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Chromatin looping and epigenetic regulation at the maize b1 locus

Louwers, M.L.D.

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Citation for published version (APA):Louwers, M. L. D. (2008). Chromatin looping and epigenetic regulation at the maize b1 locus.

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Chapter 6

Epigenetic regulation, chromatin looping and paramutation

at the maize b1 locus Marieke Louwers and Maike Stam

Adapted from: ‘Paramutation: heritable in trans effects’ The Maize Handbook: Domestication, Genetics, and Genome. Edited by Dr. Jeff L. Bennetzen and Dr. Sarah C. Hake

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Abstract Paramutation is the heritable transfer of epigenetic information from one allele of a gene to another allele of the same gene. In general, the consequence of this trans-communication is a change in gene expression. Paramutation has been observed in plants, fungi and mammals and has been extensively studied at the maize b1 locus. At b1, epigenetic regulation and chromatin looping control the b1 expression level. For decades, paramutation has been a mystery, but recent progress has shed light on the mechanisms underlying this phenomenon; RNA plays a crucial role in the trans-inactivation process, but it appears not the only player in the paramutation process. We speculate that physical in trans interactions could be involved in paramutation as well. In this chapter, potential mechanisms will be discussed in light of recent data obtained for the b1 locus.

1 Introduction Gene expression is regulated by genetic as well as epigenetic mechanisms, and through DNA sequences located in cis and in trans. Epigenetic regulation involves heritable changes in gene expression that occur without a change in DNA sequence. Epigenetic mechanisms, such as DNA methylation, histone modifications and the incorporation of histone variants are fundamental for the regulation of eukaryotic gene expression, and thus essential for normal growth and development of multi-cellular organisms. Two epialleles of the maize b1 gene provide an excellent model system to study epigenetic regulation of gene expression. Regulatory sequences affecting gene expression can be present nearby the transcription unit, but can also be located at a considerable distance. In mammals, many examples are known where gene expression is regulated by long-distance in cis interactions between promoters, enhancers and locus control regions (Carter et al., 2002; Horike et al., 2005; Murrell et al., 2004; Spilianakis and Flavell, 2004; Tolhuis et al., 2002). To our knowledge, the first example of long-range in cis interactions in plants involves the maize b1 locus. At this locus, physical in cis interactions over a distance of more than ~100kb were shown to play a role in gene expression (chapter 3). Besides in cis regulation of gene expression, there is increasing evidence that gene regulation in trans, i.e. the control of gene expression by sequences on another chromosome, is

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important in higher eukaryotes, including plants, flies and mammals (Chandler, 2007; Bacher et al., 2006; Lee and Wu, 2006; Lee et al., 2004; Lomvardas et al., 2006; Rinn et al., 2007; Spilianakis et al., 2005; Xu et al., 2006). Paramutation combines epigenetic and in trans regulation of gene expression and is defined as a mitotically and meiotically heritable change in gene expression induced by trans-interactions between homologous alleles (Chandler and Stam, 2004; Stam and Scheid, 2005). This change in gene expression is not caused by a change in DNA sequence, but rather a change in DNA methylation (for example see Meyer et al., 1993; Rassoulzadegan et al., 2002; Sidorenko and Peterson, 2001; Walker and Panavas, 2001), and/or chromatin structure (Chandler et al., 2000; Stam et al., 2002a; vanBlokland et al., 1997) and is therefore an epigenetic phenomenon. Paramutation occurs at a much higher frequency than genetic mutations and is potentially reversible. Paramutation phenomena have been observed in plants, fungi and mammals (extensively reviewed in (Louwers et al., 2005; chapter 1), but have been studied most extensively in maize thanks to the long-standing history of maize genetics. In this chapter, our recent data on the epigenetic regulation and chromatin looping of two b1 epialleles involved in paramutation will be put in perspective. Potential mechanistic models for paramutation will be discussed in light of these new data.

2 Paramutation at the b1 locus There are two types of alleles required for the paramutation process, paramutagenic (inducing) and paramutable (sensitive) alleles. When combined in one nucleus, the two alleles communicate in trans and as a result the expression level of the paramutable allele is changed by the paramutagenic allele. Paramutation at the b1 locus occurs between two epialleles, B-I and B’ (Stam et al., 2002a). B-I is transcribed approximately 10-20 times higher than B’ (Patterson et al., 1993). When combined in one nucleus, B’ and B-I alleles communicate in trans and as a result, the elevated expression level of the B-I is down-regulated by B’. The new B’ allele is in turn able to change the expression level of B-I alleles (secondary paramutation; Patterson et al., 1993). The low expressing B’ state is extremely stable; in contrast, the high expressing B-I state is not. B-I can spontaneously change into B’ with a

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frequency of 1-10% (spontaneous paramutation; Coe, 1966). Multiple 853bp repeats ~100kb upstream of the transcription start site are required for paramutation and high expression (Stam et al., 2002b). B-I and B’ contain seven copies of the 853bp sequence in a direct orientation. These are referred to as the hepta-repeat. Many b1 alleles are known (Selinger and Chandler, 1999) and the majority does not participate in paramutation, but follows the classic laws of Mendelian inheritance. These alleles are neither paramutagenic nor paramutable and are termed neutral alleles. The neutral b1 alleles analyzed contain only one copy of the 853bp sequence (Stam et al., 2002b). 2.1 Three Mechanistic Aspects Paramutation phenomena are characterized by three important mechanistic aspects: 1) gene expression, 2) paramutagenicity and paramutability, and 3) epigenetic memory. Gene expression concerns the expression level of the alleles involved in paramutation (Figure 1A). In general, the paramutagenic allele displays a low, and the paramutable allele a high expression level; this is also the case for the paramutagenic B’ and paramutable B-I. A neutral allele can display any expression level. Paramutagenicity refers to the ability of a paramutagenic allele to paramutate a paramutable allele (Figure 1B); paramutability refers to the ability of a paramutable allele to undergo paramutation. Paramutagenicity and paramutability are not necessarily the same as epigenetic memory. Epigenetic memory represents the epigenetic marks that specify the heritability of a specific epigenetic state, such as the paramutagenic state (Figure 1C). These epigenetic marks are likely to involve DNA modifications and/or a particular chromatin structure (Henderson and Jacobsen, 2007; Martin and Zhang, 2007). Epigenetic memory can be very stable. In such case, the epigenetic state of for example a paramutagenic allele does not readily change into a paramutable state. Multiple generations in the presence of a mutation affecting paramutation are needed to erase the epigenetic marks defining the paramutagenic state. We postulate that in the case of an unstable epigenetic memory, a paramutagenic state will readily change into a paramutable state in the presence of a mutation affecting paramutation.

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Figure 1. Mechanistic aspects of paramutation. A. Gene expression. Paramutation most often involves a change in gene expression. The paramutable, sensitive allele is usually highly expressed (black symbol), such as the maize B-I allele. The paramutagenic, inducing allele is mostly low expressed (dark grey symbol), such as the B’ allele. B. Paramutagenicity and paramutability. Paramutagenicity refers to the ability of a paramutagenic allele to change the expression level of a sensitive allele. Paramutability refers to the ability of a paramutable allele to undergo paramutation. In the left panel the wild-type situation is

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shown. In the right panel, a mutation preventing paramutation is present in the F1. As a result, the light expressed allele lost its paramutagenicity. C. Epigenetic memory. Epigenetic memory represents the epigenetic marks specifying the heritability of a specific epigenetic state, paramutagenic or paramutable. The epigenetic memory can be stable or unstable. In this example, the paramutagenic, low expressed allele becomes highly expressed and looses its paramutagenicity in the presence of a mutation affecting both aspects. In case of a stable epigenetic memory, once the mutation is removed the allele will revert to its original expression state and is paramutagenic again. The epigenetic memory can however be lost after a few generations in the presence of a mutation affecting paramutation. In that case, once the mutation is removed, the allele will stay in the high expression state, because it cannot ‘remember’ what the original expression state used to be. In case of an unstable epigenetic memory, already after one generation in the presence of a mutation affecting paramutation, the paramutagenic allele reverts to a high expression state and looses its paramutagenicity.

Mutations affecting paramutation (Chandler and Stam, 2004; Dorweiler et al., 2000; Hale et al., 2007; Hollick and Chandler, 2001; Hollick et al., 2005) have shown that the three aspects can be mechanistically separated from each other. For example, in maize plants homozygous for the mop1-1 mutation, the expression level of the paramutagenic B’ allele is ~3-4 times enhanced (Dorweiler et al., 2000). In the same plants, the B’ allele lost its paramutagenicity, but not its epigenetic memory. Upon removal of the mop1-1 mutation by crossings, the B’ allele behaves as B’ again: it is expressed at a low level and can paramutate B-I. This indicates that, although the expression level and paramutagenicity of B’ were altered in the mop1-1 mutant background, the B’ allele kept its epigenetic memory.

In the presence of another mutation, rmr1, the expression level of the paramutagenic maize Pl’ allele is elevated, but it can still induce paramutation (Hale et al., 2007). Similar observations have been made with the rmr6-1 mutation (Hollick et al., 2005). The use of mutations affecting paramutation makes it possible to experimentally separate the three aspects of the paramutation process, which is instrumental in unraveling the mechanisms underlying paramutation.

2.2 DNA Methylation and Chromatin Structure Epigenetic regulation of gene expression is the outcome of an intricate interplay between various epigenetic mechanisms such as DNA methylation, histone modifications and the incorporation of histone variants (DAlessio and Szyf, 2006; Henikoff and Ahmad, 2005; Klose and Bird, 2006; Tariq and Paszkowski, 2004).

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Repressed chromatin is usually characterized by DNA hypermethylation and histone modifications such as histone H3 lysine 9 and lysine 27 methylation (H3K9me & H3K27me), while active chromatin is recognized by DNA hypomethylation and histone modifications such as histone H3 lysine 4 methylation and H3 acetylation. In most paramutation systems, the transcriptionally silenced paramutagenic allele is DNA hypermethylated, and the transcriptionally active paramutable allele DNA hypomethylated. When combined in one nucleus, the trans-inactivation of the paramutable allele is associated with the acquisition of DNA methylation (Eggleston et al., 1995; Forne et al., 1997; Hatada et al., 1997; Meyer et al., 1993; Mittelsten Scheid et al., 2003; Rassoulzadegan et al., 2002; Sidorenko and Peterson, 2001; Stam et al., 2002a; Walker, 1998; Walker and Panavas, 2001). For example, in case of b1 paramutation, the B’ repeats, ~100 kb upstream of the b1 transcription start site, are DNA hypermethylated relative to the B-I repeats in all the various tissues and developmental stages examined (Haring et al., submitted). When combined in one nucleus, the acquisition of DNA methylation at the B-I repeats can already be observed a few days after germination. Importantly, simply a difference in DNA methylation level between two epialleles is not sufficient for paramutation to occur. For example, the hypermethylated, inactivated Arabidopsis SUPERMAN and FWA alleles do not trans-inactivate their hypomethylated counterparts (Jacobsen and Meyerowitz, 1997; Soppe et al., 2000; Chan et al., 2006b). What would be the role of DNA methylation in paramutation? The results described in Chapter 4 indicate that DNA methylation plays an important role in the heritability of the silenced, paramutagenic B’ epigenetic state and we propose this is true for other paramutagenic alleles. Data reported in literature on the role of DNA methylation are in line with such a hypothesis (Dieguez et al., 1998; Jones et al., 2001; Kato et al., 2003; Soppe et al., 2002). This might also explain why paramutation has not yet been described in organisms lacking extensive DNA methylation like Drosophila, C. elegans, S. pombe and S. cerevisiae. Epigenetic regulation of gene expression not only involves changes in DNA methylation, but also changes in chromatin structure. For the inactive, paramutagenic maize b1 epialleles, it has been shown that they are less accessible to nucleases than their active, paramutable counterparts (Chandler et al., 2000; Stam et al., 2002a). This also holds for the petunia A1 epialleles (vanBlokland et al., 1997).

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The role of histone modifications in paramutation is the subject of current research. Recent data indicate that the paramutagenic B’ and paramutable B-I alleles carry a different set of histone modifications at their repeats (Haring et al., submitted). Tissue-specific enhancement of B-I expression is associated with nucleosome depletion and H3 acetylation at the hepta-repeat. The B’ hepta-repeat is H3K27 methylated in a tissue-independent, and H3K9 methylated in a tissue-dependent manner. The latter is associated with nucleosome depletion. In contrast to DNA methylation, most of these histone modifications are tissue-specifically regulated. They reflect the expression level in the tissue analyzed, rather than the epigenetic state of the alleles (Haring et al., submitted). The only exception is the localization of histone H3 lysine 27 dimethylation (H3K27me2) at the repeats. This has been observed in both tissues examined (seedling shoots and husk leaves). These data fit a model in which DNA and H3K27 methylation play an important role in the epigenetic inheritance of the low B’ expression state. Analyses of the B’ epiallele in a mop1-1 mutant indicated that H3K27me2 is not required to maintain the epigenetic B’ state (chapter 4). This does not necessarily mean that H3K27me2 does not play a role at all. It is possible that, in case H3K27me2 is present at the hepta-repeat, it contributes to the inheritance of the epigenetic B’ state. In a wild-type background, the low expressing B’ epiallele is in an inactive chromatin state and the high expressing B-I epiallele in an active chromatin state (Haring et al., submitted). Interestingly, B’mop1-1 contains silent and active marks at the hepta-repeat, and also at the coding region. The data in chapter 4 of this thesis suggest that it is the interplay between active and silent marks that determines the intermediate expression level of B’ in the mop1-1 mutant.

3 Epigenetic regulation and chromatin looping Physical interactions between promoters and cis-acting regulatory sequences can determine the expression level of a gene (Fraser, 2006; Fraser and Bickmore, 2007). Recent technical developments allow the identification of long-range in cis interactions (reviewed in Simonis et al., 2007). The Chromosome Conformation Capture technique (3C; Dekker et al., 2002; Tolhuis et al., 2002) is such a technique, measuring physical interactions between chromatin regions. Multiple repeats

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~100kb upstream of the transcription start site (TSS) are required for high B-I expression (Stam et al., 2002b), suggesting long-range communication between the hepta-repeat and the b1 coding region. To test the role of long-distance in cis interactions in enhancement of b1 expression the involvement of physical interactions in regulating b1 expression, we successfully set up 3C for maize tissue (chapter 2).

3C analysis on the maize b1 locus indicated the occurrence of tissue-specific and expression level-specific long-range interactions. In both B-I and B’, the TSS region interacts with the hepta-repeat region ~100kb upstream (chapter 3). This interaction is tissue-specifically regulated; it occurs more frequently in husk than in inner stem leaves. Moreover, this interaction is in part dependent on the expression level; the interaction frequency was higher in high expressing B-I than in low expressing B’ husk tissue. Finally, multiple repeats were shown to be required for a frequent interaction with the TSS region. Additional expression level-specific interactions were observed: regions ~107kb, ~47kb, ~15kb upstream interact with the transcription start site (TSS) and hepta-region region exclusively in high expressing B-I husk tissue (chapter 3). This indicates that in B-I husk a multi-loop structure is formed between the TSS, the hepta-repeat and regions ~15kb, ~47kb and ~107kb upstream of the TSS. Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) analysis suggested that in high expressing B-I husk, regions ~15kb, ~47kb and ~107kb upstream contain active regulatory sequences. In B’ husk tissue, interactions other that the one between the hepta-repeat and the TSS were not observed, indicating only one chromatin loop at the B’ epiallele. We propose that the multi-loop conformation mediates the high B-I expression level. In contrast, the formation of a single, less stable, loop between the TSS region and the hepta-repeat in B’ husk tissue is not sufficient for high b1 expression.

Three mediator of paramutation (mop) mutants are reported to affect the B’ expression level. 3C analysis of these mutants confirmed that defined chromatin conformations are associated with particular b1 expression levels. The expression of B’ is up-regulated in mop1-1 and mop3-1 mutants and the B’ epiallele displayed a B-I-like multi-loop structure (chapters 4 and 5). The low B’ expression in mop2-1/Mop2 was associated with a B’-like single loop formation (chapter 5). The discovery of physical in cis interactions at epialleles involved in paramutation makes

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it tempting to speculate whether physical in trans contact occurs during paramutation.

4 Paramutation Models Based on recent progress in the field, two models are proposed to explain paramutation at the molecular level, an RNA model and an RNA-physical interaction model. Importantly, neither the production of a paramutagenic RNA nor physical contact would need to occur permanently, provided they last long enough to trigger a heritable change. 4.1 RNA Model In the RNA model, the in trans communication between a paramutagenic and paramutable allele, and the resulting change in gene expression are mediated by a diffusible RNA molecule (Figure 2A). RNAs derived from the paramutagenic allele affect the chromatin structure and transcriptional activity of the paramutable allele. In this RNA model, RNAs are produced from the paramutagenic allele, but not from the paramutable allele. RNAs, and especially small interfering RNAs (siRNAs), have been implicated in numerous regulatory processes and epigenetic phenomena, and recent evidence indicates they are also involved in paramutation (Alleman et al., 2006; Rassoulzadegan et al., 2006; van West et al., 1999). The outcome of siRNA-mediated silencing pathways varies from heterochromatin formation to post-transcriptional gene silencing (Grewal and Elgin, 2007; Matzke and Birchler, 2005; Mello and Conte, 2004; Vaucheret, 2006; Zaratiegui et al., 2007). Paramutation usually results in transcriptional gene silencing. Thus, RNA-mediated heterochromatin formation models would be most applicable. In such models RNAs derived from the target locus are made double-stranded by RNA-dependent RNA polymerase and processed by Dicer into siRNAs, which in turn mediate heterochromatin formation via RNA-directed DNA methylation (RdDM) and RNA-directed histone modifications (H3K9 and H3K27 methylation; Chan et al., 2006a; Liu et al., 2007; Matzke et al., 2007; Ting et al., 2005; Zaratiegui et al., 2007). This heterochromatinization process might be chromosome-associated, like observed for

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fission yeast (Grewal and Elgin, 2007; Zaratiegui et al., 2007). A paramutation-like phenomenon consistent with the RNA model is that reported by Van West et al (1999). This Phytophtera infestans system was indicated to involve transcriptional silencing and a sequence-specific, diffusible factor, which is likely to be RNA.

Figure 2. Models. In this figure, the sequences required for paramutation are depicted as directly repeated sequences (arrows). In the paramutagenic allele they have a repressed chromatin structure, in the paramutable allele they are in an active chromatin state and act as an enhancer. A. RNA model. The sequences required for paramutation are transcribed exclusively from the paramutagenic (Pg) allele and double-stranded RNA (dsRNA) is produced due to the activity of an RNA-directed RNA polymerase (RdRP). A Dicer-like protein cleaves the dsRNA into siRNAs which then act as diffusible factors and influence the chromatin structure of the paramutable (Pm) allele via RNA-dependent DNA methylation (RdDM) and RNA-directed histone modifications. Once the chromatin structure of the paramutable allele is repressed,

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enhancer complexes can no longer remain associated and silencing complexes bind instead. The allele becomes paramutated (Pt) and in this example also paramutagenic. It consequently produces RNA, which is processed into siRNAs. Repeat-derived siRNAs not only affect paramutable alleles, but also reinforce the silent state of the paramutagenic allele. B. RNA-physical interaction model. In this model, RNA as well as physical pairing between homologous sequences is required for in trans-inactivation. Both the paramutable and paramutagenic alleles are transcribed and the RNA processed into siRNAs by a Dicer-like enzyme. However, only the paramutagenic, repressed chromatin state is susceptible to silencing by siRNAs. Physical pairing between the paramutagenic and paramutable allele results in an exchange of proteins that alter the paramutable allele into a paramutated allele. The chromatin state of the paramutated allele is susceptible to the silencing effect of siRNAs.

4.2 RNA-Physical Interaction Model In the RNA-physical interaction model, the transfer in trans of the transcriptionally inactive paramutagenic state onto the paramutable counterpart is dependent on both pairing and RNAs, most likely siRNAs (Chandler and Stam, 2004; Stam and Scheid, 2005). The RNAi machinery has recently been implicated in various aspects of higher-order chromatin organization in the nucleus. For example, the clustering of the heterochromatic telomeres in S. pombe (Hall et al., 2003) and the formation of higher-order insulator complexes in Drosophila is disrupted in RNAi mutants (Lei and Corces, 2006). It has therefore been proposed that RNAs act as a ‘glue’ to promote the clustering of heterochromatic regions into higher-order structures (Grewal and Moazed, 2003). Another precedent of the RNA-physical interaction model is the trans-interactions mediated by Polycomb group (PcG) proteins (Grimaud et al., 2006). It has been shown that PcG-like proteins can mediate physical in trans-interactions (Lavigne et al., 2004) and pairing-dependent silencing in Drosophila (Bantignies et al., 2003). Importantly, the RNAi machinery is shown to be required for PcG-mediated silencing, and more specifically for the maintenance of long-range physical interactions (Grimaud et al., 2006). In female X-inactivation, non-coding RNAs are involved in silencing one of the two X chromosomes (Sun et al., 2006). Intriguingly, both X chromosomes transiently co-localize at the onset of X chromosome inactivation (Bacher et al., 2006; Xu et al., 2006). Meiotic silencing, a silencing phenomenon observed in Neurospora, involves both pairing and RNA silencing as well (Lee et al., 2004). In conclusion, in the RNA-physical interaction model, trans-interactions between paramutagenic and paramutable alleles are mediated by both the RNAi machinery and proteins involved

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in pairing, e.g. PcG-like proteins. Fluorescence In Situ Hybridization (FISH; Grimaud et al., 2006) experiments need to be performed to address the role of physical in trans interactions in paramutation. 4.3 Paramutation Models in the Context of Repeated DNA Repeated sequences, which are required for paramutation in most paramutation phenomena (see chapter 1 of this thesis), are compatible with both models proposed. Transcription of repeats, whether in a direct or inverted orientation, can lead to an efficient production of double-stranded RNA (dsRNA), whereby in the case of direct repeats the action of RdRP is required (Martienssen, 2003; Slotkin and Martienssen, 2007; Zaratiegui et al., 2007). After processing of the dsRNAs by Dicer, the resulting siRNAs mediate transcriptional silencing. On the other hand, repeated sequences have been shown to physically pair more often than single copy sequences. In Arabidopsis, transgenic lac operator arrays associate more often with each other than average euchromatic regions, and the same is observed for an inactive transgenic multicopy HPT locus (Pecinka et al., 2005). Intriguingly, DNA hypermethylation of repeated sequences is shown to increase the frequency of pairing between homologous sequences (Watanabe et al., 2005). In most paramutation systems, paramutagenic alleles have been shown to be hypermethylated (see 2.2), and might therefore display an enhanced pairing frequency, possibly facilitating paramutation. 4.4 Paramutation Models in the Context of Recent Data For long it was only possible to wildly speculate about the mechanisms underlying paramutation. The cloning of the mop1-1 mutation and other recent data (Alleman et al., 2006; Chandler, 2007) has provided a revealing glimpse into the mechanisms involved. Mediator of paramutation1 (Mop1) appeared to encode an RNA-dependent RNA polymerase (RdRP; Alleman et al., 2006; Woodhouse et al., 2006), indicating the involvement of RNA in paramutation. MOP1 is most homologous to the RDR2 protein of Arabidopsis; RDR2 is associated with the production of short interfering RNAs (siRNA) that play a role in RNA-dependent DNA methylation of repeats (RdDM; Chan et al., 2004). The mop1-1 and rdr2 mutants do however not behave exactly the same, indicating that MOP1 and RDR2 are functionally divergent

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to some extent. An rdr2 mutant for example, exhibits demethylation of the 5S ribosomal DNA (Xie et al., 2004), whereas a mop1-1 mutant does not (Dorweiler & Chandler personal communication). Consistent with the idea that RNA is involved in paramutation, the repeats are transcribed (Alleman et al., 2006) and siRNAs produced (M Arteaga-Vasquez and VL Chandler, personal communication). However, not only the repeats of B’, but also those of B-I and a neutral b1 allele are transcribed, at comparable levels (Alleman et al., 2006). In addition, repeat siRNAs are detected from all three alleles (M Arteaga-Vasquez and VL Chandler, personal communication), suggesting that although RNAs appear required, repeat transcription and siRNAs are not sufficient to establish and/or maintain a paramutagenic state. This finding is reminiscent to results reported by Chan et al. (2006), who detected tandem repeat-derived siRNAs from both the silenced and active FWA locus. Only the silenced, DNA methylated FWA locus could recruit RNA-directed DNA methylation, reinforcing its own silenced state in cis. The authors postulate that features of the siRNA-producing locus, such as the DNA methylation level and chromatin structure, determine the susceptibility to siRNA-directed DNA methylation (RdDM). In analogy, we speculate that the chromatin structure of the B-I hepta-repeat is not susceptible to RdDM, while that of B’ is. The B-I repeats contain a low level of DNA methylation, while those of B’ are heavily methylated in all tissues analyzed. In addition, the B’ repeats are H3K27 methylated in both seedling and husk tissue (Haring et al., submitted). We propose that this tissue-independent DNA and histone methylation make the B’ repeats receptive for RdDM and RNA-directed histone modifications, which in turn reinforce the repressed chromatin structure. In this model, the siRNAs derived from the B’ repeats can contribute to the maintenance of the B’ repeat DNA methylation pattern and thereby the heritable, paramutagenic state of the B’ epiallele (chapter 4), while the repeat siRNAs derived from the B-I or neutral b1 allele cannot.

The previous paragraph mainly discusses the role of siRNAs in the heritable maintenance of the B’ and B-I epigenetic states. How is the B’ state established? How does the B’ epiallele change the B-I epigenetic state into a B’ epigenetic state when both epialleles are combined in one nucleus? As discussed above RNAs are required, but they are not sufficient (Alleman et al., 2006;

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Chandler, 2007). In trans inactivation of FWA transgenes requires the Agrobacterium-mediated genetic transformation process (Chan et al., 2006b). What are the additional requirements for b1 paramutation? We propose that pairing, stimulated by the DNA hypermethylated B’ repeats, is such a requirement (Figure 2B). The physical pairing could allow the transfer of proteins mediating paramutation from the B’ to the B-I allele. The presence of H3K27 methylation at the B’ repeats suggests that besides RNAs, pairing mediated by PcG-like proteins or other pairing proteins, could be involved. H3K27 methylation has been implicated in both RNAi-dependent heterochromatin formation and PcG-protein-mediated chromatin silencing (Bantignies and Cavalli, 2006; Liu et al., 2007; Peters and Schubeler, 2005; Weinberg et al., 2006). Further studies are needed to investigate if this is actually the case. Alternatively, as a result of the different chromatin structure, the B’ repeat siRNAs obtain specific features that the siRNAs derived from B-I and the neutral b1 allele lack. These features would enable the B’ siRNAs to affect a paramutable allele in trans. Yet another option would be that a specific set of not yet detected B’ repeat siRNAs are involved in paramutation. The presence of these siRNAs, which should only be present in B’ and not in B-I plants, may be limited to a narrow developmental time window, a specific tissue or a set of cells not yet investigated.

5 Concluding Remarks and Future Directions The b1 paramutation system has been extensively studied. The identification of MOP1 as an RNA-dependent RNA polymerase shows that RNA plays a crucial role in the trans-inactivation process. However, RNA appears not the only player in the paramutation process; physical interactions may be involved in paramutation as well. In this chapter we discussed two models: an RNA model and a combined RNA-physical interaction model, but other models might apply as well. To reveal the mechanisms involved, cloning and thorough characterization of the players involved is crucial, also for their effects beyond paramutation. By now, multiple trans-acting mutations implicated in various aspects of paramutation have been isolated (Dorweiler et al., 2000; Hollick and Chandler, 2001; Hollick et al., 2005)

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and excitingly, two of them have been cloned recently (Alleman et al., 2006; Hale et al., 2007) and cloning of others is underway.

An important aspect for future research is the timing of paramutation. The change from a paramutable into a paramutagenic allele takes place after combining both alleles in one zygote, but does not necessarily occur immediately. Furthermore, the change in epigenetic state most likely involves multiple events such as the change in expression state of the paramutable allele, and the imposition of the epigenetic marks representing the epigenetic memory. Studies on mutations affecting paramutation have shown that these aspects can be separated from each other (Dorweiler et al., 2000; Hale et al., 2007; Hollick and Chandler, 2001; Hollick et al., 2005). In case of b1 paramutation, the change in expression can be monitored at the seedling stage (Chandler et al., 2000), while the epigenetic memory might only be established in plants carrying ten expanded leaves (Coe, 1966; Ed Coe, personal communication). Besides the timing, the tissue or cell type in which paramutation occurs needs to be determined. Knowledge about the time and place of paramutation is crucial in order to pinpoint the mechanisms involved in paramutation. The RNA-physical interaction model discussed in this chapter implicates a role for physical pairing in paramutation. 3C is an excellent technique to study in trans interactions between homologous sequences. At present, there is even a plant-specific 3C protocol available (chapter 2). Unfortunately, we cannot apply 3C to study the existence of in trans interactions between the B-I and B’ hepta-repeats during paramutation. To design primers specific for each allele, sequence diversity is required. As B-I and B’ are epialleles, this cannot be done. No other alleles are available that are both involved in b1 paramutation and exhibit sequence differences. High resolution Fluorescent in situ hybridization (FISH), such as cryo-FISH (Branco and Pombo, 2006), in which the hepta-repeat is fluorescently labeled can be applied to study the co-localization of the B-I and B’ hepta-repeats. References Alleman, M., Sidorenko, L., McGinnis, K., Seshadri, V., Dorweiler, J.E., White, J., Sikkink, K. and

Chandler, V.L. (2006) An RNA-dependent RNA polymerase is required for paramutation in maize. Nature, 442, 295-298.

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UvA-DARE (Digital Academic Repository) Chromatin looping and epigenetic regulation … · important in higher eukaryotes, including plants, flies and mammals (Chandler, 2007; Bacher