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Genetic Comparisons Yield Insight Into the Evolution of Enamel Thickness During Human Evolutionq

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Genetic Comparisons Yield Insight Into the Evolution of Enamel Thickness During Human Evolutionq Journal of Human Evolution 73 (2014) 75e87 Contents lists available at ScienceDirect Journal of Human Evolution journal homepage: www.elsevier.com/locate/jhevol Genetic comparisons yield insight into the evolution of enamel thickness during human evolutionq Julie E. Horvath a,b,c,d, Gowri L. Ramachandran c, Olivier Fedrigo d, William J. Nielsen e, Courtney C. Babbitt d,e, Elizabeth M. St. Clair c, Lisa W. Pfefferle e, Jukka Jernvall f, Gregory A. Wray c,d,e, Christine E. Wall c,*,1 a North Carolina Museum of Natural Sciences, Nature Research Center, Raleigh, NC 27601, USA b Department of Biology, North Carolina Central University, Durham, NC 27707, USA c Department of Evolutionary Anthropology, Duke University, Durham, NC 27708, USA d Duke Institute for Genome Sciences and Policy, Duke University, Durham, NC 27708, USA e Department of Biology, Duke University, Durham, NC 27708, USA f Institute for Biotechnology, University of Helsinki, Helsinki, Finland article info abstract Article history: Enamel thickness varies substantially among extant hominoids and is a key trait with significance for Received 6 June 2013 interpreting dietary adaptation, life history trajectory, and phylogenetic relationships. There is a strong Accepted 9 January 2014 link in humans between enamel formation and mutations in the exons of the four genes that code for the Available online 5 May 2014 enamel matrix proteins and the associated protease. The evolution of thick enamel in humans may have included changes in the regulation of these genes during tooth development. The cis-regulatory region in Keywords: the 50 flank (upstream non-coding region) of MMP20, which codes for enamelysin, the predominant Primate comparative genomics protease active during enamel secretion, has previously been shown to be under strong positive selection MMP20 AMELX in the lineages leading to both humans and chimpanzees. Here we examine evidence for positive se- 0 fl 0 fl ENAM lection in the 5 ank and 3 ank of AMELX, AMBN, ENAM, and MMP20. We contrast the human sequence AMBN changes with other hominoids (chimpanzees, gorillas, orangutans, gibbons) and rhesus macaques (outgroup), a sample comprising a range of enamel thickness. We find no evidence for positive selection in the protein-coding regions of any of these genes. In contrast, we find strong evidence for positive selection in the 50 flank region of MMP20 and ENAM along the lineage leading to humans, and in both the 50 flank and 30 flank regions of MMP20 along the lineage leading to chimpanzees. We also identify pu- tative transcription factor binding sites overlapping some of the species-specific nucleotide sites and we refine which sections of the up- and downstream putative regulatory regions are most likely to harbor important changes. These non-coding changes and their potential for differential regulation by tran- scription factors known to regulate tooth development may offer insight into the mechanisms that allow for rapid evolutionary changes in enamel thickness across closely-related species, and contribute to our understanding of the enamel phenotype in hominoids. Ó 2014 Elsevier Ltd. All rights reserved. Introduction Abbreviations: UTR, untranslated region; kb, kilobase; DNA, deoxyribonucleic Thick enamel distinguishes modern humans from chimpanzees acid; mRNA, messenger ribonucleic acid; miRNA, micro ribonucleic acid; mya, and gorillas and has long been a central feature in discussions of the 0 fl 0 fl million years ago; 5 anking, upstream non-coding region; 3 anking, down- origins and evolution of humans (Molnar and Gantt, 1977; Martin, stream non-coding region; Gene names are italicized and are uppercase for humans (e.g., MMP20), but only the first letter is uppercase for mice or rats (e.g., Mmp20), 1983; Martin, 1985; Grine and Martin, 1988; Schwartz, 2000; Protein names follow the same naming convention for humans and mice, but are Teaford and Ungar, 2000; Ungar et al., 2006; Teaford, 2007). not italicized. Teeth are frequently preserved in the fossil record and enamel q Accession number KF509948 was generated for this manuscript. thickness has measurable links to tooth function and development, * Corresponding author. conferring a strong potential for variation in enamel thickness to E-mail address: [email protected] (C.E. Wall). 1 Department of Evolutionary Anthropology, Box 90383 Duke University, provide insights into the inter-relations among dietary adaptation, Durham, NC 27708, USA. phylogeny, behavioral ecology, and life history during human http://dx.doi.org/10.1016/j.jhevol.2014.01.005 0047-2484/Ó 2014 Elsevier Ltd. All rights reserved. 76 J.E. Horvath et al. / Journal of Human Evolution 73 (2014) 75e87 evolution (Dean, 1985, 1987; Smith, 1991, 1993, 1994; Smith and that disrupts mRNA splicing results in hypomineralized (i.e., weakly Tompkins, 1995; Dean, 2000; Dean, 2006; Lacruz et al., 2008; mineralized) and hypoplastic (i.e., thin) enamel, demonstrating Dean and Smith, 2009; Guatelli-Steinberg, 2009; Dean, 2010; that normal MMP20 expression is essential for proper enamel for- Schwartz, 2012). Our interest in the thick enamel phenotype of mation (Kim et al., 2005; Lu et al., 2008; Wright et al., 2009). The humans stems from our efforts to uncover the genetic basis for the role of MMP20 in enamel formation is likely to be shared across origin of diet-related traits in humans (Haygood et al., 2007; Babbitt mammals as mice lacking functional Mmp20 have hypoplastic and et al., 2011; Fedrigo et al., 2011; Pfefferle et al., 2011). A critical step hypomineralized enamel (Wright et al., 2009). Amelogenin, ame- in expanding our understanding of pattern and process in human loblastin, and enamelin are the major structural EMPs (Wright, evolution is to integrate genetic, comparative anatomical, and 2006) and are encoded by AMELX, AMBN, and ENAM, respectively. paleontological information. Enamel thickness is a trait that pro- Together, these proteins and their products account for about 97% vides an ideal opportunity to combine these types of data. of the enamel extracellular matrix prior to maturation (Fincham Comparative genomics is a powerful method to identify con- et al., 1991). Although it is evident from both genetic and geno- nections to phenotypic trait differences (for a review see Babbitt typeephenotype association studies on primate and mouse denti- et al., 2011), as well as functional elements (ENCODE et al., 2007; tions that tooth development involves the actions of many genes Margulies et al., 2007) and evolutionary changes in gene architec- (Jernvall and Thesleff, 2000; Hlusko et al., 2004; Hu et al., 2005), the ture (Horvath et al., 2011; Lindblad-Toh et al., 2011). Comparative genetic and phenotypic evidence suggests that the four genes methods can identify potential cases of positive selection in one investigated here are excellent candidates for beginning to under- species relative to two or more others, based on sequence changes stand the genetic underpinnings of the evolution of enamel thick- that accumulate faster than would be expected through the neutral ness in humans (Hu et al., 2005; Wright, 2006; Gibson, 2011; process of genetic drift. This elevated rate of sequence change Moradian-Oldak, 2012). suggests a selective advantage to the organism that derives from Here we expand the Haygood et al. (2007) analysis of MMP20 by changes in the function of a protein or the regulation of a gene. The considering more species (gorillas, orangutans and gibbons) in specific identity of genomic regions under positive selection can addition to humans, chimpanzees and rhesus macaques. We also offer insight into how those changes are related to phenotypic investigate three additional genes (AMELX, AMBN, and ENAM) and evolution. Positive selection in protein-coding regions has been additional genic regions (coding and 30 flank). Given the strong link studied extensively at the scale of single genes (Evans et al., 2004a, between enamel formation and mutations in these four genes in 2004b; Al-Hashimi et al., 2009; Fedrigo et al., 2011; Pfefferle et al., humans (Hart et al., 2003; Pavlic et al., 2007; Wright et al., 2011), 2011) and the entire genome (Clark et al., 2003; Vallender and Lahn, we predict that the evolution of thick enamel is linked, at least in 2004; Bustamante et al., 2005; Cooper et al., 2005; Lindblad-Toh part, to genetic changes in the cis-regulatory and/or coding regions et al., 2005; Nielsen et al., 2005; Sabeti et al., 2006; Kosiol et al., of these four genes. The comparative species span a range of 2008; Enard et al., 2010). More recently, methods have been enamel thickness phenotypes from thin (chimpanzees and gorillas) developed to test for positive selection in putative regulatory re- to intermediate (orangutans, gibbons and rhesus macaques) as gions of genes, which have been applied throughout the genome measured by relative enamel thickness (Martin, 1985; Smith et al., (Pollard et al., 2006; Prabhakar et al., 2006; Haygood et al., 2007). 2003, 2005; Olejniczak et al., 2008a). Our goal is to determine King and Wilson (1975) predicted that many of the trait differ- whether there are signals of positive selection in the cis-regulatory ences distinguishing humans and chimpanzees are due to changes regions contrasted with the coding regions in humans as compared in gene regulation rather than protein structure. In support of this with other extant hominoids. We test the null hypothesis that the hypothesis, Haygood et al. (2007) detected at least as much evi- genomic signals of natural
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