlyhave shaped the hominid origins (Andrews, 1992;Gibbons, 2002a,b). Positive darwinian selection atthe molecular level has now been extensively docu-mented in primate genomes (Messier & Stewart, 1997;Wyckoff, Wang & Wu, 2000; Johnson et al., 2001;Schaner et al., 2001; Ding et al., 2002). Obviously,the relationship between these molecular changes andselection of function and/or speciation remains elu-sive. However, establishing a link between genomeevolution, Anthropoid speciation and environmentvariations require accurate age estimate.

Chromosomal rearrangements

Comparative chromosome banding analysis has re-vealed a wide variety of rearrangements in prim-ates including translocations, insertions/deletions,pericentric inversions and modifications of hetero-chromatin, as illustrated during evolution of the hu-man chromosome 5 (Figure 2). Chromosome fissionsamong Cercopithecoidea and pericentric inversions in

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Hominoidea were dominant. Interestingly, reconstit-ution of the karyotype of the last common ancestorof Hominoidea and Cercopithecoidea indicated thatorangutan carry the closest karyotype to this of thisextinct primate (Dutrillaux, 1979). Furthermore, while18 of the 23 pairs of chromosomes are very close inhumans and Apes, chromosomes X, 4, 9 and 12 con-tained large segments that have been reshuffled afterchimpanzee speciation (Hacia, 2001).

Molecular cytogenetics analysis using chromo-some painting technique confirmed most of the re-arrangements suggested by chromosome banding andpointed out to particular inter- and intrachromosomalrearrangements. Indeed, the well-described fusion-origin of human chromosome 2 and the reciprocaltranslocation in the gorilla between chromosomes ho-mologous to human chromosomes 5 and 17 wereestablished (Hacia, 2001). Numerous translocationswere mapped in hylobatid karyotypes and found as-sociated with a high rate of chromosome evolution bycomparison with other Catarrhini species (Jauch et al.,1992). Recently, the use of subchromosomal probesallowed to identify new intrachromosomal rearrange-ments and showed extensive disturbance of gene orderin the well-known syntenic block, such as the chromo-some 3/chromosome 21 synteny (Müller et al., 2000).

Only two molecular mechanisms of gene duplic-ation were originally associated with the emergenceof new genes during evolution: tandem duplicationof ‘template’ gene and whole-genome duplication(polypoidy) (reviewed by Lundin, 1993; Lynch &Conery, 2000). However, combinatorial analysis ofFISH studies and sequence data from the Human Gen-ome Project revealed the existence of a new genomicduplication, that is, the so-called segmental duplica-tion. Based on the seminal works by J. Korenberg,J. Lupski and E. Eichler laboratories, it is now wellrecognized that these large, nearly identical genomicsegments are involved both in chromosomal instabilityin humans and in shaping the primate genome (re-viewed by Eichler, 2001; Emanuel & Shaikh, 2001;Samonte & Eichler, 2002; Stankiewicz & Lupski,2002). The segmental duplications possess strikingfeatures:

(1) They represent about 5% of the whole gen-ome; this high proportion of recent duplicationsclearly distinguishes human genome from othersequenced genomes.

(2) They involve blocks of DNA ranging in size fromone to several hundred kb that share very high se-

quence identities (90–100%) and that are localizedto a subset of human chromosomes.

(3) They can be divided into interchromosomal du-plications that represent the largest part of thesegmental duplications found located in peri-centromeric and subtelomeric regions and intra-chromosomal duplications that comprise complexmosaic of near identical segments, also called lowcopy repeat (LCR) sequences, conspicuously in-volved in recurrent rearrangements associated withhuman diseases.

(4) The smallest genomic module that constitutesLCR, also named ‘duplicon’ (Eichler, 1998),is composed of fragments of unrelated non-processed genes. This essential feature of du-plicons distinguishes them from other classes ofrepetitive sequences in the human genome.

(5) A major consequence of the juxtaposition of vari-ous exonic–intronic sequences is that new fu-sion/chimeric transcripts may be produced. In thecase of chimeric mRNA carrying functional open-reading frame (ORF), it might be a powerful wayto promote domain accretion and to enhance pro-teome complexity. Indeed, several examples ofchimeric transcript genes have been documentedin the last 2 years (see below).

Gene novelties through DNA-mediatedmechanisms

There is compelling evidence that duplication and lossof gene mechanisms (the ‘birth-and-death’ process;Nei, Gu & Sitnikova, 1997) have been major driv-ing forces in shaping the eukaryotic genomes (Lynch& Conery, 2000). Sequence analysis of the humangenome confirmed the importance of the process ofgene duplication and revealed that the major differencebetween human and other eukaryotes resides withinthe increased complexity of building block of pro-tein modules rather than the number of genesperse. Several examples of gene families that appeareddifferently amplified in primate genomes have beenreported (reviewed in Gagneux & Varki, 2001) but alink between gene/module expansion and functionaldifferences among primates remained elusive.

The acquisition of a phenotypic trait such as thetrichromatic color vision represented a major im-provement for detecting yellow/red fruits or leaves(Moffat, 2002). This fundamental event happenedabout 35 Mya in a common ancestor of Old World

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monkeys and apes (Jacobs et al., 1996). Cattarhinimonkeys and Hominoids carry an autosomal gene thatencodes the blue-sensitive pigment as well as dupli-cated genes on X chromosome that encode the redand green-sensitive pigments (Nathans, Thomas &Hogness, 1986). The X-linked genes arose from a geneduplication after divergence of New World monkeys.These primates possess, but howler monkeys, a singleX-linked gene and one autosomal color pigment gene(opsin) (Boissinot et al., 1998).

Most of the genes encoding proteins involved insperm formation, playing a role in male–female mat-ing or human germ cell development, evolved rapidlyin primate lineages. Generally, this fast evolution pro-cess operated under positive darwinian selection andparticipated likely in the recent evolution of male orfemale reproductive traits. However, the genetic basisfor these adaptive evolution processes did not involvegene noveltiesper se (Ulvsbäck & Lundwall, 1997;Wyckoff, Wang & Wu, 2000; Klonisch et al., 2001;reviewed in Hacia, 2001). In contrast, phylogeneticanalyses of the DAZ gene family indicated that newfunctions arose with duplicated genes during verte-brate evolution (Xu, Moore & Pera, 2001). Indeed, theancestral human BOULE gene (Boule gene in flies)performs an essential meiotic function, the duplicatedgene DAZL regulates early germ cell development andthe lately evolved DAZ genes are still genes ‘in searchof function’ in human testis.

Exon shuffling through illegitimate recombin-ation (Bergen et al., 1998), LINE-1 mediatedretrotransposition (see below; Moran, DeBarardinis &Kazazian Jr., 1999) and segmental duplication expan-sion (Samonte & Eichler, 2002) provided powerfulmechanisms to generate chimeric mRNA-encodinggenes in primates. A spectacular example of gene fu-sion through a new mechanism concerns the humanKua-UEV gene (Long, 2000; Thomson et al., 2000). InCaenorablitis elegans andDrosophila melanogaster,a single UEV gene encoding proteins involved inprotein ubiquitination or error-free DNA repair wasfound, whereas inHomo sapiens two genes (UEV1andUEV2) were mapped onto distinct chromosomes.Strikingly, UEV1 gene was found associated on chro-mosome 20 with a hitherto unknown gene, namedKua to produce a fused transcript only in humans.This transcript may potentially encode a fused pro-tein with theKua domain at its amino-terminus andtheUEV domain at its carboxy terminus. The genera-tion of theKua-UEV chimeric transcript would resultfrom combination of transcriptional readthrough at the

termination site of theKua gene and alternative spli-cing to skip exon 6 of theKUA gene (containing thestop codon) and exon A of theUEV gene (containingthe translation initiation codon). This complex mech-anism led to the production of a new fused protein,likely involved in an emerging function in primates.

Conversely, loss of gene functions may have alsocrucial impacts on the appearance of new phenotypes.In this context, a human-specific exon deletion in theCMP-sialic acid hydroxylase gene led to a drasticreduction in the synthesis ofN-glycolylneuraminicacid (Neu 5Gc) in human tissues, while this moleculeis very abundant in all organs, except the brain, ofother mammals, including Great Apes (Chou et al.,1998; Gagneux & Varki, 2001). Interestingly, be-sides the well-known example of reduction of fetalγ-globin genes to one in New World monkeys (Chiuet al., 1996), reduction of functions such as smell anddietary selection were found associated respectivelywith the selective loss in different Antropoid lineagesof functional genes encoding the olfatory receptors(Rouquier, Blancher & Giorgi, 2000) and with a de-crease of mitochondrial targeting of glyoxylate aminotransferase (GAT) (Holbrook et al., 2000). Theseexamples illustrated molecular adaptations as a con-sequence of positive selection pressure, a process wewill find also involved when emergence of new genefamilies will be examined (see below).

Gene novelties through RNA mediatedmechanisms

In vertebrate genomes the majority of middle inter-spersed repetitive elements was generated by reversetranscription of an RNA molecule (small non-codingRNA or protein coding mRNA) into a DNA copyand integration throughout the genome (reviewedin Kazazian Jr. & Moran, 1998; Brosius, 1999;Nekrutenko & Li, 2001). While most of these ele-ments are functionally inactive, the retroposition pro-cess may lead to active genes when retroelements(also named retronuons; Brosius & Gould, 1992)recruit flanking sequences at the insertion site andeither become part of a fusion (or chimeric) mRNAor contribute to build up a new promoter region.

The human major histocompatibility complex(MHC) class I and II regions represent classical ex-amples of genome plasticity due to multiple retroele-ment integrations during primate evolution (Hughes,1995; Andersson et al., 1998). Furthermore, long

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terminal repeats (LTRs) of human endogenous retro-viruses (HERVs) were found involved into the tran-scriptional activation of adjacent genes. For instance,the parotid-specific expression of the amylase gene inprimates results from the presence of a HERV-LTR inthe promoter region of the amylase gene (Emi et al.,1988; Samuelson et al., 1988; Samuelson, Phillips &Swanberg, 1996).

BC200 RNA is a representative example of anAlu sequence-derived retrogene that emerges duringprimate evolution. The founder Alu-element has beenexapted 35–55 Mya as a gene encoding a small RNAexpressed in the brain and involved likely in the trans-lational regulation of dendritic protein biosynthesis(reviewed by Brosius, 1999).

Alu-sequences are rarely found in protein-codingexons but are rather common within the non-codingregions of mRNAs and could play a role in mRNAstability and/or translational efficiencies (reviewed inBatzer & Deininger, 2002). Because Alu sequencecontained regions of high sequence identities withthe donor and acceptor splice sites, the insertionof such element in introns of protein-coding genesmay also contribute to alter the normal mRNA splic-ing and protein expression (reviewed in Mighell,Markham & Robinson, 1997). Furthermore, Alu-sequence retroposition could influence transcriptionof their neighboring genes when inserted into reg-ulatory regions. Indeed, several examples of Aluelements in promoter regions with dramatic impacton gene transcription control have been now docu-mented (reviewed in Mighell, Markham & Robinson,1997; Batzer & Deininger, 2002). A recent reportaddressed the issue of the tissue-specific expressionof ABH antigenes in primates and revealed the pres-ence of an Alu-Y element in a critical regulatorysequence of intron 1 of theα2-fucosyltransferase H(FUT1) gene only in Anthropoid apes and humans(Apoil et al., 2000). The insertion took place atthe time of human and African apes divergence andcould be therefore the founder event that allowed re-stricted expression of ABH antigenes on red cells inhominids.

Processed genes made by retroposition of mes-senger RNA from RNA polymerase II-transcribedgenes represent 10,000–20,000 copies in the humangenome (Gonçalves, Duret & Mouchiroud, 2000;IHGSC, 2001; Venter et al., 2001). Many of theseprocessed genes are inactive due to integration into‘silent’ transcriptional region or have accumulatedmutations, including missense/nonsense substitutions

or insertion/deletions, that preclude translation of theoriginal coding region. They represent overt pseudo-genes. However, part of these retrogenes are transcrip-tionally active. These genes often exhibit differentpatterns of expression than the founder gene or evenlead to emergence of a new function by exaptation andsecondary selection (Brosius & Gould, 1992). Thereare accumulating examples of human ‘expressed’ pro-cessed retrogenes and I recommend to consult thereview of Brosius (1999) and appended web site.I would mention here two representative examples,the Phosphoglycerate kinase 2 (Pgk-2) gene and thePhosphoglycerate mutase processed gene (PGAM3).Historically, Pgk-2 was one of the first examples ofintronless retrogenes that maintained the original openreading frame (ORF) with similar function but dis-played a testis-specific expression, different from theconstitutive expression of the parental gene (McCarrey& Thomas, 1987; McCarrey et al., 1996). Con-versely,PGAM3 was originally described as a human-specific pseudogene without functionality (Dierick,Mercer & Glover, 1997). Very recently, Betrán et al.(2002) reported a strong positive selection at theprotein-coding level acting onPGAM3 during prim-ate evolution and revealed gene expression in hu-man leukocytes suggesting therefore thatPGAM3 isunder rapid fixation, probably corresponding to anexaptation.

The retroposition of RNA sequences requires acellular production of reverse transcriptase. The mostobvious source in mammalian cells is the ORF2 pro-tein of L1 (LINE-1) elements that contains an en-donuclease domain (EN) in its N-terminus and areverse transcriptase (RT) domain in its C-terminus.Furthermore, the proposed mechanisms of L1 retro-transposition may represent a general model to un-derstand the Alu or mRNA retroposition processes(Kazazian Jr. & Moran, 1998). Indeed, although bind-ing of ORF2 protein displays acis preference to theRNA of the template L1 element, there is strongevidence thattrans-complementation may occur withAlu or messenger RNAs (Boeke, 1997; Dhellin,Maestre & Heidmann, 1997; Esnault, Maestre &Heidmann, 2000). Interestingly, a sub family of L1elements (T-1) were found arising 4 Mya and accu-mulating in human (Boissinot, Chevret & Furano,2000), suggesting that L1 retrotransposition is stillan ongoing process that contributes to both shapingthe human genome and producing endogenome sourceof reverse-transcriptase to help retroposition of otherpoly A-containing RNAs.

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Molecular evolution of PMCHL1/PMCHL2 genes

A powerful and rational approach to investigate theissue of the emergence of novel genes is the identi-fication and careful characterization of a gene, youngenough to have kept the trace of the early events thatcontribute to its birth in a restricted species lineage(Long, 2001). In looking for the evolutionary historyof a neuropeptide-encoding gene, namedMelanin-concentrating hormone (MCH) gene, we were luckyenough to discover such a ‘Holy Grail’ in the primategenomes.

MCH is a cyclic peptide expressed predomi-nantly in the lateral hypothalamus and zona incertaof mammalian brains (Nahon et al., 1989; Bittencourtet al., 1992). This neuropeptide acts as neurotrans-mitter/neuromodulator and regulates a broad array offunctions (reviewed in Nahon, 1994). Data of in-tracerebral injections of MCH in the rat or mousebrains, joined to analysis of gene expression patternsin obese and lean animals and transgenic mouse mod-els lacking or over-expressing theMCH gene or one ofits receptor (MCH1 or SLC1), have revealed a majorrole for MCH in the regulation of feeding behaviorand energy homeostasis (Qu et al., 1996; Shimadaet al., 1998; Marsh et al., 2002; reviewed in Tritos &Maratos-Flier, 1999). A singleMCH gene was foundin rat and mouse genomes (Thompson & Watson,1990; Breton et al., 1993a) and the human ortholog-ous gene was mapped to chromosome 12q24 (Vialeet al., 1997). The authenticMCH gene has a character-istic three exons–two introns structure with the MCHsequence overlapping exon II and III (Figure 3). Inthe mid-1990s, we identified two other loci on chro-mosome 5p14 and 5q13,PMCHL1 and PMCHL2,respectively, that encompassed a truncated versionof the genuineMCH gene, we named at this timethe variantMCH gene (Breton, Schorpp & Nahon,1993b; Pedeutour, Szpirer & Nahon, 1994). This geneexhibited several intriguing features: (1) It was com-posed of exon II–intron B–exon III and lacks exon I(Figure 3(B)). (2) It displayed 92–95% sequence iden-tity with the humanMCH gene, suggesting a recentevolutionary story. (3) It showed a pattern of expres-sion in the human brain that was markedly differentfrom this of the genuineMCH gene. Further functionalanalysis (Miller, Burmiester & Thompson, 1998; Vialeet al., 1998, 2000) demonstrated that both sense andantisense unspliced RNAs from thePMCHL1 genewere transcribed in various areas of the developing hu-man brain. Conversely, no transcript of thePMCHL2

gene could be identified in the human brain. Mostinterestingly, the brain-expressedPMCHL1 gene en-coded a putative protein of 8 kDa with a nucleus-localization signal (NLS) in its N-terminus, (Vialeet al., 2000) (Figure 3(B)).

When (and how) did a neuropeptide-encodinggene, the authenticMCH gene, switch to a gene en-coding a putative nuclear protein? Reconstitution ofthe evolutionary history of the variantMCH genepointed out a complex scenario. The first phylogen-etic studies (Viale et al., 1998) suggested that thePMCHL1 gene arose during primate evolution by afounder event of transposition/truncation from the an-cestral chromosome 12 to the ancestral chromosome5p at the time ofCatarrhini and Platyrrhini diver-gence, that is, 25–35Mya. Then, a duplication in acommon ancestor of Hominids, i.e. 5–10 Mya, pro-ducedPMCHL2 gene on the chromosome 5q13. Thisattractive evolutionary model turned out to be toosimplistic. Parallel studies on theMCH gene expres-sion regulation in pheochromocytoma (PC12) cells(Presse et al., 1997) shed light on the presence of nat-ural antisense RNAs to theMCH gene transcripts. Theexpression of both sense and antisense MCH RNAsappeared tightly regulated in PC12 cells and numer-ous rat tissues, including testis. Detailed structural andfunctional studies allowed the discovery of a complextranscriptional unit, we namedAROM for Antisense-RNA-Overlapping-MCH (Borsu, Presse & Nahon,2000), that may produce two classes ofMCH gene-antisense RNAs: (i) large spliced-variant mRNA initi-ated at cap sites 1 and 2 and encoding new RNA/DNAbinding proteins and (ii) short unspliced untranslatedRNAs beginning at cap sites 3–5 and strictly com-plementary to most of the MCH primary transcriptsequence (Figure 4(A)). Close inspection of the regionof insertion at thePMCHL1 locus (Figure 4(A)) re-vealed several features (including a poly A track andflanking short direct repeats) that could be explainedby the triggering event that allowed the emergence ofthe PMCHL1 gene was the retroposition of a shortunspliced RNA transcribed from theAROM gene ac-cording to the scheme proposed in Figure 4(B). Thisrepresents the first and, at the present time, uniqueexample of exon shuffling mediated by an antisenseRNA (Courseaux & Nahon, 2001). However, a recentsurvey of the human genome database has revealed theexistence of about 800 genes that could encode bothsense and antisense transcripts (Lehner et al., 2002).Therefore, it can be anticipated that more examples of‘human-specific’ genes that have been shaped through

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retroposition of antisense RNAs will be reported in thenear future.

We carried on the characterization ofPMCHL1/PMCHL2 genes and made additional discoveries(Courseaux & Nahon, 2001). First, by combininginsilico screening of ESTs, RACE-PCR and RT-PCR ex-periments, we identified a large spectrum of alternativespliced-variant mRNAs generated exclusively fromthePMCHL1 when found in the human brain or frombothPMCHL1 andPMCHL2 genes when found in thehuman testis (Figure 5(A)). Structure of the completePMCHL1/PMCHL2 gene was finally established bysequencing BACs covering the 5p14 and 5q13 regionsand including the novel exons and introns mapped inthe 3′ flanking region of the nested variantMCH gene(Figure 5(A)). Second, phylogenetic studies disclosedthat splice donor and acceptor sites between exons4 and 5 and polyadenylation signal sequences of theancestralPMCHL gene were created as a result ofnucleotide substitutions at the time of divergenceof Hominoids (Figure 5(B)). Finally, we establishedthat thePMCHL2 gene emerged later at the time ofdivergence of Hominids, that is, 5–10 Mya, throughpericentromeric transposition of a large genomic re-gion from 5p14 to 5q13. Overall, the moleculardissection of the structural features of thePMCHL1/PMCHL2 genes during primate evolution revealed anamazing degree of combinations of known but raregenetic events (de novo creation of splice sites) andunanticipated mechanisms (retroposition of antisensetranscripts and truncation event unmasking new ORF).This study casted doubt about comparative sequenceanalysis using algorithms aimed at predicting exon–intron structures from genome sequences. Indeed,none of the exon prediction programs described so farwere able to identify the complex organization of thePMCHL1/PMCHL2 genes.

Evolution of chimeric gene families at segmentalduplications: the ‘gene nursery’ hypothesisrevisited

As mentioned above, segmental duplications are likelyto be the sites of intensive genomic remodeling duringlate primate evolution that allow emergence of novelprotein-encoding genes by accretion of unrelated pro-tein domains. There is a growing list of segmentalduplications found in the human genome (Bailey et al.,2001) and I would recommend to refer to recentexhaustive reviews (Eichler, 1998, 2001; Ji et al.,

2000; Emanuel & Shaikh, 2001; Samonte & Eichler,2002; Stankiewicz & Lupski, 2002). I will focushere on the description of three representative ex-amples of emergence of gene families at segmentalduplications.

In a landmark paper published recently, Eichlerand collaborators (Johnson et al., 2001) described thestructure, evolution and expression of a segmentalduplication, termed LCR 16a. This duplication wasmapped on human chromosome 16 and comprised 15copies of a 20 kb-long duplicon that shared high levelof sequence identity in humans. Combination of FISHand sequence analysis studies revealed that the ge-nomes of Great Apes and humans displayed a dramaticexpansion of the LCR 16a duplicon. Conversely,a single or few copies could be identified in OldWorld monkeys. Even more striking was the discoverythat the coding exons of the human paralogous andhominid orthologous were hypervariable (10% nucle-otide divergence) and that a peak of positive selectionoccurred at the time of divergence of Pongidae andHominidae lineage. The functional relevance of thisextraordinary enhancement of amino-acids replace-ment is presently unknown. However, at least onemember of these gene families would encode a proteinassociated with the nuclear pore complex, a subcel-lular localization that appears reminiscent of this ofanother putative ‘human-specific’ protein, namely theVMCH-p8 derived from thePMCHL1 gene (Vialeet al., 2000).

A global analysis of segmental duplications onhuman chromosome 22 was recently performedand allowed the identification of 11 novel mosaicgenes (Bailey et al., 2002). An intriguing featureof these segmental duplications was the discovery

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 3. Structures of the human ‘authentic’MCH gene (A) andthe ‘variant’ MCH gene (B). Position of exons (Exon I–III; ExIIv–Exon IIIv) and introns (Intron A and B; Intron Bv) are indicated.mRNAs and proteins generated from theMCH genes are shown(Start: position of the translation initiation codon; Stop: positionof the translation termination codon).

Figure 4. Model of antisense MCH RNA retroposition. (A)Exon–intron structures of theMCH, AROM and PMCHL1 genes.Sense and antisense DNA strands are shown at theMCH/AROMlocus andPMCHL1 locus. CS 1–5: cap sites 1–5 of theAROMgene; poly A 1–3: polyadenylation sites 1–3 of theAROM gene.Colored dot lines indicate the boundaries of regions of homologyamongMCH and PMCHL1 genes (from Borsu, Presse & Nahon,2000). (B) A model for the birth of the variantMCH gene (fromCourseaux & Nahon, 2001).

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Figure 3

Figure 4

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Figure 5. Structure and evolution of thePMCHL1 andPMCHL2 genes. (A) Structure of thePMCHL1/PMCHL2 genes with combinations ofalternative splicing found in human brain and testis. The extend of the nested variantMCH gene (including the retroposon shown in Figure 4) isindicated by a double-arrow. Exons and introns numbering are described in Courseaux and Nahon (2001). A 1–4: positions of polyadenylationsites 1–4. (B) A hypothetical model for the emergence ofPMCHL1 andPMCHL2 genes during primate evolution. AMCH/AROM gene-derivedsequence was inserted (together with one Alu sequence) by retroposition in an equivalent region of the human chromosome 5p at the timeof divergence of Cercopithecoidae (25–30 Mya). Subsequent mutations led to the formation of functional intron–exon splice junctions andpolyadenylation sites before divergence of Hylobatidae (18 Mya). Finally, a large block of chromosome 5p14, encompassing thePMCHL1gene, was duplicated on chromosome 5q13 at the time of divergence of Hominidae, 5–10 Mya (from Courseaux & Nahon, 2001).

Figure 6. Gene organization at the SMA locus on chromosome 5q13. YAC contigs spanning the 5q13 region and location of genes (SMN1and 2, NAIP, BFT2p44t and c,. . .), ‘expressed’ pseudogenes or chimeric genes (PMCHL2, gene 1/6, gene 1/4, gene 2/5-10,. . .) and repetitiveDNA sequences (MW3) are shown. The centromeric (Cent.) and telomeric (Tel.) copy of duplication at the SMA locus is indicated in red (fromLewin, 1995; Courseaux et al., 2003).

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Figure 7. Hypothetical model for the emergence of ‘human-specific’ genes in the Antropoid lineage. Gene duplications at intermediate ac-cceptor loci (sub-telomeric and pericentromeric regions, in yellow) as well as RNA retropositions at ‘gene nurseries’ (or other loci; dottedlines) occurred in an ancestor to Catarrhini (25–30 Mya). Intrachromosomal duplications, duplicon expansion (partial, dotted [ ]; complete, [ ])and pericentromeric translocation operated in an ancestor to Hominidae, 5–10 Mya.

of a gradient of pericentromeric duplications, themost recent localized close to the centromere. Thechimeric genes arose from both inter- and intra-chromosomal duplications and they were generatedthrough very different mechanisms, including in-tact gene duplication and conservation with slightchanges in the original protein sequence, par-tial duplication with extensive modifications ofthe template gene and full mosaicism of exonsfrom different donor genes. Extrapolation of thesedata to the entire genome predicts, in the most con-servative hypothesis (Bailey et al., 2002), that hu-man and chimpanzee would differ by about 150–350genes.

Proximal spinal muscular atrophy (SMA) is anautosomal recessive lethal childhood neuropathy inits most severe form due to deletions/mutations in

the telomeric copy of thesurvival motor neuron gene(Lefebvre et al., 1995). The twoSMN genes (SMN1,telomeric copy;SM2, centromeric copy) are carriedby an inverted repeat element of about 500 kb thatmaps to chromosome 5q13 (Lewin, 1995; Figure 6).Other functional genes were also found in this invertedduplication, such as genes encoding the transcriptionfactor BFT2p44 and NAIP. Interestingly,PMCHL2gene was assigned to chromosome 5q13.3 region,that is, on the telomeric side of the critical regionof the SMA locus (Figure 6). Numerous ‘expressed’pseudogenes have been found within the SMA locusinterval (Thompson et al., 1995). These genomicelements contained exons–introns cassettes highly ho-mologous to functional genes located on differentchromosomes. We recently investigated the structure,chromosomal organization and evolution of three of

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these atypical genomic sequences, namely gene 1/6and gene 1/4 which encoded two untranslated RNAsand gene 2/5-10 that carried putative functional ORFs(Courseaux et al., 2003; see Figure 6). Clusters ofgene 1/6, gene 1/4 and gene 2/5-10 were localizedon chromosome 5p14 and 5q13, in close proximityto PMCHL1 andPMCHL2 genes, respectively. Poly-morphism in number and structural organization wasfound among humans and could contribute to the dif-ferences in chromosomal rearrangements associatedwith the gradual severity of SMA. Phylogenetic stud-ies suggested that all three genes appeared at the 5p14locus at the time ofCatarrhini divergence 25–30 Myaand then duplicated at the 5q13 locus in the hominidlineage, in close agreement with the evolutionary his-tory of thePMCHL1 andPMCHL2 genes. However,a marked difference in the number of the gene 1 andgene 2-derived copies was observed among hominids,indicating lineage-specific expansion events. We arecurrently testing whether positive selection have op-erated on the protein coding gene 2/5-10 family thatcould be indicative of functional exaptation.

On the basis of accumulating data on segmentalduplications and retroposons as described above, Ipropose that ‘human-specific’ genes arose duringprimate evolution according to a general two-stepprocess (Figure 7). First, transposition events me-diated through DNA or RNA sequences took placelikely between the divergence of the Platyrrhini andCatarrhini, 25–30Mya. At this step, RNA retroposonscould have been inserted directly in core chromosomalregions (gene nurseries) or been targeted to gene-richsubtelomeric and pericentromeric regions. These re-gions (named here as intermediate acceptor loci inFigure 7) served also as safe haven for duplicatedDNA sequences and be considered as gene remodelinglaboratory where ‘genic tinkering’ could operate. In-deed, striking features of these acceptor loci are thevery rapid turnover of inserted sequences on theseregions and their capabilities to generate genomicfragments that transpose further throughout the ge-nome. Second, intrachromosomal duplications arosemainly at the time of divergence of Hominidae, that is,5–10 Mya, with concomitant or subsequent species-specific spreading of the duplicons. Obviously, pri-mate genomes did not remain static in betweenthese two periods of major genomic alterations butchanges concerned mainly nucleotide mutations andlocal chromosome rearrangements when consideringa single chromosome. As pointed out by Dubouleand Wilkins (1998) “evolution is neither inherently

gradualistic nor punctuational but progresses from oneextreme to the other”. The molecular evolutionarymodel I suggest here fits perfectly with the conceptof ‘transitionism’ put forward by these authors.

Concluding remarks

Over the past 5 years, the concept of ‘young’ species-specific gene has progressively been recognized asvalid with now several well-documented examples inDrosophila and rodents (Long, 2000). In the context ofhuman evolution, the search for the small number ofgenes that could have evolutionary differentiated earlyhominids and modern human from other primates was,and still remains, hampered by our inability to geta direct access to the genomic information stored inthe bones of our extinct ancestors. Nevertheless, asshown above, candidate ‘human-specific’ genes havebeen identified by comparing primate and human gen-omes. At this point, it should be kept in mind thatseveral specific human features depend more on altera-tion of gene expression, whether they are quantitative,spatial (tissue-specific expression) or temporal (het-erochrony), than on the recent acquisition of human-specific genes. Thus, mankind had not obligatorily torely on duplication of coding sequences, and manyexamples exist of striking evolutionary changes thatwere promoted by just phenomenon of heterochronyfor example (Slack & Ruvkun, 1997; Klingenberg,1998). It remains that the discovery of ‘gene nursery’,places where genetic remodeling is very high, wouldbe an obvious source of new genes, the raw materialfor selection upon new adaptive constraints. Ongoingfunctional studies and distribution analysis in humanpopulation should shed light on the roles they couldhave played at the different stages of human evolution,from bipedism and tool designing until the recent ap-pearance of creative thinking, artistic expression andlanguage.

I described here different strategies that have beenused in order to tackle the ‘human-specific’ geneissue: large-scale comparison of primate and hu-man genomes (the Primate Genome Project), analysisof chromosomal rearrangements that operate only inprimates (segmental duplications) and detailed anal-ysis of a gene family that emerged during primateevolution (PMCHL1/PMCHL2 gene paradigm). Cer-tainly a promising way to find candidate genes inthe future will be to search for susceptibility genesfor human-specific diseases. Indeed, a gene named

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FOXP2 was recently identified by screening familieswhose affected members have severe deficit in ar-ticulation and language processing (Lai et al., 2001;Bishop, 2002). While this gene encodes a transcrip-tion factor that exhibits a strong conservation amongmammals, it contains two amino-acid changes fixedin human populations, that highly suggest strong se-lection during recent human evolution (Enard et al.,2002b). However, its expression does not appear to berestricted to the brain. Therefore, this gene does notcontrol language on its own (this would be very sur-prising) but should interact with other genes to regu-late crucial neuronal pathway involved in the languageacquisition. Furthermore, panic/phobic disorders inhumans appeared to be associated with an interstitialduplication at chromosome 15q24-26 (named DUP25)flanked by duplicons and encompassing several genesand ‘expressed’ pseudogenes (Gratacòs et al., 2001).Using genome-wide scanning methods, no gene hasyet been clearly found associated with major psy-chiatric disorders such as schizophrenia, bipolar dis-order, autism and diseases linked to food consumption(bulimiaor anorexia nervosa) (DeLisi et al., 2000).No doubt that once the ‘human-specific’ genes willbe further characterized in terms of tissue distribu-tion and functions, they will become obvious targetsfor investigations in the field of the neuropsychiatricdiseases.

A central question remains about the role ‘human-specific’ genes play in the expression of human char-acters that contribute to unique phenotypic traits. Atthe cellular level, the example ofMorpheus gene(Johnson et al., 2001) indicates that the protein en-coded by this ‘human-specific’ gene is preferentiallytargeted to the nucleus or associated with the scaf-fold and could play a role in gene transcription and/ormRNA processing/nuclear import–export. However,beside the selection of new coding sequences, othermechanisms may have played a role in human evo-lution. Indeed, the human genome sequencing projecthas revealed that the vast majority of the ‘transcrip-tome’ in the higher organisms is composed of non-protein-coding RNAs (IHGSC, 2001; Venter et al.,2001; Kapranov et al., 2002). The conceptual revolu-tion which now emerges predicts that phenotypic vari-ation in complex organisms will result essentially fromthe differential use of a set of ‘control’ untranslatedRNAs (Mattick & Gagen, 2001) rather than changesin the core proteome, considered now as fairly stable(Maslov & Sneppen, 2002). These ‘control’ RNAswill participate in a network control architecture that

will contribute to variability of phenotypes in com-plex eukaryotes. It is likely that most of the mosaicgenes created by retroposon insertion and/or dupliconexpansion during primate evolution would not encodeproteins. It is tempting to speculate therefore that theseuntranslated RNAs would operate as control elementsto synchronize developmental patterns during fetal lifeand/or complex integrative task in adulthood. Numer-ous examples of non-protein-coding RNAs with im-portant functions are now well documented (reviewedby Erdmann et al., 1999; Kelley & Kuroda, 2000;Storz, 2002) and included theXIST gene (involved infemale X-chromosome inactivation) and theH19 one(involved in tumor development), both being imprint-ed (Frevel, Hornberg & Reeve, 1999; Avner & Heard,2001). The evolution of complex organisms requiredthe appearance of sophistically regulatory mecha-nisms that allowed fine tuning of expression of well-conserved key stable molecules (proteins) involved inbasic functions such as gene transcription and signaltransduction. ‘Human-specific’ genes would encodetherefore few but essential multitasked proteins anda large spectrum of untranslated RNA, some partici-pating in the genome renewal and creation of noveltyby tinkering-driven mechanisms (Long, 2000) and/orcontrolling the cellular network architecture (Mattickand Gagen, 2001). If this hypothesis is correct, Ican predict that differences among primates in theexpression patterns of conserved mammalian genes(Enard et al., 2002a,b) would be under the control of‘human-specific’ genes.

Acknowledgements

I thank very much P. Vernier (UPR 2197-CNRS,Gif-sur-Yvette, France) for helpful discussions onthe manuscript and the reviewer for his insight-ful comments. I am grateful respectively to W.Ferlin and A. Patel (IPMC, Valbonne) for criti-cism in writing the review and to J. Kervellaand F. Aguilla for assistance in the prepara-tion of this manuscript. I apologize to the au-thors of uncited relevant articles that could havebeen missed. The works from my own laborat-ory were supported by grants from the AssociationFrançaise contre les Myopathies (AFM) (ASI 1996–1998) and the Centre National de la RechercheScientifique (CNRS), France (Programme OHLL,2002).

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