Downloaded from genome. cshlporg on June 20, 2011-Published by Cold Spring Harbor Laboratory Press Kaessmann from a"parental"source gene is reverse transcribed into a com- likely to be redundant) than gene copies arising from DNA-based plementary DNA copy, which is then inserted into the genome duplication mechanisms. Indeed, a number of new retrogenes (Fig. 1B). The enzymes necessary for retroposition(in particular with intriguing functions have been identified. Detailed analyses the reverse transcriptase)are encoded by different retrotranspos. of these retrogenes uncovered novel mechanisms underlying the able elements in different species. In mammals, LINE-1 retro- emergence of new gene functions. For example, analyses of young transposons provide the required enzymatic machinery(Mathias retrogenes in primates not only revealed that retrogenes have et al. 1991; Feng et al. 1996; Esnault et al. 2000). Given that the contributed to hominoid brain evolution, but dentified dif- resulting intronless retroposed gene copies (retrocopies) only ferent molecular levels at which new genes may adapt to new contain the parental exon information (i.e,, they usually lack pa- functions Namely, in addition to evolving new spatial expressic rental introns and core promoter sequences), retrocopies were long patterns relative to the parental source genes, the proteins encoded thought to be consigned to the scrapheap of genome evolution by these retrogenes evolved new biochemical properties (Burki and and were routinely labeled as"processed pseudogenes"(Mighell Kaessmann 2004)and/or subcellular localization patterns(Burki et al. 2000). However, after anecdotal findings of individual func- and Kaessmann 2004; Rosso et al. 2008a, b). The latter process, tional retrocopies(so-called retrogenes) in the 1980s and 1990s, a dubbed subcellular adaptation or rele n, could be estab- uprising number of retrogenes could be discovered with the ad- lished and generalized as a new trajectory for the evolution of new vent of the genomics era. Notably, detailed analyses of this strip- gene functions after these observations (Marques et al. 2008 ped-down type of new genes have revealed previously unknown Kaessmann et al. 2009) echanisms underlying the appearance of new genes and their Other interesting retrogenes have recently been unveiled that functions and demonstrated that new retrogenes have contributed exemplify the sometimes unexpected and curious pathways of to the appearance of lineage-specific phenotypic innovations evolutionary change. An example is a mouse retrocopy of a ribo- Kaessmann et al. 2009 somal protein gene(Rps23), of which there are hundreds in mammalian g Sources of regulatory elements retropseudogenes, consistent with the idea that duplication of The observation of numerous functional retrogenes in various these genes is usually redundant and/or is subject to dosage bal- genomes(detailed below) immediately raises the question of how ance constraints. Yet the Rps23 retrocopy evolved a completely w function, not by changes in the pr retrocopies can obtain regulatory sequences that allow them to by being transcribed from the reverse strand and the incorporation become transcribed-a precondition for gene functionality. Stud- es that sought to address this question uncovered various sources of sequences flanking its insertion site as new(coding and of retrogene promoters and regulators and therefore also provided oding) exons(Zhang et al. 2009). This gave rise to a new protein general insights into how new genes can acquire promoters and (completely unrelated to that encoded by its parental gene), which had profound functional implications in that it conferred in- evolve new expression patterns(Kaessmann et al. 2009) First, it creased resistance in mice against the formation of Alzheimer- was shown that the expression of new retrogenes often benefits from preexisting regulatory machinery and expression capacities causing amyloid plaques. of genes in their vicinity. Thus, retrogenes profited from the open illustrates the far-reaching and immediate phenotypic conse. hearby genes, directly fused to host genes into which they inserted quences a retroduplication event may have. Parker et al.(2009) found that a retrocopy derived from a growth factor gene(fgf4)is (also see below), or captured bidirectional promoters of genes solely responsible for the short-legged phenotype characteristic of in their proximity (Vinckenbosch et al. 2006; Fablet et al. 2009; everal common dog breeds. Remarkably, the phenotypic impact Kaessmann et al. 2009). Second, retrogenes recruited CpG di- of the fsf 4 retrogene seems to be a rather direct consequence of the nucleotide-enriched proto-promoter sequences in their genomic vicinity not previously associated with other genes for their tran- FGF4 expression during bone development), given that its coding et al. 2009). Fourth, unexpectedly, retrogenes also seem to fre- immediately lead to phenotypic innovation (in this case o r s of of retrocopy insertion sites were shown to have provided retro- sequence is identical to that of its parental gene. The analy genes with regulatory potential(Zaiss and Kloetzel 1999; Fablet morphological trait)merely thro parental transcripts that gave rise to them(Okamura and Nakai 2008; Kaessmann et al. 2009). Finally, basic retrogene promoters Retrogenes and meiotic sex chromosome inactivation may sometimes have evolved de novo through small substitu- tional changes under the influence of natural selection(Betran and Numerous other illuminating cases of retrogenes known to 2007), Remarkably, the process of promoter and flies to plants have recently been described (for review, se lated exon-intron structure Kaessmann et al. 2009). However, global surveys of retroposition distances between the recruited promoters and retrogene insertion conducted in mammals and fruit flies have also identified a com- sites(Fablet et al. 2009) mon theme uniting a significant subset of new retrogenes in these species: expression and functionality in testes. while these retro- genes seem to have evolved a variety of functional roles(a process New retrogene functions hat may have a mechanistic basis and was likely influenced by Given that retrocopies usually need to acquire regulatory elements sexual selection, see below), the functions of a disproportionately for their transcription, retrocopies that eventually do become high number among them are apparen associated with the transcribed-a surprisingly frequent event (Vinckenbosch et al. transcriptional inactivation of the sex chromosomes in the male 2006) -are much more prone to evolve novel functions(and less germline during and(to a lesser extent)after meiosis (Turner 2007) 1316 Genomefrom a ‘‘parental’’ source gene is reverse transcribed into a com￾plementary DNA copy, which is then inserted into the genome (Fig. 1B). The enzymes necessary for retroposition (in particular the reverse transcriptase) are encoded by different retrotranspos￾able elements in different species. In mammals, LINE-1 retro￾transposons provide the required enzymatic machinery (Mathias et al. 1991; Feng et al. 1996; Esnault et al. 2000). Given that the resulting intronless retroposed gene copies (retrocopies) only contain the parental exon information (i.e., they usually lack pa￾rental introns and core promoter sequences), retrocopies were long thought to be consigned to the scrapheap of genome evolution and were routinely labeled as ‘‘processed pseudogenes’’ (Mighell et al. 2000). However, after anecdotal findings of individual func￾tional retrocopies (so-called retrogenes) in the 1980s and 1990s, a surprising number of retrogenes could be discovered with the ad￾vent of the genomics era. Notably, detailed analyses of this strip￾ped-down type of new genes have revealed previously unknown mechanisms underlying the appearance of new genes and their functions and demonstrated that new retrogenes have contributed to the appearance of lineage-specific phenotypic innovations (Kaessmann et al. 2009). Sources of regulatory elements The observation of numerous functional retrogenes in various genomes (detailed below) immediately raises the question of how retrocopies can obtain regulatory sequences that allow them to become transcribed—a precondition for gene functionality. Stud￾ies that sought to address this question uncovered various sources of retrogene promoters and regulators and therefore also provided general insights into how new genes can acquire promoters and evolve new expression patterns (Kaessmann et al. 2009). First, it was shown that the expression of new retrogenes often benefits from preexisting regulatory machinery and expression capacities of genes in their vicinity. Thus, retrogenes profited from the open chromatin state and accessory regulators (enhancers/silencers) of nearby genes, directly fused to host genes into which they inserted (also see below), or captured bidirectional promoters of genes in their proximity (Vinckenbosch et al. 2006; Fablet et al. 2009; Kaessmann et al. 2009). Second, retrogenes recruited CpG di￾nucleotide-enriched proto-promoter sequences in their genomic vicinity not previously associated with other genes for their tran￾scription (Fablet et al. 2009). Third, retrotransposons upstream of retrocopy insertion sites were shown to have provided retro￾genes with regulatory potential (Zaiss and Kloetzel 1999; Fablet et al. 2009). Fourth, unexpectedly, retrogenes also seem to fre￾quently have directly inherited alternative promoters embedded in parental transcripts that gave rise to them (Okamura and Nakai 2008; Kaessmann et al. 2009). Finally, basic retrogene promoters may sometimes have evolved de novo through small substitu￾tional changes under the influence of natural selection (Betran and Long 2003; Bai et al. 2007). Remarkably, the process of promoter acquisition sometimes involved the evolution of new 59 untrans￾lated exon–intron structures, which span the often substantial distances between the recruited promoters and retrogene insertion sites (Fablet et al. 2009). New retrogene functions Given that retrocopies usually need to acquire regulatory elements for their transcription, retrocopies that eventually do become transcribed—a surprisingly frequent event (Vinckenbosch et al. 2006)—are much more prone to evolve novel functions (and less likely to be redundant) than gene copies arising from DNA-based duplication mechanisms. Indeed, a number of new retrogenes with intriguing functions have been identified. Detailed analyses of these retrogenes uncovered novel mechanisms underlying the emergence of new gene functions. For example, analyses of young retrogenes in primates not only revealed that retrogenes have contributed to hominoid brain evolution, but also identified dif￾ferent molecular levels at which new genes may adapt to new functions. Namely, in addition to evolving new spatial expression patterns relative to the parental source genes, the proteins encoded by these retrogenes evolved new biochemical properties (Burki and Kaessmann 2004) and/or subcellular localization patterns (Burki and Kaessmann 2004; Rosso et al. 2008a,b). The latter process, dubbed subcellular adaptation or relocalization, could be estab￾lished and generalized as a new trajectory for the evolution of new gene functions after these observations (Marques et al. 2008; Kaessmann et al. 2009). Other interesting retrogenes have recently been unveiled that exemplify the sometimes unexpected and curious pathways of evolutionary change. An example is a mouse retrocopy of a ribo￾somal protein gene (Rps23), of which there are hundreds in mammalian genomes and that usually represent nonfunctional retropseudogenes, consistent with the idea that duplication of these genes is usually redundant and/or is subject to dosage bal￾ance constraints. Yet the Rps23 retrocopy evolved a completely new function, not by changes in the protein-coding sequence, but by being transcribed from the reverse strand and the incorporation of sequences flanking its insertion site as new (coding and non￾coding) exons (Zhang et al. 2009). This gave rise to a new protein (completely unrelated to that encoded by its parental gene), which had profound functional implications in that it conferred in￾creased resistance in mice against the formation of Alzheimer￾causing amyloid plaques. Another intriguing recent case of new retrogene formation illustrates the far-reaching and immediate phenotypic conse￾quences a retroduplication event may have. Parker et al. (2009) found that a retrocopy derived from a growth factor gene (fgf4) is solely responsible for the short-legged phenotype characteristic of several common dog breeds. Remarkably, the phenotypic impact of the fgf4 retrogene seems to be a rather direct consequence of the gene dosage change associated with its emergence (i.e., increased FGF4 expression during bone development), given that its coding sequence is identical to that of its parental gene. The analysis of fgf4 in dogs thus strikingly illustrates that gene duplication can immediately lead to phenotypic innovation (in this case a new morphological trait) merely through gene dosage alterations. Retrogenes and meiotic sex chromosome inactivation Numerous other illuminating cases of retrogenes known to have evolved diverse functions in species ranging from primates and flies to plants have recently been described (for review, see Kaessmann et al. 2009). However, global surveys of retroposition conducted in mammals and fruit flies have also identified a com￾mon theme uniting a significant subset of new retrogenes in these species: expression and functionality in testes. While these retro￾genes seem to have evolved a variety of functional roles (a process that may have a mechanistic basis and was likely influenced by sexual selection, see below), the functions of a disproportionately high number among them are apparently associated with the transcriptional inactivation of the sex chromosomes in the male germline during and (to a lesser extent) after meiosis (Turner 2007). Kaessmann 1316 Genome Research www.genome.org Downloaded from genome.cshlp.org on June 20, 2011 - Published by Cold Spring Harbor Laboratory Press