Epigenetics: the Second Genetic Code

CHAPTER THREE

Epigenetics: The Second Genetic Code

Nathan M. Springer* and Shawn M. Kaeppler†

Contents 1. Introduction 60 2. Molecular Mechanisms of Epigenetic Inheritance 60 2.1. DNA methylation 61 2.2. Histone modifications 62 2.3. Chromatin structure 63 2.4. Role of RNA in heritable silencing 64 2.5. Interactions among DNA methylation, histone modifications, and chromatin structure 65 3. Epigenetic Phenomena in Plants 65 3.1. Phenotypic examples of epigenetic inheritance 66 3.2. Genomic and molecular genetic examples of epigenetic variation 70 4. Epigenetic Inheritance and Crop Improvement 71 4.1. Epigenetics in quantitative inheritance and selection response 72 4.2. Epialleles and gene discovery 73 References 73

Abstract Plant breeders utilize directed selection and transgenics to produce novel cultivars of diploid and polyploid species. DNA sequence is clearly important in these processes, but growing evidence implicates epigenetics as an important factor in controlling genetic variation and gene/transgene expression. In this article, we focus on epigenetic variation defined as mitotically and meiotically heritable but reversible states of gene expression that are not conditioned by differences in DNA sequence. We summarize mechanisms underlying epigenetic states of expression, and discuss implications of epigenetics in cultivar development.

* Department of Plant Biology, University of Minnesota, Saint Paul, Minnesota 55108, USA { Department of Agronomy, University of Wisconsin, Madison, Wisconsin 53706, USA

59 60 Nathan M. Springer and Shawn M. Kaeppler

1. Introduction

Plant breeders have made tremendous progress towards altering plant phenotypes to provide more productive crops. The majority of these gains have been made through selection of variation that is present within a species. While it is clear that phenotypic selection has resulted in improved plant characteristics, the molecular basis of the selected variation remains largely unknown. Traditionally, it was assumed that the majority of heritable variation was due to genetic sequence differences. However, a growing body of evidence suggests that intraspecific expression differences among genotypes can also be caused by epigenetic variation. To some researchers, epigenetics is used as a catchall term to describe any variation that does not seem to follow Mendelian inheritance patterns. In this article, we use the term epigenetics to specifically describe meiotically or mitotically heritable differences in gene expression that are not caused by sequence differences. We will begin by describing some of the molecular changes that are associated with epigenetic variation and then proceed to discussing some of the well characterized examples of epigenetic variation. Finally, we will discuss the potential impact of epigenetic inheritance in crop improvement.

2. Molecular Mechanisms of Epigenetic Inheritance

Epigenetics involves changes in heritable phenotypes without changes in DNA sequence. With the exception of protein-based inheritance such as prions, epigenetic differences are the result of altered gene expression levels. Research on the molecular mechanisms of epigenetic inheritance has identified several complementary pathways for the stable regulation of gene expression without sequence changes. The basis of epigenetic inheritance is the manner in which DNA is packaged and modified. DNA in plant cells contains four standard bases, cytosine, guanine, thymine, and adenine, but can also contain the modified base 5-methylcytosine. Most often, presence of 5-methylcytosine is associated with repressed gene expression. DNA in the cell is packaged by various sets of proteins. The first level of packaging involves wrapping DNA around cores of histone octamers containing two each of the proteins: Histone 2A (H2A), Histone 2B (H2B), Histone 3 (H3), and Histone 4 (H4). The histone proteins in the octamer can contain various modifications. The most relevant modifications for epigenetic expression are acetylation and methylation, which occur on the tails of H3 and H4. The tails of these histones extend outside of the DNA/protein core, and the modifications affect how those cores are packaged into a higher order structure. Epigenetics: The Second Genetic Code 61

Various proteins are involved in methylation of the DNA, modification of the histones, recognition of chromatin state, and in energy-dependent remodeling of one chromatin state to another. Following is a brief summary of some of the most important players in this process, chosen largely because one or more members have proven effects on gene expression in plants.

2.1. DNA methylation DNA methylation is found in the genomes of many plant and animal species. In eukaryotic genomes, DNA methylation refers primarily to 5-methylcytosine although some evidence for methylated adenines has been reported. The methyl moiety is added to cytosine residues present in double stranded DNA by a group of enzymes referred to as DNA methyltransferases. The majority of DNA methylation is found in CpG dinucleo- tide (plants and animals) and CpHpG trinucleotide (plants only) sequence contexts. Plant genomes encode several different DNA methyltransferase enzymes that fall into three different functional categories (reviewed by Chan et al., 2005). The Domains Rearranged Methyltransferases (DRM) encode de novo methyltransferases. These enzymes are capable of adding methyl groups to DNA that is unmethylated. The other two categories, DNA methyltransferases (MET) and chromomethylases (CMT), encode maintenance methyltransferases. Following DNA replication, the parent strand retains 5-methylcytosine but all cytosines within the daughter strand are unmethylated. This hemimethylated substrate is the target of the maintenance methyltransferases. Hemimethylated CpGs are the target of the MET class of enzymes while hemimethylated CpHpGs are methylated by CMTs. The targeting of a DRM protein to a specific genomic location will result in methylation of all cytosines within the region. If the targeting signal is no longer present then only the CpG and CpHpG methylation will be maintained by maintenance activities. These are likely oversimplifications of the activities and preferences for these enzymes as there appears to be some redundancy and locus-specific activities for these classes (reviewed by Chan et al., 2005). In general, DNA methylation is associated with transcriptional silencing of a locus. DNA methylation can result in silencing by directly interfering with the binding of transcriptional activators or by recruiting proteins that bind to methylated DNA and recruit transcriptional repressors (Bird and Wolffe, 1999; Klose and Bird, 2006). DNA methylation is often associated with centromeres and repetitive elements (Zhang et al., 2006). Recently, the application of microarrays and high-throughput sequencing approaches have provided a view of genome-wide DNA methylation patterns in Arabi- dopsis and the relationship of DNA methylation and gene expression (Cokus et al., 2008; Lister et al., 2008; Zhang et al., 2006; Zilberman et al., 2007). DNA methylation is quite high in transposon sequences and loss of CpG 62 Nathan M. Springer and Shawn M. Kaeppler methylation often results in transcriptional activation of these sequences. Two different types of genic methylation were noted (Lister et al., 2008; Zhang et al., 2006; Zilberman et al., 2007). A significant proportion of genes (33%) exhibit methylation within the coding region and a much smaller proportion of genes (5%) exhibit promoter methylation. However, a relatively small number of genes (500) exhibit altered expression when DNA methylation is reduced (Zhang et al., 2006). The majority of genes which are sensitive to DNA methylation exhibit promoter methylation. Interestingly, most of the genes controlled by CpG methylation are pseudogenes located within peri- centromeric heterochromatin while the genes regulated by CpHpG methylation are spread throughout euchromatic portions of the genome (Zhang et al., 2006). There is also evidence for altered expression of antisense and nc RNAs in plants with reduced DNA methylation levels suggesting that DNA methylation may be required to reduce transcriptional ‘‘noise.’’

2.2. Histone modifications The histone proteins exhibit remarkable sequence conservation in eukaryotes within the globular head domain that interacts with other histones and DNA as well as within the ‘‘tail’’ domain that protrudes from the central octomer– DNA complex. Recent research has identified a number of posttranslational modifications that occur to the histone tails including acetylation, methylation, SUMOlation, ubiquitination, and others (reviewed by Kouzarides, 2007; Pfluger and Wagner, 2007). These histone modifications can provide a variety of functions including transcriptional activation, transcriptional repression, efficient assembly into chromatin, and DNA replication. The theory of a histone code, in which each modification indicates a specific meaning and the combinations of modifications result in interpretations of chromatin state, was proposed ( Jenuwein and Allis, 2001). However, most current research suggests that there are actually a limited number of chromatin states and that the many of the modifications can act in a redundant manner (Kouzarides, 2007; Peterson and Laniel, 2004). Histone modifications can have both direct and indirect effects upon transcription. The presence of modifications, such as acetylation, may affect the ability of adjacent nucleosomes to interact. Histone modifications can also provide binding sites for other proteins which in turn may act as corepressors or coactivators (Jenuwein and Allis, 2001; Kouzarides, 2007; Peterson and Laniel, 2004). The epigenetic information of histone modifications is generally thought to be less stable than that of DNA methylation. The mechanisms for maintenance of DNA methylation patterns following replication are well understood. However, the mechanisms for maintaining histone modification patterns following dispersive replication of chromatin are unclear. In addition, most histone modifications are Epigenetics: The Second Genetic Code 63 reversible and the equilibrium for a particular locus is controlled by the access of the modifiers and demodifiers. Recent studies have provided a genomic view of the distribution of certain histone modifications. Cytogenetic studies have documented the chromosomal distribution for several histone modifications (Baroux et al., 2007; Fuchs et al., 2006; Houben et al., 2003; Jackson et al., 2004; Jasencakova et al., 2003; Soppe et al., 2002). A higher level of resolution has been provided by studies that combine chromatin immunoprecipitation and microarray hybridization (Bernatavichute et al., 2008; Gendrel et al., 2005; Turck et al., 2007; Zhang et al., 2007). Trimethylation of lysine 27 of histone H3 (H3K27me3) is present at 18% of genes and is enriched at transcription factors and developmental regulators that are not expressed in the tissues being studied (Zhang et al., 2007). H3K27me3 was often enriched near the promoter of genes. Interestingly, another histone modification that is associated with silencing, H3K9me3, does not tend to colocalize with H3K27me3 (Turck et al., 2007). In general, H3K9me3 is more prevalent at constitutive heterochromatin such as transposons while H3K27me3 is found at loci with more dynamic tissue-specific regulation. The mutually exclusive presence of these modifications suggests two different types of silencing that can be conditioned by histone methylation.

2.3. Chromatin structure Alterations to chromatin structure are also important in epigenetic regulation. Chromatin structure alterations can include chromatin structure changes caused by histone variants (reviewed by Henikoff and Ahmad, 2005; Williams and Tyler, 2007) or the physical remodeling of chromatin structure. Histone variants are critical for the epigenetic definition of centromeres in plants (Dalal et al., 2007; Dawe and Henikoff, 2006; Zhang et al., 2008). In addition, histone variants are also involved in epigenetic processes such as vernalization and plant immunity (Deal et al., 2007; March-Diaz et al., 2008). Relatively little is known about the genomic distribution of histone variants in plant cells. Studies on animal genomes have revealed that the H3.3 variant histone is deposited in a replication independent manner at regions with active transcription (Mito et al., 2005). A recent study found differences in H3.3 distributions within the developing embryo and endosperm of Arabidopsis (Ingouff et al., 2007). Chromatin structure can be altered by a family of enzymes that utilize ATP to physically alter chromatin (reviewed by Jerzmanowski, 2007; Kwon and Wagner, 2007). Arabidopsis encodes a large family of ATP-dependant chromatin remodeling enzymes ( Jerzmanowski, 2007; Verbsky and Richards, 2001). Several of these genes have been identified in genetic screens for epigenetic or developmental regulators ( Jerzmanowski, 2007; Kwon and Wagner, 2007). Very little is known about how these chromatin 64 Nathan M. Springer and Shawn M. Kaeppler remodeling enzymes are targeted to specific genomic regions. However, it is clear that these chromatin remodeling activities are critical for epigenetic regulation. In most cases, the chromatin remodeling activities appear to be important for gene regulation in response to environmental cues, not long-term changes in expression (Kwon and Wagner, 2007). A model that links histone modifications, histone variants and chromatin structure to nucleasome stability was recently proposed by Henikoff (2008).

2.4. Role of RNA in heritable silencing Posttranscriptional gene silencing (PTGS) or RNAi is a well-characterized process by which RNA is transcribed, but then degraded before translation. While PTGS is important in processes including development, transgene silencing, and plant responses to the environment, it does not fit the definition of epigenetics that we have used in this review, and will therefore not be addressed. However, recent results provide evidence that RNA can also play a critical role in establishing and maintaining heritable chromatin states. Evidence for the role of RNA in establishing and maintaining heritable chromatin states come from several types of studies. Research utilizing mutants in RNAi genes found that plants mutant for genes in this pathway displayed altered heritable DNA methylation patterns (Chan et al., 2004, 2006; Lippman et al., 2003; Tran et al., 2005; Zilberman et al., 2003). The mop1 mutant in maize is an example of the role of RNA in multiple types of heritable silencing. The mop1 gene encodes an RNA-dependent RNA polymerase 2-like protein that is involved in paramutation (Alleman et al., 2006), heritable transgene silencing (McGinnis et al., 2006), and transposon silencing (Lisch et al., 2002). While the complete mechanism of RNA-directed heritable silencing has not been completely elucidated, several important players in this pathway have been identified. Plants contain a unique family of RNA polymerase, PolIV. These polymerases contain component proteins of the NRPD1 and NRPD2 classes, and play an important role in RNA directed DNA methylation via the production of 24 nt siRNAs (Pikaard et al., 2008). Produc- tion of these siRNAs involves RNA-dependent RNA polymerase 2 type proteins which produce dsRNA that is processed by RISC complexes containing Argonaute 4 or Argonaute 6 (Vaucheret, 2008) homologs. Also involved in the process is SGS3, a protein of currently unknown function. An excellent summary of the various roles of RNA pathways in transcriptional and posttranscriptional silencing is provided by Shiba and Takayama (2007). Epigenetics: The Second Genetic Code 65

2.5. Interactions among DNA methylation, histone modifications, and chromatin structure The general descriptions of the molecular mechanisms of epigenetic inheritance provided above suggest discrete pathways for epigenetic regulation. However, it is clear that there is significant cross-talk and redundancy between these molecular mechanisms. The first plant mutant with reduced DNA methylation levels was Arabidopsis ddm1 (Vongs et al., 1993). The DDM1 gene actually encodes an ATP-dependent chromatin remodeling protein that is required for proper DNA methylation patterns ( Jeddeloh et al., 1999). A similar relationship between DNA methylation and chromatin remodeling has been noted in mammals (Dennis et al., 2001). This suggests that chromatin remodeling activities are required for proper DNA methylation patterns. There is also evidence for a strong relationship between histone methylation and DNA methylation in plants. Mutations in the H3K9 histone methyltransferase KRYPTONITE affect CpHpG methylation ( Jackson et al., 2002; Malagnac et al., 2002). There is evidence that alterations in DNA methylation patterns affects the histone methylation patterns (Gendrel et al., 2002; Johnson et al., 2002; Lawrence et al., 2004; Lindroth et al., 2004; Soppe et al., 2002; Tariq et al., 2003). The CMT DNA methyltransferase exhibit preferential binding to chromatin containing both H3K9 and H3K27 methylation (Lindroth et al., 2004). Several of the plant H3K9 methyltransferases contain an SRA domain which exhibit methyl- DNA specific binding activities ( Johnson et al., 2007; Woo et al., 2007, 2008). These molecular properties of the DNA and histone methyltransferases lead to complementary reinforcement of CpHpG DNA methylation and H3K9 histone methylation. Proper histone acetylation patterns are also required for maintenance of DNA methylation (Aufsatz et al., 2002; Probst et al., 2004). Mutations in chromatin remodeling genes can also affect histone modification patterns (Gendrel et al., 2002; Kanno et al., 2004, 2005; Lippman et al., 2003). The epigenetic mechanisms for gene regulation exhibit a high level of redundancy and interdependence. It is likely that these relationships strengthen the heritability and help to preserve this epigenetic information following DNA replication and cell division.

3. Epigenetic Phenomena in Plants

As discussed above there are a number of different molecular mechanisms for storing epigenetic information. Similarly, epigenetic inheritance exhibits a variety of different types of inheritance. In some cases, an epigenetic difference can be quite stable and can appear to follow Mendelian segregation patterns. In other cases epigenetic states can be programmed by 66 Nathan M. Springer and Shawn M. Kaeppler the parent of origin or exposure to other alleles. We will begin by discussing several examples of epigenetic inheritance in plants. This discussion will be limited to examples of epigenetic inheritance that can be meiotically transmitted. There are examples of epigenetic gene regulation during development, such as vernalization (Schmitz and Amasino, 2007), but these do not affect phenotypes in the off-spring. A survey of studies on epigenetic variation within plant species will also be presented.

3.1. Phenotypic examples of epigenetic inheritance 3.1.1. Imprinting Imprinting is a form of epigenetic regulation in which the maternal and paternal alleles exhibit differential expression following fertilization. At an imprinted locus, there are two alleles with identical, or nearly identical, sequences in the same nucleus, yet these two alleles exhibit differential expression. To date, all examples of imprinting in plants occur in endosperm tissue (Huh et al., 2007). The endosperm exhibits unique epigenetic states relative to other plant tissues (Baroux et al., 2007; Lauria et al., 2004). Most imprinted genes exhibit expression only from the maternal allele but several examples with paternal expression have also been identified (reviewed by Huh et al., 2007). Imprinting can result in phenotypic differences between reciprocal hybrids. For example, the phenotype conditioned by some r1 locus haplotypes differs depending upon maternal or paternal transmission. The maternally transmitted haplotype provides solid kernel coloration while the paternally transmitted allele conditions a mottled pattern (Kermicle, 1970, 1978; Kermicle and Alleman, 1990). Interestingly, while some r1 haplotypes are subject to imprinting, the majority do not exhibit any evidence of imprinting (Kermicle and Alleman, 1990). The number of plant genes that exhibit complete, or binary, imprinting is relatively small. Many of the imprinted plants genes including Medea, Fis2, ZmFie1, and Mez1, have sequence homology to proteins in the Polycomb repressive complex2 (PRC2) (Kohler and Makarevich, 2006). There is evidence that the imprinting mechanism involves DNA methylation and histone modifications (reviewed by Huh et al., 2008). In plants, the mechanism for imprinting at the Arabidopsis Medea (MEA) locus is understood in the greatest detail. MEA encodes a SET domain protein that can perform methylation of H3K27 (Baroux et al., 2006; Gehring et al., 2006; Grossniklaus et al., 1998; Jullien et al., 2006). DNA methylation establishes a default silenced state at the MEA locus and maintenance of this silent state requires MET1 (Xiao et al., 2003). A DNA glycosylase enzyme, DEMETER (DME ) is expressed in the central cell and removes DNA methylation from the maternal allele of MEA (Choi et al., 2002; Gehring et al., 2006; Kinoshita et al., 2004). Upon fertilization the maternal allele is Epigenetics: The Second Genetic Code 67 unmethylated and active while the paternal allele is methylated. During endosperm growth and development the MEA protein expressed from the maternal allele is required for maintenance of silencing of the paternal allele (Baroux et al., 2006; Gehring et al., 2006; Jullien et al., 2006). The maternally produced MEA protein is recruited to the promoter of the paternal MEA allele and catalyzes H3K27me3. There is evidence for differential DNA methylation of the maternal and paternal alleles of FWA (Kinoshita et al., 2004), Fis2 ( Jullien et al., 2006), ZmFie1 (Gutierrez-Marcos et al., 2006; Hermon et al., 2007), and Mez1 (Haun et al., 2007). Three imprinted maize genes, Mez1, ZmFie1, and Nrp1 also exhibit evidence for H3K27me3 enrichment at the promoter of the silenced paternal allele and H3 acetylation and H4 acetylation enrichment within the coding region of the maternal allele (Haun and Springer, 2008).

3.1.2. Paramutation Paramutation is a form of epigenetic inheritance that involves the commu- nication of two alleles. Paramutable alleles can be heritably altered by being exposed to a paramutagenic allele in a heterozygote (reviewed by Chandler, 2007; Chandler and Stam, 2004; Hollick et al., 1997). Paramutation has been well-characterized at the r1, b1, p1, and pl1 loci in maize (Brink, 1956; Coe, 1966; Hollick et al., 1995; Sidorenko and Peterson, 2001). The b1 locus of maize provides the best characterized example of paramutation. The b gene encodes a transcription factor that regulates the production of anthcyanin. The B-I allele provides dark pigmentation of several vegetative tissues while the B’ allele provides very light pigmentation of these tissues. The B-I allele is dominant over loss-of-function b alleles (Coe, 1966; Patterson et al., 1993, 1995). However, plants that are heterozygous for B-I/B’ exhibit light pigmentation similar to B’/B’ homozygotes. Self- pollination of B-I/B’ heterozygotes produces only off-spring with light- pigmentation. The paramutable B-I allele is affected by the paramutagenic B’ allele in the heterozygote such that the B-I allele is transformed into a B’ allele. The transition of B-I to B’ can happen spontaneously at low rates but is 100% when exposed to B’ in a heterozygote. This transition of B-I to B’ does not involve any sequence changes at the B locus. A series of elegant genetic experiments have identified a series of direct repeats 100 kb 50 of the B gene that are required for paramutation (Stam et al., 2002a,b). Character- ization of mutants that are impaired in paramutation reveals that RNAi and chromatin remodeling are important components of paramutation (Alleman et al., 2006; Dorweiler et al., 2000; Hale et al., 2007). There is no evidence that cytosine DNA methylation is required for paramutation at the B locus. However, altered DNA methylation patterns have been associated with paramutation at the R and P1 loci (Sidorenko and Peterson, 2001; Walker et al., 1998). There is genetic evidence that a common mechanism 68 Nathan M. Springer and Shawn M. Kaeppler might underlie paramutation at these distinct loci (Dorweiler et al., 2000; Hollick and Chandler, 2001; Hollick et al., 2005). There is some evidence for paramutation-like interactions between transgenes in other plant and animal species (reviewed by Chandler and Stam, 2004). It is unclear whether paramutation is limited to a small number of loci or might be acting at many genomic locations.

3.1.3. Naturally occurring epialleles Stable epigenetic alleles have also been identified by studies on intraspecific variation. In the process of analyzing floral development mutants, Jacobsen and Meyerowitz (1997) identified several alleles of the SUP locus. These alleles were mapped to the SUP locus and were confirmed by complemen- tation tests. However, no sequence differences were identified at the SUP locus. These clark kent alleles exhibit higher methylation levels than the wild-type SUP allele and reduced expression. While the clark kent alleles generally exhibit stable inheritance, 1–3% of progeny reverted to wild-type phenotype and these revertents had reduced, wild-type, methylation levels ( Jacobsen and Meyerowitz, 1997). The epigenetic silencing of SUP requires CpHpG DNA methylation (Lindroth et al., 2001), histone methylation ( Jackson et al., 2002) and RNAi components (Zilberman et al., 2003). The PAI gene family of Arabidopsis also exhibits epigenetic variation (Bender and Fink, 1995). The four copies of PAI present in the WS ecotype are methylated and transcriptionally silenced while the three PAI genes in Col are unmethylated and expressed (Bender and Fink, 1995). This silencing in WS is triggered by the inverted repeat arrangement of the PAI1 and PAI4 genes (Melquist and Bender, 2004; Melquist et al., 1999) and requires CpHpG methylation (Bartee et al., 2001) and histone methylation (Ebbs et al., 2006; Malagnac et al., 2002). There is also evidence that a disease resistance gene cluster exhibits naturally occurring epigenetic variation (Yi and Richards, 2007). There are several examples of naturally occurring epigenetic variants in other plant species as well. Linneus described a variant of Linaria vulgaris that had altered floral morphology (Cubas et al., 1999). This natural variant has been stably maintained for over 250 years and exhibits relatively stable genetic transmission. Molecular characterization revealed that the altered floral morphology is due to increased cytosine methylation at the Lcyc gene (Cubas et al., 1999). There is a mutation that affects tomato fruit development and ripening that is caused by epigenetic changes at the Cnr locus (Manning et al., 2006). There are naturally occurring alleles of several maize genes affecting seed or plant pigmentation levels, including Pl1 (Della Vedova et al., 2005; Hoekenga et al., 2000); P1 (Chopra et al., 2003; Sekhon et al., 2007; Sidorenko and Peterson, 2001) and R (Kermicle et al., 1995; Ronchi et al., 1995; Walker and Panavas, 2001), that exhibit epigenetic regulation. Epigenetics: The Second Genetic Code 69

3.1.4. Polyploid formation Epigenetic regulation may also play an important role in the success of polyploids (Hegarty and Hiscock, 2008; Liu and Wendel, 2003). Polyploidy involves an alteration in gene dosage (autopolyploidy) or a fusion of two complete genomes (allopolyploidy). In both instances, a situation is created in which the majority of genes are redundant and novel functions or expression patterns can be sampled. There are numerous examples of altered epigenetic states in recently formed polyploids. Newly formed polyploids often show substantial genomic instability, however the exact type of changes observed varies in different species. There is evidence for sequence elimination in wheat and Tragopogon polyploids (Feldman et al., 1997; Shaked et al., 2001; Tate et al., 2006). Chromosomal translocations and transposon insertions are common in Brassica polyploids (Song et al., 1995) while mainly gene expression changes are observed in Arabidopsis and cotton (Adams, 2007; Adams et al., 2003; Lee and Chen, 2001; Wang et al., 2004). Many polyploids also exhibit altered DNA methylation patterns upon hybridization of the two paternal genomes (reviewed by Liu and Wendel, 2003). The alteration of DNA methylation patterns following polyploidiza- tion suggests that epigenetic changes may be common in newly formed allopolyploids. Indeed, a number of studies have provided evidence that epigenetic changes cause altered gene expression in newly formed polyploids (Comai, 2000; Comai et al., 2000; Kashkush et al., 2002; Lee and Chen, 2001; Madlung et al., 2002). Madlung and Comai (2004) proposed that the formation of a polyploidy causes a high level of genomic stress which results in relaxation of epigenetic silencing and expression of nor- mally suppressed sequences. As the epigenetic systems are reestablished, novel epigenetic states are formed in the polyploids relative to the parental genomes. This system allows for novel expression states to be sampled and selected in polyploids.

3.1.5. Nucleolar dominance Ribosomal RNA genes in plants are highly repeated and occur in long contiguous stretches. Interestingly, only a portion of these genes are nor- mally expressed in any cell, and the silenced portion is in a contiguous stretch indicating a mechanism to simultaneously silence megabase segments of DNA. Furthermore, when multiple clusters of ribosomal genes are introduced into an organism by hybridization, the phenomena of nucleolar dominance is observed. Nucleolar dominance is achieved during an interaction of gene clusters on different chromosomes in which an entire cluster on one chromosome is silenced and the other remains active. The mechanism underlying this process is still being characterized, but the current state of knowledge is provided in Preuss and Pikaard (2007). 70 Nathan M. Springer and Shawn M. Kaeppler

3.2. Genomic and molecular genetic examples of epigenetic variation In addition to the previously described examples of epigenetic inheritance, there are several studies that address the prevalence of epigenetic variation within plant species. In general, it is quite difficult to attribute variation to epigenetic mechanisms. However, by studying the mechanisms of epigenetic inheritance, such as DNA methylation, it is possible to identify epigenetic variation. It is likely that these studies represent only the tip of the iceberg and that the application of genomic technologies will provide a more detailed understanding of epigenetic variation.

3.2.1. Natural variation for methylation of repetitive elements One approach towards the identification of epigenetic variation within a species is to simply monitor DNA methylation levels in populations. Eric Richards and colleagues have used Southern blot analysis of DNA methylation at repetitive sequences to monitor variation in Arabidopsis (Riddle and Richards, 2002, 2005; Woo et al., 2007). The methylation of rDNA ranged from 20% in some Arabidopsis ecotypes to over 90% in others (Riddle and Richards, 2002). There was a correlation between the methylation level of ribosomal DNA and the copy number for rDNA. A QTL analysis of rDNA methylation identified two major QTL located at the genomic locations of the rDNA that explain 50% of the variation in DNA methylation levels. It is likely that much of the methylation variation contributed by these QTL was due to inheritance of parental DNA methylation patterns at these loci (Riddle and Richards, 2002). In addition, several trans-acting QTL were identified on chromosomes 1, 3, and 5 (Riddle and Richards, 2002). A larger screen of DNA methylation levels in Arabidopsis ecotypes revealed that the Bor-4 ecotype exhibits low levels of DNA methylation at the 180-bp repeats but not at other loci (Woo et al., 2007). This reduced methylation segregated as a simple Mendelian trait and map-based cloning identified the VIM1 gene (Woo et al., 2007). There is a large deletion within the Bor-4 allele of VIM1 (Woo et al., 2007). The intraspecific variation for VIM1 function therefore leads to intraspecific variation for centromeric methylation in Arabidopsis. There is no evidence for functional significance for the variation of DNA methylation levels at centromeres or rDNA in Arabidopsis.

3.2.2. Natural variation for genic epigenetic variation Several approaches have been used to identify genes that exhibit variation for DNA methylation levels within a population. Rangwala et al. (2006) used microarray profiling to identify natural epigenetic variants in Arabi- dopsis. One noncoding transcript that may be derived from a retrotransposon was characterized in detail. This Sadhu element (At2g10410) is methylated Epigenetics: The Second Genetic Code 71 in most of the accessions tested, but in Col and N13 this sequence is highly expressed and hypomethylated (Rangwala et al., 2006). Multiple members of the Sadhu family of retroelements exhibit natural epigenetic variation (Rangwala et al., 2006). Interestingly, different Sadhu elements are controlled through different epigenetic mechanisms (Rangwala et al., 2007) and these members of this family do not exhibit coordinate regulation (Rangwala et al., 2006). Vaughn et al. (2007) used chromosome 4 tiling microarrays to characterize the DNA methylation patterns in two Arabidopsis accessions. This approach identified numerous examples of variable DNA methylation in Ler and Col ecotypes. A limited analysis of the stability of DNA methylation patterns found that some polymorphisms were quite stable while others exhibit high reversion frequencies in F2 families (Vaughn et al., 2007). A survey of sixteen loci in 96 different ecotypes revealed high levels of methylation polymorphism within Arabidopsis. Using a different approach Zhang et al. (2008) identified high levels of DNA methylation among Arabidopsis ecotypes. Methylation polymorphisms were more common near gene ends than within the coding region and were correlated with expression differences in some examples (Zhang et al., 2008). Another recent study identified widespread epigenetic natural variation for RNAi targets in Arabidopsis (Zhai et al., 2008). There is also evidence for natural epigenetic variation in maize. Makarevitch et al. (2007) used microarray profiling to identify the targets of CpHpG methylation in the maize inbreds B73 and Mo17. Over 100 genes sensitive to CpHpG methylation were identified by comparing expression isogenic wild-type inbred and zmet2-m1 mutant lines. The majority of the genes are sensitive to CpHpG methylation only in one of the two inbred lines. In most cases, these genes exhibit different expression levels in wild-type B73 and Mo17 and this variation maps to the gene itself and is controlled by DNA methylation (Makarevitch et al., 2007). A survey of the methylation and expression levels for several of these genes provided evidence for stable natural epigenetic variation in eight different inbred lines (Makarevitch et al., 2007).

4. Epigenetic Inheritance and Crop Improvement

Heritable transcriptional gene silencing is common in transgenic experiments. Transgene silencing reduces the efficiency of the transformation process and increases the work necessary to identify good events. Epigenetics also has a role in somaclonal variation (Kaeppler et al., 2000). Somaclonal variation generally is detrimental to the processes of transformation and clonal propagation, but in some instances can produce useful phenotypes. 72 Nathan M. Springer and Shawn M. Kaeppler

One vastly underexplored area of epigenetics is the potential role of heritable states of expression as a mechanism of quantitative variation, and as a rapid-response reservoir of variation that can contribute to selection response. In the following section, we will discuss the potential role of epigenetic inheritance in the process of plant breeding.

4.1. Epigenetics in quantitative inheritance and selection response Recent research provides evidence for the following interesting attributes of epigenetic inheritance. 1. Naturally occurring epialleles have been documented in numerous species and are relatively stable, sometimes over many generations. 2. Epialleles have reversion frequency 2–3 orders of magnitude greater than changes in the primary sequence. In the case of silenced alleles, reversion is to the active state in contrast to reversion of base sequence to a prior state which is very rare. 3. Stability and formation of epigenetic states can be influenced by the environment. 4. Epigenetic states of expression are not simply on or off, but stable intermediate levels of expression can be established. 5. Introduction of trans-acting alleles in ‘‘chromatin genes’’ can cause transient (one generation) or permanent alteration in state. For example, an epiallele could be stably maintained over many generations, but could revert to activity after a single generation interaction in a loss-of- function methyltransferase mutant. 6. Hybridization/heterozygosity is required for establishment of some epigenetic states such as paramutation, so epiallelic variation is enhanced in outcrosses and may increase in proportion to diversity. The molecular basis of allelic variation for quantitative traits in plants is just starting to be characterized, so it is difficult to provide examples for which quantitative models based on sequence information would be insufficient to explain the variation present. In fact, the stability of epialleles would make them appear no different than any sequence variant within the temporal context of most plant breeding experiments. However, long-term selection experiments indicate an amazing ability of plants to respond to selection in short periods of time. The Illinois long- term selection program is one unique example of selection in plants (Dudley, 2007). An interesting attribute of this program was the strong response to reverse and switchback selection. Especially intriguing is the ability of populations selected in the low direction (e.g., for low oil) to respond to selection for increases in the biochemical components. The reverse selection in the low oil population was initiated at a cycle when it Epigenetics: The Second Genetic Code 73 appeared that selection response had dramatically reduced and that there was little genetic variation remaining. While there are various sequence-based genetic mechanisms that could cause this result, it is intriguing to speculate that epiallelic variation may maintain a reservoir of variation that might be selected upon in a situation such as this. In natural populations, such a reservoir of variation could allow population responses to rapid changes in environment for traits with seemingly little phenotypic variation.

4.2. Epialleles and gene discovery Substantial effort is currently being devoted in plants to characterizing the sequence variation that underlies quantitative phenotypic variation. This is accomplished by associating sequence-based haplotypes with phenotypic performance to determine causal phenotypes. Epigenetic variation that contributes to phenotypic variation would complicate this effort. Epigenetic molecular variation would not be detected by normal sequence-based approaches to haplotype characterization. Methylation mapping, using a technique such as bisulfite sequencing, or chromatin mapping would be required to characterize the epigenetic states of target genes. Alternatively, epialleles might be predicted by detection of transcription states that cannot be explained by sequence haplotype variation. A further complication of epigenetics in this process is that epialleles may occur in multiple lineages in a comparatively short time frame. Whereas sequence variants occur in a logical progression with a founder sequence giving rise to a series of accumulating variants over the course of times, epialleles may appear and disappear at any point within this process, and will likely occur independently of most sequence polymorphisms. Therefore, epialleles may at best cause noise in the process of searching for causal sequence polymorphisms, and in extreme cases may be more important for specific genes in determining phenotype than any sequence polymorphism. Growing documentation of the presence of stable epialleles in numerous species suggests that epigenetic variation needs to be considered in the process of associating molecular variation with phenotype. Advancing technology may allow chromatin patterns to be included with sequence as another layer of information that can be included in the analysis complex traits in plants.

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