Current Zoology

58 (1): 138145, 2012

The influence of mitochondria in epigenetics revealed through naturally occurring fish cybrids
Bernard ANGERS*, Antoine DALLAIRE, Simon VERVAET, Francis VALLIRES, Annie ANGERS
Department of Biological Sciences, Universit de Montral, C.P. 6128, succ. Centre-Ville, Montral, Qubec, Canada, H3C 3J7

Abstract Epigenetic processes are important mechanisms for phenotypic changes that occur in response to the environment. As
such, it is expected that the alteration of cytoplasmic composition (the immediate environment of nuclei) results in the modification of the methylome and the expression of the nuclear genome. Cytoplasmic hybrids (or cybrids) are an ideal model to study the influence of mitochondria on gene expression. In this study, we take advantage of the natural co-occurrence of two biotypes that have a similar nuclear genome type Chrosomus eos, but harbor mitochondria from different species (C. eos in wild type or C. neogaeus in cybrids) to assess the effects of mitochondria on DNA methylation profiles and protein expression of the nuclear genome. Comparison between these biotypes is particularly relevant given their recent divergence and their low level of genetic differentiation. Variations of DNA methylation assessed on tissues from different embryonic origins revealed the distinct profiles of cybrid and wild type populations. Differences are more pronounced between wild type and cybrids than between populations of a given biotype. The proteome is also more different between biotypes than within a given biotype. These results indicate a strong influence of mitochondria on the nuclear genome, which remains detectable in different genetic and environmental contexts. These changes in the methylome and proteome of cybrids are expected to reflect the adjustments imposed by the coexistence of nuclear and mitochondrial genomes from different species [Current Zoology 58 (1): 138145, 2012].

Keywords

Epigenetics, Mitochondria, Chrosomus, Phenotype

Epigenetic processes are increasingly recognized as the underlying mechanisms responsible for many of the phenotypic responses to environmental changes (Angers et al., 2010). In particular, it has been shown that DNA methylation contributes to phenotypic variation by modifying gene expression (Bird, 2002; Jaenisch and Bird, 2003). Unlike DNA mutations, changes in DNA methylation can be influenced by the environment (Weaver et al., 2004; Blewitt et al., 2006; Manning et al., 2006; Crews et al., 2007; Kucharski et al., 2008; Jablonka and Raz, 2009; Massicotte et al., 2011) and allows for phenotypic variability without having to rely on genetic variation. It is expected that these environmentally induced phenotypes may have ecological and evolutionary significance (Angers et al., 2010; Beldade et al., 2011). The study of epigenetics in nature is especially complex because the environmental conditions that are likely to affect the epigenome are difficult to control. It has been shown that epigenetic profiles are influenced by numerous environmental signals, such as diet (KuReceived July 30, 2011; accepted Dec. 4, 2011. Corresponding author. E-mail: bernard.angers@umontreal.ca 2012 Current Zoology

charski et al., 2008), temperature (Sheldon et al., 1999), behaviour (Weaver et al., 2004), and exposure to chemicals (Crews et al., 2007). As a result, it may be extremely difficult to disentangle the effects of such a large number of variables. Mitochondria are part of the cellular environment surrounding the nucleus. These organelles are essential components at the center of vital processes, such as sugar metabolism, cellular respiration, and thermoregulation. Their size, number, and specific protein content vary greatly by type of tissue and also in response to changing environmental conditions, stress, and metabolic state (Attardi and Schatz, 1988). Despite the fact that the mitochondrial genome is several orders of magnitude smaller than the nuclear genome, it encodes essential mitochondrial proteins that must interact with proteins encoded by nuclear DNA to form functional oxidative phosphorylation complexes. Consequently, extensive crosstalk between both organelles is required to ensure that the cell metabolic needs are fulfilled. In fact, mitochondrial dysfunction is a major cause in seve-

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ral human diseases (Schapira, 2006). Genomic expression of mitochondrial protein genes has been demonstrated to have a high degree of variation, which is important to be able to accommodate the metabolic needs of different tissues. This strongly suggests that mitochondria can influence nuclear gene expression. Indeed, epigenetic marks have been detected in a wide variety of mitochondrial protein genes and their methylation state is correlated with tissue metabolism (Takasugi et al., 2010). Given their intimate relationship, coadaptation of mitochondrial and nuclear genes is expected (King and Attardi 1989; Blier et al., 2001; Rand et al., 2004). Demonstration of the influence of the mitochondrial genome on nuclear gene expression or the metabolic performance of cells generally relies on experimental manipulations (Blier et al., 2001). These may be performed with cytoplasmic hybrids (cybrids), in which the fusion of an enucleated cell with either a whole cell or a cell deprived of its own mitochondria can result in the coexpression of mitochondrial and nuclear genomes from different species in the same cell (Kenyon and Moaes, 1997; James and Ballard, 2003; McKenzie et al., 2003). Such experiments provide evidence that cell viability and mitochondria performance decrease sharply with increased genetic distance between the cytoplasmic and nuclear donors (McKenzie et al., 2003; Bayona-Bafaluy et al., 2005). Epigenetic modifications have not been directly measured in cybrids, but they do occur alongside strong modification of gene expression in cells deprived of mtDNA, further demonstrating the importance of mitochondrial control on nuclear gene expression (Delsite et al., 2002; Smiraglia et al., 2008). In this study, we take advantage of the natural co-occurrence of two northern redbelly dace (Chrosomus eos, Cyprinidea, Pisces; formerly Phoxinus (Strange and Mayden, 2009)) biotypes in geographically close lakes in the Laurentian region (Quebec, Canada). One of the biotypes is a stable cybrid originating from the cross-fertilization of C. eos-neogaeus x eos triploid females by a C. eos male, which resulted in the production of fish bearing diploid C. eos nuclei and C. neogaeus mitochondria (Dawley et al., 1987; Goddard et al., 1998; Binet and Angers, 2005; Angers and Sclosser, 2007). The DNA sequence of mitochondrial cytochrome oxidase subunit I (CO I) from C. eos differ from that of P. neogaeus by 9.6 % (Angers and Schlosser, 2007). These fish are found in close proximity with normal C. eos populations (hereafter referred to as wild type), thereby allowing for the direct comparison of the bio-

types harboring distinct cellular environments. Comparative studies between wild type and cybrids can be useful to disentangle the genetic contribution of the mitochondrial genome from that of the nuclear genome. Our objective was to determine the modification of DNA methylation profiles on the nuclear genome linked to a foreign mitochondrial genome and assess the consequences of such changes on protein expression in natural conditions. We also assess the relative effect of mitochondria versus the surrounding environment by sampling wild type and cybrid individuals from different lakes.

Material and Methods

1.1 The biological model The cybrids of the study system occurred naturally. They most likely originated from the particular hybridization process characterizing the Chrosomus eos-neogaeus unisexual complex (Dawley et al., 1987; Goddard et al., 1998). Such cybrids, with C. eos nuclear genome but C. neogaeus mitochondrial DNA, were first reported by Dawley et al. (1987). The proposed mechanism leading to the natural occurrence of cybrids is outlined in Fig. 1. Chrosomus eos-neogaeus diploid hybrids resulted from hybridization between females of finescale dace C. neogaeus and males of northern redbelly dace C. eos. While these hybrids generally produce unreduced eggs and reproduce clonally via gynogenesis (Goddard et al., 1998; Binet and Angers, 2005), triploid hybrids C. eos-neogaeus x eos may occur when the nuclear genome of the C. eos sperm is incorporated into the eggs diploid genome (Dawley et al., 1987; Binet and Angers, 2005). It is expected that, during gametogenesis of such triploid individuals, the unmatched set of chromosomes would be discarded and meiosis would occur on a diploid genome, therefore producing haploid eggs (Goddard et al., 1998). If a male of the same parental species fertilizes one of these haploid eggs, a pure nuclear diploid genome of C. eos is restored. The individual will, however, inherit solely the female C. neogaeus mitochondrial genome. The cybrids may thereafter reproduce sexually, as does the wild type. 1.2 Sampling and biotypes identification Firstly, we determined which biotypes were present in each of the four lakes (Fig. 2, Table 1). The biotype of 15 to 20 individuals per lake was determined using diagnostic nuclear and mitochondrial markers (Binet and Angers, 2005). The nuclear composition of individuals was characterized by the presence of

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Fig. 1

Proposed mechanism for the formation of natural cybrids

1. Gynogenetic hybrids result from hybridization between female Chrosomus neogaeus and male C. eos. 2. Triploid hybrids occur when the nuclear genome of the C. eos sperm is incorporated into the unreduced genome of the egg. 3. Cybrids occur when the haploid egg from triploid hybrid is fertilized by the C. eos sperm. E and N refer to nuclear genome of C. eos and C. neogaeus, respectively, superscript to mitochondrial genome.

Fig. 2

Geographic mapping of the sampled lakes in the Laurentian region (Quebec, Canada)

Letters refer to wild type (W) and cybrid (C) populations; numbers refer to the drainage system.

Table 1
Lake W1 W2 C1 C2

Characteristics of the lakes sampled in this study

Geographic coordinates 4559 14 N, 7400 30 W 4552 12 N, 7408 58 W 4557 10 N, 7356 05 W 4555 29 N, 7403 52 W Biotypes Wild type Wild type Cybrids Cybrids Genetic 15 20 15 20 Epigenetic 4 (M, E, I) 3 (E) 3 (E) 4 (M, E, I) Proteins 4 (M) 4 (M)

Geographic localization of the sites, genetic identification of the individuals sampled, and the number of individuals analyzed for genetic, epigenetic, and protein surveys. M, E, and I refer to muscle, eye, and intestine, respectively.

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chromosomes from C. eos and/or C. neogaeus species, which allowed for distinction between each parental species and hybrids. A similar procedure was performed on the CO I gene in order to assess the origin of the mitochondrial genome that differentiated cybrids from wild type. We assessed the evolutionary relationship between wild type and cybrid populations from the genetic variability measured from six highly variable microsatellite loci (Pho-1, 2, 60, 61, Seat-12, 412), as detailed in Binet and Angers (2005) and Angers and Schlosser (2007). Factorial correspondence analysis (FCA) was applied on microsatellite multilocus genotypes using Genetix version 4.05.2 (Belkhir et al., 19962004). Tests of population differentiation and hierarchical analyses of genetic variance were performed using Arlequin version 3.5.1.2 (Schneider et al., 2000). 1.3 Epigenetic analysis A survey of the DNA methylation variation was performed on three or four individuals per lake, for two lakes per biotype (Table 1). These fish were sampled during the breeding season over the period of two days. The most geographically distant lakes (W2 vs C1, Fig. 2) are separated by less than 25 km (straight line distance). Three different tissues were chosen for their distinct embryonic origin: muscle (mesoderm), eye (ectoderm and mesoderm), and intestines (endoderm). Total DNA of each tissue was extracted by proteinase K digestion followed by phenol-chloroform purification and ethanol precipitation (Sambrook et al., 1989). We investigated epigenetic variation by controlling for the genetic polymorphism at CCGG motif using a methylationsensitive amplified polymorphism analysis (MSAP; Xiong et al., 1999). Fragments that displayed methylation polymorphism with the methylation sensitive treatment (HpaII) were considered only when the non-sensitive (MspI) treatment provided monomorphic bands across individuals (i.e. when there was no nucleotide polymorphism for the CCGG site). Selective amplifications were performed using four primer combinations (EcoRI-AGC/HpaII-ATC; EcoRI-ACG/HpaIIACC; EcoRI-AAG/HpaII-AGC; EcoRI-AGG/HpaIIACG). Amplification products were separated on a denaturing 6% polyacrylamide gel (19:1 acrylamide: bis-acrylamide) and revealed using silver nitrate staining (Bassam et al., 1991). Euclidian distance (Danielsson, 1980) was then computed from the presenceabsence matrix of MSAP bands for every tissue of individuals with R version 1.12.2 software. Distance was then used to infer a dendogram using the

neighbor-joining method (Saitou and Nei, 1987). Relative support for the different groups was assessed by bootstrap analysis (1000 replicates) using PHYLIP package version 3.69 (Felsenstein, 1989). 1.4 Protein analysis A partial survey of the proteome was performed on four individuals of each biotype (Table 1). Muscle tissue was mechanically homogenized and proteins were extracted in 8M urea, 4% Triton X100. Total protein concentration was determined using a modified Bradford method (Zor and Selinger, 1996). Total protein (250 g) was separated using two-dimensional electrophoresis (Klose, 1975; OFarrell, 1975). Isoelectric focusing was performed using IPG strips (pH 47) (GE Healthcare) with an EttanTM IPGphorTM Isoelectric Focusing System (GE Healthcare). Separation by molecular weight was then performed on a SDS polyacrylamide gradient gel (8-20%). 2D-gels were stained with silver nitrate coloration (Heukeshoven and Dernick, 1988). The presence/absence of proteins among individuals was assessed using the MELANIE 2D gel analysis software version 7.05 (Swiss Institute of Bioinformatics).

Results

The results of the genetic identification confirmed the presence of cybrids bearing a nuclear genome of C. eos but a mitochondrial genome of C. neogaeus in two lakes (Table 1). A striking feature of the present survey was the allopatry between wild type and cybrids; each lake harbored either one or the other biotype. Genetic analyses performed on nuclear genome with microsatellite markers did not support the hypothesis that wild type and cybrids represent two distinct groups. As represented on FCA (Fig. 3), allelic composition of

Fig. 3 Factorial correspondence analyses (FCA) applied on microsatellite multilocus genotypes

W1-W2 and C1-C2 refer to wild type and cybrid populations, respectively (Figure 1, Table 1). Axes 1 and 2 account for 81% of the variance.

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both biotypes overlapped, especially for W1 and C1 from the same drainage system. Tests of population differentiation were significant, except between W1-C1 (P=0.1789). Hierarchical analyses revealed that the estimated variance between biotypes was very low and not significant (1.8%, P=0.34), while the variance among populations within biotype was higher and significant (5.3%, P<0.00001). Out of a total of 141 loci screened for epigenetic variation, 36 (25.5%) did not display variation. The remaining loci provided distinct methylation profiles within and/or among tissues. Once controlled for genetic variants, clustering analysis revealed the similarity of epigenetic profiles per tissue, supported by high bootstrap values (Fig. 4). More importantly, for a given tissue, cybrids and wild type displayed distinct epigenetic profiles, supported by bootstrap values higher than 54% (Fig. 4). A total of 14 methylated bands that differentiated wild type from cybrids were detected over all of the tissues: 5 bands were detected in muscles, 3

bands were detected in eyes, and 8 bands were detected in intestines (one band was shared between muscles and intestines and another band was shared between eyes and intestines). To determine if wild type and cybrid profiles could be differentiated in other environments, MSAP analyses were performed in eye samples of additional wild type (W2) and cybrid (C2) populations. Epigenetic profiles obtained from these different populations showed clear clustering of the biotypes, consistent with the strong influence of different mitochondria genomes on epigenetic marks (Figure 4). However, it appears that the environment of the lake also influences the epigenetic marks, as individuals of a given population also clustered together, especially in the case of cybrids. We then examined the potential effect on gene expression of the differential epigenetic marking of cybrids versus wild-type individuals. Analysis of a subsample of 405 unambiguously scored proteins from muscle extracts of individuals from two lakes revealed a

Fig. 4

Dendogram inferred from the different epigenetic profiles of tissues for wild type and cybrid individuals

Numbers in italic refer to bootstrap support (in percentage); values below 50% were not reported. W1-W2 and C1-C2 refer to wild type and cybrid populations, respectively (Fig. 1, Table 1).

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low variability within biotypes. Pairwise comparisons of differences among individuals varied from 0.8 to 1.4% for wild type and from 0 to 0.8% for cybrids. However, the differences between biotypes were more marked; between 7.5 and 8.7% of the 405 proteins analyzed were different between wild type and cybrids.

3 Discussion
The comparison of DNA methylation and proteome between wild type and cybrid populations of C. eos revealed a marked difference related to the origin of the mitochondrial genome. While sharing a similar C. eos nuclear genome, biotypes both displayed distinct epigenetic profiles (regardless of the tissue analyzed) and expressed different proteins in muscle. However, prior to attributing the detected epigenetic differences to the expression of foreign mitochondrial DNA, we must rule out the role of genetic divergence and environmental conditions, which may also account for this variability. Genetic analyses performed on populations of different biotypes suggest that they do not represent distinct lineages separated over a long evolutionary period. For instance, differentiation between wild type populations is higher than between wild type (W1) and cybrids (C1) from the same drainage system. Since no differences were found between mitochondrial DNA of the hybrid C. eos/neogaeus, the C. neogaeus, or the cybrids (Angers and Schlosser, 2007), the origin of cybrids is relatively recent. Interestingly, wild type and cybrids sampled in this study display an allopatric distribution, as reported in most of the populations analyzed thus far (Angers and Schlosser, 2007). This suggests two non-mutually exclusive scenarios for the formation of cybrids. Firstly, cybrids could have occurred during the Pleistocene and colonized the current lakes following deglaciation. Consequently, the divergence among populations of a given biotype would have occurred approximately 10,000 years ago (end of Pleistocene glaciation), while divergence between wild type and cybrids would have occurred more than 10,000, but less than 50,000, years ago (the origin of hybrids) (Angers and Schlosser, 2007). Alternatively, the formation of cybrids may have occurred in the current lakes, in which case the competition or random extinction following partition of cybrids would explain the allopatry. In this latter explanation, the biotypes would have diverged less than 10,000 years ago. The very low divergence detected between cybrids and wild type populations supports the last scenario, but further investigation is required. More importantly, genetic analyses confirmed

that the populations of both biotypes separated only recently and, as such, divergence cannot be the factor causing the differences observed on epigenetic profiles and the proteome. Comparison of the epigenome of each biotype from different lakes allows for the disentangling of the effects of mitochondrial genome from the different environmental conditions of the lakes. Environmental conditions of the lakes influenced the epigenetic marks as individuals of a given population clustered together. Due to the allopatry of wild type and cybrids analyzed in the present study, individuals were sampled in different locations. We could not rule out the hypothesis that environmental conditions were more similar among populations of a given biotype than between biotypes. Analyses of biotypes in sympatric conditions or common garden experiments may ensure that variance among locations do not induce the observed epigenetic and proteomic patterns. However, the similarity in a given biotype of the methylation profiles of the eye, which remains detectable in different genetic and environmental contexts indicates that these changes did not occur at random. The clear distinction between the methylation profiles of wild type and cybrids detected on all of the tissues analyzed confirmed the strong influence of mitochondria on the nuclear genome. In addition, an important difference was observed between the muscle proteome of wild type and that of cybrids (7.5%8.7%). By comparison, a similar analysis between C. eos and C. neogaeus (nuclear and mitochondrial donor species respectively) revealed a difference of 11.9% over 580 proteins (F. Vallires, unpublished data). The differences between C. eos and C. neogaeus species were expected to reflect the mutations accumulated since speciation. However, wild type and cybrid populations have only been recently separated and share the same nuclear genome. These changes in the methylome and the proteome of cybrids were expected to reflect the adjustments imposed by the coexistence of nuclear and mitochondrial genomes from different species. In regards to the changes observed in the cybrids, this suggests a major rewiring of gene expression related to the foreign mitochondria. Mitochondrial proteomes vary depending on the type of tissue (Mootha et al., 2003; Forner et al., 2006). Such tissue-specific differences in organelle composition are linked to tissue-dependent mitochondrial functions. It has been shown that epigenetic regulation of nuclearencoded mitochondrial genes is tissue-dependent. Liver,

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brain, and heart tissues of mice present different methylation profiles on numerous nuclear mitochondrial genes according to the mitochondrial functions of these tissues (Takasugi et al., 2010). Previous experiments with artificial cybrids also showed disruption of specific coadapted nucleo-mitochondrial complexes with increasing evolutionary distance between nuclear and mitochondrial genomes, resulting in a decrease in metabolic activity (Kenyon and Moaes, 1997; James and Ballard, 2003; McKenzie et al., 2003). The 9.6% genetic divergence between C. eos and C. neogaeus (Angers and Schlosser, 2007) falls within the range of normal metabolic activities in primate and murine cybrids (Kenyon and Moaes, 1997; McKenzie et al., 2003). However, McKenzie et al. (2003) detected metabolic differences even in murine cybrids obtained from fusion of closely related species. If, as observed in other cybrids (e.g. Rawson and Burton, 2002), such metabolic differences exist between wild type and cybrids of C. eos, they could be associated with different epigenetic regulation of the expression of nuclearencoded mitochondrial proteins. This may explain, at least partially, the rewiring of gene expression across tissues related to the foreign mitochondria. In conclusion, this comparative study of Chrosomus eos wild type and cybrids provides broad insights on the effects of mitochondrial genome on epigenetic processes. These results, while preliminary, appear to confirm the implication of the mitochondrial genome in regulating DNA methylation and gene expression of the nuclear genome. To our knowledge, this represents the first attempt at assessing the interplay between mitochondrial DNA and epigenetics in natural conditions. While cybrids can be constructed in the laboratory, naturally occurring cybrids provide a unique opportunity because these individuals are adapted to their environment and have adjusted to their "foreign" mitochondria.
Acknowledgements We are particularly grateful to M. Perez, G. Desrochers, C. Leung, and F. Cyr for their invaluable field and laboratory assistance and to Root Gorelick and two anonymous reviewers for their helpful comments on the manuscript. This research was supported by a research grant from the Fonds Qubcois pour la Recherche sur la Nature et les Technologies (FQRNT) to BA and AA.

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