Paramutation: Epigenetic Instructions Passed Across Generations

Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove PARAMUTATION is the fascinating ability of specific DNA sequences to communicate in trans to establish meiotically heritable expression states. Intriguingly, newly silenced sequences continue to issue instructions to naïve alleles in subsequent generations. The term “paramutation” was first coined in the 1950s by Alexander Brink to describe this puzzling phenomenon at the r1 locus in maize (Brink 1956); an interaction between specific alleles in heterozygotes led to heritable decreases in gene expression of one allele. Not only was the reduced expression state stable through meiosis, but also the low-expressing allele could induce silencing of another high-expressing allele in subsequent generations. The frequency of the change was 100% and the stability of the change was lower than typical mutations; hence the term “paramutation.” A few years later, Ed Coe, Jr., described another maize example in which interaction between alleles at the b1 locus also led to heritable silencing (Coe 1959) and Rudolf Hagemann described interactions at the sulfurea locus in tomato (Hagemann 1969). Since that time other examples of paramutation have been identified in maize and in other species (reviewed in Chandler and Stam 2004; Stam and MittelstenScheid 2005; Chandler 2007), yet the two maize loci where paramutation was initially described, r1 and b1, remain the most extensively characterized and best understood. The r1 and b1 loci encode closely related, functionally equivalent transcription factors that activate the anthocyanin pigment biosynthetic pathway (Goffet al. 1990; Ludwiget al. 1990). They are likely related to each other through a duplication resulting from an ancient allotetraploidization event during maize evolution (Gaut and Doebley 1997). The two loci have multiple alleles with distinct expression patterns, which regulate the distribution of anthocyanin pigments during development (Styleset al. 1973; Coe 1979). Recent work demonstrates a key role for RNA in mediating both r1 and b1 paramutation, as the mop1 gene that encodes an RNA-dependent RNA polymerase (RDR; Allemanet al. 2006) is absolutely required for paramutation at both loci (Dorweileret al. 2000). Yet, there are striking differences in the properties of r1 and b1 paramutation, which hint at distinct mechanisms. In this article, the most striking differences between r1 and b1 paramutation are described and potential mechanisms are discussed relative to our current understanding of the role of RNA interference (RNAi) in mediating transcriptional silencing. In spite of the close relationship, r1 and b1 have quite distinct gene organizations and there is no evidence of paramutant interactions between the loci (Brinket al. 1960). Specific alleles at each locus participate in paramutation. At r1, all known alleles induce paramutation, respond to paramutation, or cause reversal of paramutation. The alleles that cause paramutation are generally structurally distinct from the alleles that become silenced (reviewed in Chandleret al. 2000; diagrammed in Figure 1). The r1 haplotypes that participate in paramutation are expressed in the aleurone layer of the seed and the paramutant phenotype is strongest in this tissue (reviewed in Brinket al. 1968; Brink 1973). All r1 “alleles” that undergo paramutation contain multiple copies of the coding region (Figure 1) and as such are referred to as haplotypes. In contrast, the b1 alleles that participate in paramutation have a single coding region and identical DNA sequences and are thus epialleles (Stamet al. 2002a,b). These b1 epialleles are expressed in the epidermal layer of the plant body, but not in the seed (Figure 2). Paramutation at r1. The structures of the paramutable (R-r) and paramutagenic (R-st) alleles are diagrammed as well as the crosses and phenotypes used to monitor paramutation. The open boxes represent the r1 genes with the small arrows indicating the direction of transcription: P stands for plant and is a gene expressed in vegetative plant tissues; S1 and S2 are two genes expressed in the seed that are organized in an inverted repeat; Sc stands for self-color and is the most highly expressed gene in the R-st complex; Nc1–Nc3 stand for near colorless, three r1 genes that are expressed at low levels in R-st. I-R indicates the transposable element inserted into Sc, which is responsible for the spotted phenotype. The thick arrows in R-st represent the large repeats spanning the r1 genes. Only the repeats associated with the coding regions are shown. There are additional small, related sequences related to transposable elements that are not diagrammed, but are described in detail with primary references cited in Chandleret al. (2000). A comprehensive list of r1 haplotypes with their tissue-specific expression patterns and paramutation properties can be found in Neufferet al. (1997). Paramutation at b1. The paramutable (B-I) and paramutagenic (B′) epialleles have identical DNA sequences, but distinct chromatin structures represented by different proteins (circles) associated with the seven tandem repeats (arrowheads) that mediate paramutation and are located 100 kbp upstream of the coding region (open box). The arrows below the gene boxes indicate the direction of transcription and the thickness of the arrows indicate the relative transcription levels observed with the two epialleles. The crosses and phenotypes used to monitor paramutation are diagrammed with B′*, indicating a B′ allele that was B-I in the previous generation. B′ and B′* are indistinguishable in their ability to paramutate B-I. When the R-r allele, which confers dark purple seeds, is crossed to R-stippled (R-st), which confers purple spotted seeds (because of a transposon insertion that is not involved in paramutation), R-r is heritably changed such that it confers lightly pigmented seeds upon subsequent outcrosses (designated R-r′), while the R-stippled allele segregates unchanged (Figure 1). Intriguingly, when R-r′ is crossed to R-r, R-r can be changed to R-r′; this is referred to as secondary paramutation to distinguish it from the primary paramutational interaction between R-st and R-r. At the b1 locus, the low-expressing B′ allele (which can also derive spontaneously from the high-expressing B-I allele) changes B-I into B′ (new B′ alleles are designated B′*) with the silencing observed in the F1 (Figure 2). The newly altered B′* allele is indistinguishable from B′ in its ability to paramutate naïve B-I alleles in subsequent generations. Thus, at b1, secondary paramutation is indistinguishable from primary paramutation. This contrasts with r1 paramutation; the strength of R-r′ secondary paramutation (the ability of R-r′ to paramutate a naïve R-r allele) is fully penetrant only when R-r′ has been heterozygous with R-st for multiple generations (Brown and Brink 1960). Two assays are routinely used to monitor paramutation: (1) the ability of a paramutagenic allele to cause a heritable change in the expression of a paramutable allele (as measured at r1 and b1 by a reduction in pigment); and (2) the heritable alteration of the paramutable allele into a paramutagenic allele. At b1, these two phenotypes always occur simultaneously and completely. In contrast, at r1, the extent of paramutagenicity measured as the level of R-r pigmentation depends on the haplotype and the circumstances of the crosses (discussed in the next section and reviewed in Chandleret al. 2000). Specific nomenclature is used to describe the various steps in the paramutation process. Establishment describes the trans-interactions between alleles that produce the distinct expression states. In the case of both r1 and b1, the new states show reduced expression (designated R-r′ and B′), and the ability to maintain that silencing phenotype is referred to as “maintenance of silencing.” The extent to which the silencing and ability to cause paramutation is maintained in subsequent generations is referred to as heritability. The reason for distinguishing among establishment, maintenance, and heritability is that certain situations or genetic backgrounds can influence these processes differentially. The frequent association between paramutation and genes involved in pigmentation is likely to reflect ascertainment bias, because of the ease of scoring visible pigment phenotypes and the dispensable nature of anthocyanin for plant development. The r1 and b1 systems differ in several properties, which hint at basic mechanistic differences. One dramatic example is the extreme stability of B′ as compared with the instability of the R-r′ silenced state. B-I is truly converted to a B′ epiallele whereas R-r′ states exist as a continuum, depending on the counter-allele and the genetic history. In wild-type genetic backgrounds, B-I is always changed to B′* (Coe 1966). B′ and B′* are indistinguishable, and both are extremely stable; they have never been observed to change back to B-I in wild-type genetic backgrounds and this is true whether they are maintained as homozygous or heterozygous with an allele that does not participate in paramutation (Coe 1966). Spontaneous changes of B-I to B′ occur at high frequencies, often 1–10% (Coe 1966), and spontaneous B′ alleles are as fully paramutagenic as B′* alleles segregating from B′/B-I plants (Figure 2). Thus, the low-expression state associated with B′ is invariably associated with strong paramutagenicity (Coe 1966). In contrast, R-r′ is unstable with its pigment level and reversion frequency dependent on the strength of the R-st derivative to which it was crossed, the number of generations of heterozygosity with R-st, or when R-r′ is homozygous. When R-r′ is exposed to repeated generations of crosses with paramutagenic haplotypes, its seed pigmentation level continues to decrease to almost colorless and the haplotype becomes more paramutagenic (Mikula 1961; McWhirter and Brink 1962). Furthermore, expression of R-r is more reduced following passage through trisomics containing two doses of the paramutagenic allele relative to passage through disomic heterozygotes with one dose of the paramutagenic allele (Kermicleet al. 1995). Paramutation can also be reversed gradually if R-r′ is maintained as hemizygous or heterozygous with alleles that contain a single copy of the r1 locus, such that seed color becomes even darker than nonparamutant R-r (Styles and Brink 1966, 1969). This set of experiments, together, suggests that there is communication between the various r1 haplotypes and that this communication influences expression levels. One common feature of r1 and b1 paramutation is that tandem repeats are involved in paramutation at each locus, yet the actual sequences and properties of these repeats are quite distinct between the two loci. Within the r1 paramutagenic haplotypes, the repeats are of unknown length but each is likely quite large, spanning the r1-coding regions (Figure 1; Egglestonet al. 1995). The regions associated with paramutagenicity have been mapped for two r1 haplotypes using unequal crossing over between the multiple r1 genes in paramutagenic and nonparamutagenic haplotypes to generate a series of alleles with differing numbers of tandem repeats (Kermicleet al. 1995; Kermicle 1996; Panavaset al. 1999). Intriguingly, these results suggest that no specific region is required for r1 paramutation, but that only repeat number matters: paramutagenicity is reduced as repeat number is reduced and paramutagenicity is increased as repeat number is increased. The region within R-r required to respond to paramutation has also been mapped and lies within the inverted repeat structure of the S component (Brown and Brink 1960; Robbinset al. 1991; Kermicle 1996). Again, the r1 genes themselves lie within the sequences associated with paramutation. The results are quite different at b1 (Figure 2), in which specific sequences, an array of seven tandem repeats of 853 bp each, are required for paramutation; these are located >100 kb upstream of the single b1-coding region and share no sequence identity with the coding region (Stamet al. 2002a). Analyses of recombinant alleles differing only in the number of the upstream b1 tandem repeats demonstrated that tandem repeats are required for paramutation and the high expression of B-I (Stamet al. 2002a). Alleles that do not participate in paramutation have a single copy of this sequence, which is unique in the maize genome, and thus is not shared with other genes that also undergo paramutation, such as r1. The observation that tandem repeats are associated with paramutation might suggest a mechanism of repeat expansion and contraction. However, as discussed below, the numbers of repeats do not change with paramutation. What might be the basis for the allele and haplotype communication? Two possibilities are the RNA-based model or the DNA pairing model, but these are not mutually exclusive (reviewed in Chandler and Stam 2004). Both Brink and Coe explored various mechanisms for the cause of r1 and b1 paramutation using the genetic approaches available to them at the time. Both were on the right track in understanding that paramutation must involve direct or indirect contact between paramutagenic and paramutable alleles. Brink understood that during paramutation R-st and R-r must communicate early in development in new heterozygotes. By attempting to block paramutation through the use of translocated chromosomes that would disrupt pairing, he tested whether chromosome pairing might mediate the communication. The experiment did not lead to conclusive results because the prior genetic history of the alleles used confounded the interpretation (J. Kermicle, personal communication). Brink also tested for the existence of a “cytoplasmic particle,” loosely interpreted as a “trans-acting” or “communication molecule” that would be produced by R-st and transmit paramutation to R-r, but found no evidence for such (Brinket al. 1964). On the basis of studies at the b1 locus, Coe favored a model in which the paramutagenic allelic transferred a physical entity, which he hypothesized could be DNA or RNA (Coe 1968). We now know that there are no genetic changes among b1 alleles, but there is good evidence that RNA signals are involved in allele interactions (see below). At the b1 locus, the full penetrance and stability of paramutation is conferred by the seven tandem repeats, as paramutation with b1 alleles with three tandem repeats was less penetrant and less stable (Stamet al. 2002a). This result suggests that there is a mechanism for counting the numbers of repeats. Potential counting mechanisms could involve chromatin marks such as DNA and histone modifications, number of specific proteins bound, or RNA signals, whose level depends on the number of repeats. Brinket al. (1968) suggested that the r1 haplotypes exist in a wide continuum of states and that the ability to move along the continuum is an inherent property of the haplotype itself with the extent and direction of movement along the continuum influenced by the nature of the other allele present. Brink proposed that the r1 locus had two components: (1) the gene complex encoding the protein involved in anthocyanin synthesis and (2) a heterochromatic segment assumed to consist of varying numbers of a repeating unit called a metamere, which functioned to repress r1. He further proposed that the degree of repression was proportional to the number of metameres, which could change through misreplication during somatic mitosis (Brinket al. 1968). This hypothesis sprang from Brink's being influenced by the description of position-effect variegation (PEV) in Drosophila (reviewed in Lewis 1950). The influence likely resulted from the mosaic pattern of PEV in Drosophila eyes being strikingly similar to the mottled phenotype of paramutant R-r′ kernels and the idea that both PEV and paramutation were caused by aberrations in gene expression systems. Brink clearly sought a hypothesis that incorporated the essence of heterochromatin and repeated sequences without the requirement for large centric blocks that were known to be part of the PEV phenomenon. He attempted to avoid these inconsistencies with an alteration of terminology, for example, “ortho” and “parachromatin,” in which parachromatin is the part of the genome responsible for chromatin condensation and gene silencing (Brinket al. 1960). He called parachromatin the “nexus between chromosomal heredity and development” and provided that it could be “altered in accordance with the cellular substrate and could react to extra-chromosomal stimuli that define developmental progression.” This description sounds provocatively similar to RISC-complex-mediated gene silencing (Zaratieguiet al. 2007). The large tandem repeats associated with paramutagenic alleles are consistent with the metamere hypothesis, but molecular studies have demonstrated that the number of repeats does not change during plant development (M. Alleman, unpublished data); thus differences in r1 expression are likely to be mediated by epigenetic mechanisms (such as changes in DNA methylation and histone modifications). Potentially, the larger number of tandem repeats in paramutagenic alleles facilitates the communication with the inverted repeat at the sensitive locus—either directly through pairing or through some type of RNA-signaling molecule. At r1, a larger number of repeats could result in an increase in the amount of communication signal sent by paramutagenic haplotypes or an increased frequency of direct pairing. The inverted repeat nature of the paramutable haplotypes may make them particularly receptive to this signal. Characterization of trans-acting factors required for paramutation has begun to provide mechanistic clues. Recent results strongly suggest that the communication signal is likely to be RNA molecules produced and interpreted by genes involved in the RNAi transcriptional silencing pathway. Several trans-acting factors that affect paramutation have been identified (Dorweileret al. 2000; Hollick and Chandler 2001; Hollicket al. 2005). Two of these genes have been cloned: mop1, which encodes an RDR (Allemanet al. 2006), and rmr1, which encodes a putative protein al. 2007). Both of these proteins are closely related to proteins involved in transcriptional gene silencing in that the RNAi pathway mediating heritable chromatin structures is required for paramutation. The tandem repeats mediating b1 paramutation are on both (Allemanet al. 2006) and is required for an increased of from these repeats (M. and unpublished In to paramutation at multiple loci (Dorweileret al. the protein also influences the epigenetic of al. al. 2005; al. and certain silenced al. 2006), processes associated with RNAi mechanisms in multiple Both of paramutation and of the transcriptional silencing at B′ absolutely on mop1 (Dorweileret al. a role for RNA in both However, mop1 has a more role in the heritability of the reduced expression state. When mop1 are to type to the wild-type most of the have B′ expression levels (Dorweileret al. 2000). This result suggests that the protein can the epiallele from the mop1 is that the allele that it was B′ in the previous in spite of its high expression by the of and is expressed at a low level in the The hypothesis would that some type of heritable remain at the B′ allele even when it is at a B-I level and would that to be through when of in the of the that at a low frequency the B′ epiallele is changed to a B-I state that is heritable and to in the of the This contrasts with wild-type backgrounds in which B′ has never been observed to change to B-I. in the of the a can be such that it for multiple generations even in the of the protein by outcrosses al. This result is consistent with the of chromatin states that are to silencing. is also absolutely required to establish r1 paramutation (Dorweileret al. as R-r is not changed to R-r′ by R-st in homozygous mop1 Intriguingly, the protein is not required to maintain the reduced expression associated with R-r′ (J. Kermicle, personal communication). There are several potential an RNA mechanism may not be involved in the silencing of R-r. at r1, the silencing might be through the more DNA methylation that A could be that the R-r′ allele does not the RDR to maintain an RNA the inverted repeat structure at R-r (Figure 1) could generate RNA in This hypothesis is similar to is with the locus of which a RNA to the fully silencing it al. this silencing does not al. to the role of other RNAi pathway whether an RNAi pathway is involved in R-r′ silencing. in the r1 result is that the phenotype is observed only in alleles, which are to and 2000). DNA methylation within the inverted repeats in R-r does with its silencing and this methylation may to the of silencing in the of the pathway. is that the silencing mediated by is to the silencing mediated by and thus is the dramatic differences between r1 and b1 paramutation, it is striking that both involve repeated sequences of some type and that an RNA the two silencing and heterochromatin silencing in multiple species also involve repeated sequences, and chromatin changes are mediated by of the RNAi pathway and al. 2007). similar proteins are involved in maize paramutation and DNA methylation in it is that paramutation has yet to be described in The locus in is the most similar to b1 paramutation in that tandem repeats from a silenced locus can communicate with an to it and 2007). a silenced locus does not an allele. is the locus to silencing while the is additional What the extremely and highly heritable associated with paramutation in What is the nature of the RNA that are tandem repeats What are the heritable molecules or does paramutation exist and is it on some of these are discussed in Chandler approaches additional key genes required for paramutation and understanding the between tandem repeat RNA and chromatin structure for understanding this process. Paramutation studies in the Chandler were by to from the and the of a from the We Kermicle for