Briefings in Functional Genomics Advance Access published July 2, 2015 Briefings in Functional Genomics, 2015, 1–10 doi: 10.1093/bfgp/elv025 Review paper

The roles of cross-talk epigenetic patterns in Arabidopsis thaliana Jingjing Wang, Xianwen Meng, Chunhui Yuan, Andrew P. Harrison, and Ming Chen

Abstract The epigenetic mechanisms, including histone modifications, DNA cytosine methylation, histone variants and noncoding RNAs (ncRNAs), play a key role in determining transcriptional outcomes. Recently, many studies have demonstrated that the different epigenetic mechanisms interplay with each other rather than work independently. In this article, we outline a framework for how different epigenetic mechanisms work with each other in Arabidopsis thaliana. We build a network of cross-talk between chromatin marks based on six classes of cross-talk interactions. The first pattern details coordinated modifications that act together to enhance or repress gene expression. The second pattern details bivalent modifications that act antagonistically toward gene expression. The third pattern is for unilateral promotion of one modification by the existence of another modification. The fourth pattern is for unilateral inhibition of one modification by another modification. The fifth pattern is for mutual inhibitory patterns. The sixth pattern is for epigenetic modifications that appear independent. We also explore the mutual regulation between chromatin marks and ncRNAs in various ways. These regulations can be divided into six parts: how ncRNA affects the binding of chromatin mark, such as miR2Epi, siR2Epi and lncR2Epi; how chromatin mark regulates ncRNA, such as Epi2miR, Epi2siR and Epi2lncR. A comprehensive network of cross-talk between different epigenetic mechanisms will help in fully understanding the functional roles and biological impacts of epigenetic regulation. Key words: histone modification; DNA methylation; histone variant; noncoding RNA; cross-talk network

Introduction In eukaryotes, the DNA is packaged into a complex structure known as chromatin, whose fundamental repeating unit is the nucleosome [1, 2]. A nucleosome is composed of 147 base pairs of DNA, wrapped around an octamer of four core histones, H2A, H2B, H3 and H4 [3, 4]. Chromatin is divided into different

functional regions by posttranslational modifications of histones, histone variants and DNA methylation in the form of 5methylcytosine. All of these chromatin marks have a substantial effect on chromatin structure and gene function [1, 5]. There are a variety of histone modification types, such as methylation, acetylation, phosphorylation and ubiquitination

Jingjing Wang is a PhD student in Ming Chen’s laboratory in Zhejiang University. Her research focuses on epigenetic regulation network in plants and animals. Xianwen Meng is a Master student in Ming Chen’s laboratory in Zhejiang University. His research focuses on transcription factors regulation network in plants and animals. Chunhui Yuan is a PhD student in Ming Chen’s laboratory in Zhejiang University. Her research focuses on noncoding RNAs regulation network in plants. Andrew Harrison is a Senior Lecturer in the Department of Mathematical Sciences, University of Essex. His research concentrates on identifying biases in scientific data sets that are available in the public domain. Ming Chen is a Professor in the Department of Bioinformatics, College of Life Sciences, Zhejiang University. His current research focuses on construction of small RNA-mediated regulatory networks and establishing useful web servers and platforms to help biologists browse and analyze massive biological data sets. C The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected] V

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Correspondence author. Ming Chen, Department of Bioinformatics, State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China. Tel.: þ86-571-88206612; Fax: þ 86-571-88206612; E-mail: [email protected]

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Epigenetic distribution, establishment and their relationship with gene transcription Histone modifications The genomic distributions pattern of mono-, di- and trimethylation of histone H3 at lysine 4 (H3K4me1, H3K4me2 and H3K4me3) have revealed similar localization patterns between Arabidopsis and rice [10, 11]. However, the three methylation forms of H3K4 distribute differently from each other (Figure 1). H3K4me1 is enriched within the gene body with an apparent 30 -end bias [2, 11]. However, both H3K4me2 and H3K4me3 accumulate in the promoters and 50 genic regions, with the distribution of H3K4me3 slightly upstream of H3K4me2. H3K4 methylation is known as a euchromatic mark and H3K4me3 is often linked to active genes [12, 13]. Histone H3 di-methylation at lysine9 (H3K9me2) is enriched in both the promoter and gene body (Figure 1), which has been found in a limited number of repressed genes in Arabidopsis [14]. Another methylation of H3K9, H3K9me3, shows a different distribution, enriched at the 50 and 30 end of genes, and tends to mark some highly expressed genes in plants [15]. However, compared with H3K4me3, H3K9me3 is a weak activator. Moreover, the results for Arabidopsis are unlike those in animals, where H3K9me3 is mostly associated with repressive chromatin states [5]. Mono-, di- and tri-methylation at lysine 27 of histone H3 (H3K27me1, H3K27m2 and H3K27me3) prefer to mark across transcribed regions (Figure 1). H3K27me1 is highest more centrally but with a relatively low level [15–18]. Moreover, the three forms of H3K27 tend to be associated with tissue-specific and developmentally regulated genes. H3K27me1 and H3K27me2/3 target largely nonoverlapping sets of genes with different ontologies [5]. H3K27me1 is largely enriched in heterochromatic

chromocenters, whereas H3K27me2 and H3K27me3 are both mainly enriched in euchromatic regions [18, 19]. Di- and tri-methylation of lysine 36 (H3K36me2 and H3K36me3) are both abundant in highly expressed genes. However, they exhibit different distributions from H3K4me3, since H3K36me2 and H3K36me3 accumulate across the transcribed region (Figure 1) [16, 20]. Acetylation at different lysine residues on histone H3 has a similar distribution pattern, with peaks that are always enriched within genes with a bias toward 50 -end of genes and peaking around the transcriptional start site (TSS) [14, 15, 20]. This is in contrast to methylation of the H3 lysine residues, whose distribution patterns differ for each of the methyl groups added. Over the past decade, studies have found that acetylation is often associated with active transcription [1]. In Arabidopsis thaliana, H2B monoubiquitination (H2Bub) can be induced by both the ubiquitin-conjugating enzyme 1 (UBC1), UBC2 pathway and the HISTONE MONOUBIQUITINATION 1(HUB1), HUB2 pathway [21–23]. H2Bub is another chromatin mark that links to transcriptional activation, showing an even distribution across genic regions, especially the central part [5]. There is no relationship between gene downregulation and H2Bub levels [24].

Histone variants Histone variants also regulate the repertoire of chromatin by affecting nucleosome stability and histone–protein interactions [25, 26]. In the Arabidopsis genome, there are two main types of histone H3, namely H3.1 and H3.3 [27]. H3.3 differs only in four or five amino acids from the canonical H3.1 [26]. Both H3 variants are more enriched in gene bodies compared with their 50 and 30 flanking regions [26, 28]. The H3.3 signal is also enriched at the downstream of the 30 end. In contrast, H3.1 does not display distinct occurrences at either gene end [26, 28]. H2A.Z may replace the canonical H2A in some cases [29]. Genome-wide localization experiments in fungi, animals and plants have all shown that H2A.Z is preferentially enriched within the TSS of genes [30]. In Arabidopsis, many genes also display considerable enrichment of H2A.Z across their coding regions [30]. H2A.Z is also related with either transcriptional regression or activation [31, 32]. There is a negative correlation between transcription and H2A.Z enrichment within gene bodies in Arabidopsis, with the lowest expressed genes showing the greatest gene body enrichment of H2A.Z. However, the relationship between promoter H2A.Z enrichment and transcription shows a roughly parabolic distribution, with H2A.Z at its highest in moderately transcribed genes [30].

DNA methylation Methylation of cytosines in nuclear DNA is a common form of epigenetic modification, which is involved in many biological processes in both animals and plants [33, 34]. There is a striking difference in DNA methylation patterns between plants and mammals, as methylation can occur at any cytosine in plants— CG, CHG and CHH (where H ¼ A, C or T)—but primarily occurs at CpG dinucleotides in mammals [35]. Distinct methyltransferases are involved in the regulation of methylation in each of the three sequence contexts in plants (CG, CHG and CHH). METHYLTRANSFERASE1 (MET1), the homolog of the mammalian DNA methyltransferase1 (DNMT1), maintains the methylation at CG sites [36, 37], whereas the plant-specific DNA methyltransferase CHROMOMETHYLASE3

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[6]. Moreover, histone H3 and H2A variants have been reported to exist in mammals and several plant species. The diversity of histone modifications and variants highlight their considerable diversity of functions. However, the main effect of DNA methylation is to repress transcription or protect genomes against both endogenous selfish DNA elements (transposable elements or TEs) and exogenous virus invasions, proposed as a genome defense system [7]. Other than chromatin marks, another important epigenetic marks is the more recently discovered noncoding RNAs (ncRNAs). NcRNAs are functional RNA molecules that do not encode information for protein [8]. According to length, ncRNAs can be divided into long noncoding RNAs (lncRNAs) that have >200 nt, and the rest ones that are named ‘small’ RNAs (smRNAs). Generally, smRNAs include many different types, such as microRNAs (miRNAs), trans-acting small interfering (tasiRNAs), small nucleolar RNAs and piwiRNAs [8, 9]. In recent years, evidence has been accumulating that epigenetic marks modulate the impact of other epigenetic marks, providing cross-talk regulatory patterns. That is, the occurrence of one epigenetic mark can have an effect on the activity of one or more subsequent marks. Here, we discussed the distribution of epigenetic marks, their establishment and maintenance, as well as their roles in modulating transcription. We further summarized the complex cross-talk between different chromatin marks and mutual regulation between ncRNAs and chromatin marks, and addressed how these epigenetic marks interplay with each other. Finally, we analyzed the characters of crosstalk network and the different effects of different cross-talk patterns on gene transcription.

The roles of cross-talk epigenetic patterns

TSS

TTS 3’

5’

Epigenetic marks

| 3

Enzyme/direct regulator

Distribution

UBC1, UBC2, HUB1, HUB2

H3K4me1

JMJ14/PKDM7B

H3K4me2

ATX2, SDG4, JMJ14/PKDM7B

H3K4me3

ATX1, ASH2R, JMJ14/PKDM7B

H3K9me2

KYP/SUVH4, CMT3, IBM1

H3K9me3

SUVR4

H3K27me1

ATXR5, ATXR6

H3K27me2/3

PCR2, REF6

H3K36me2/me3

SDG8, SDG4, ATXR7

H3K18/23ac

IDM1, HDA6

H2A.Z

ARP6, PIE1, SEF, HTA4/8/9/11

H3.3

HTR4, HTR5, HTR8

mCG

MET1, V1M1, DDM1, HDA6

mCHG

CMT3, KYP/SUVH4

mCHH

CMT3, DRM1, DRM2

Figure 1. The patterns of distributions of chromatin marks in expressed genes and related modifying enzymes and genes. The chromatin marks are mapped on an arbitrary gene. Color indicates the relation of this mark and active transcription: green refers to associate with transcriptional activation; red refers to associate with transcription repression; yellow refers to not precise or little correlation to active transcription.

(CMT3) plays an important role in the methylation of CHG sequence context [38, 39]. The de novo methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE 1/2 (DRM1/ DRM2), similar to the mammalian DNMT3a/b, is responsible for establishing methylation in all sequence contexts, especially for CHH sites, which works through the RNA-directed DNA methylation (RdDM) pathway (Figure 2C) [40, 41]. Within the RdDM pathway, smRNAs bound by Argonaute4 (AGO4) can guide DNA methylation at homologous loci through DRM1/DRM2 [42]. Previous methylation studies in Arabidopsis have revealed that the genome-wide methylation levels at the CGs, CHGs and CHHs are 24%, 6.7% and 1.7%, respectively [43]. Genes are usually methylated within the promoters (so-called ‘promotermethylated’) or within the transcribed regions away from the 30 and 50 end (so-called ‘body-methylated’) (Figure 1) [44]. Generally, promoter-methylation is usually associated with transcriptional silencing, whereas body-methylation prefers exons and likely plays a role in exon definition during splicing [45, 46].

Noncoding RNAs MiRNA typically 21 nt in length is a class of smRNA molecules that is involved in transcription gene silencing through the specifically binding messenger RNAs based on sequence complementation at the posttranscriptional level. Similar to protein-coding genes, miRNAs are transcribed from a transcriptional unit by RNA Polymerase II (Pol II) to produce pri-miRNAs

(Figure 2A) [47]. Pri-miRNAs are processed by the double-strand RNA endonuclease DICERLIKE1 (DCL1) to generate miRNA duplexes; subsequently, these miRNA duplexes undergo a ‘maturation’ process mediated by HUA ENHANCER 1 (HEN1) in which a methyl group is added to their 30 end [47]. The mature miRNAs are loaded onto ARGONAUTE 1 (AGO1) to form a silencing effector complex [47]. In addition to miRNA processing, DCL4 is also required for the biogenesis of a few miRNAs in Arabidopsis [48]. Small interfering RNA (siRNA) typically 24 nt in length is another class of smRNA molecules, which is involved in heterochromatin formation and posttranscriptional silencing. Data suggest that Pol IV transcripts are copied into double-stranded RNA (dsRNA) by RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) (Figure 2B) [9, 49]. The dsRNAs are cleaved by DCL3 into 24 nt siRNAs; in turn, the siRNAs are added a methyl group by HEN1. These are loaded to RNA-induced silencing complex assembly, which includes AGO4 proteins as a key component [9, 49]. According to the genome location, lncRNAs can be classified into intronic lncRNAs, intergenic lncRNAs and antisense exonic lncRNAs [50]. Intronic lncRNAs locate inside the introns of a coding gene; intergenic lncRNAs locate in the interval regions between two coding genes, which are named lincRNAs; antisense exonic lncRNAs overlap with exon(s) of other loci on the opposite strand [50–52]. So far, studies have revealed that a subset of lncRNAs is produced by Pol IV and/or Pol V [53], whereas researchers also found two lncRNAs transcripts by Pol III in Arabidopsis [54].

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H2Bub

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Pol IV

Pol II

Pol V DRM2

RDR2

Pol II lncRNA

TF

dsRNA

pri-miRNA

DCL3

DCL1

miRNA duplex

siRNA HEN1

HEN1

methylated miRNA duplexes

methylated siRNA

AGO1

AGO4

Figure 2. Small RNA biogenesis and model of ncRNA involved RNA-mediated transcriptional gene silencing. (A) miRNA biogenesis. A miRNA gene is transcribed by Pol II to produce pri-miRNA, Pri-miRNA is processed by DCL1 to generate miRNA duplex, miRNA duplex is added a methyl group to its 30 end by HEN1 and mature miRNA is loaded onto AGO1. (B) siRNA biogenesis. Transcripts depend on Pol IV are copied into dsRNA by RDR2 after transcription, dsRNAs are cleaved by DCL3 into 24nt siRNAs, the siRNAs are added a methyl group by HEN1 and Methylated siRNAs are loaded to AGO4 proteins. (C) Model of the RdDM pathway. Pol V produces lncRNA, which is a scaffold for AGO4-siRNA binding. AGO4 recruit DRM2 to the target locus, DRM2 establishes CHH methylation and CHH methylation represses the binding of TFs and negatively affects Pol II transcription.

Evidence for cross-talk among different epigenetic marks Although we have learnt more about the location, occurrence and function of individual chromatin marks (Figure 1) and ncRNAs (Figure 2), it is not enough to determine a gene’s expression status. Therefore, additional research has added layers of complexity, and explored how the different epigenetic marks ‘communicate’ with each other (Supplementary Figure S1) [55–57].

Cross-talk among different histone modifications In A. thaliana, H2Bub deposition helps to recruit COMPASS (Complex Proteins Associated with Set1), which is involved in the binding of H3K4me3, H3K36me2 and H3K36me3 (Figure 3A) [58]. The links between H2Bub and H3K4me3, between H2Bub and H3K36me2 and between H2Bub and H3K36me3 are called ‘unilateral promotion’. Researchers have observed that H3K9ac and H3K27ac show coordination and tend to co-occur under all stages of growth, in a non-tissue-specific manner [15]. The studies also found positive correlation at the same loci between H3K9ac and H3K4me2, as well as between H3K9ac and H3K4me3 [5, 59]. Genes with coordinated modifications of two or more active marks have a higher level of expression compared with genes targeted only by one active mark [15, 59]. On the whole, the pattern of coordinately histone modifications has a stacking effect on the regulation of gene expression: the combination of histone marks with transcriptional activation reinforces the positive effect on

gene expression. We label this a coordinated modification pattern. A ‘bivalent’ histone modification pattern is defined as links between particular modifications that have antagonistic biological functions. The links between H3K4me3 and H3K27me3 and those between H3K4me2 and H3K27me3 have been found in whole Arabidopsis seeding [5]. However, the frequency of comarking between H3K4me3 and H3K27me3 is lower than that between H3K4me2 and H3K27me3. This phenomenon can also be explained by transcriptional repression of H3K4me demethylases. H3K4me3 demethylases could physically interact with H3K27me3 methyltransferase complex Polycomb repressive complex (PRC) 1 and/or PRC2 in Arabidopsis and other plants, so that a fraction of the H3K4me2, the demethylation product of H3K4me3, can co-localize with H3K27me3 [11]. H3K9me3 and H3K27me3 tend to co-localize in wild-type seeding. Genes targeted by H3K9me3, but not by H3K27me3, expressed at higher levels than those targeted by both H3K9me3 and H3K27me3 [15]. H3K9me2 and H3K9ac are also found to colocalize in the body regions of some specific genes in Arabidopsis [14]. Likewise, genes bearing both H3K9ac and H3K9me2 have lower expression levels than those that were solely targeted by H3K9ac [14]. Considering the overall situation, the modification pattern of bivalent histone modifications has an important role in the regulation of transcriptional outcomes, which strongly suggests H3K9me2 and H3K27me3 can repress the positive effect of H3K9ac, H3K9me3, H3K4me3 and other active modifications.

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AGO4

The roles of cross-talk epigenetic patterns

C

B

A H3K9ac

| 5

H3K27me3

H3K27me3

H3K9me2

H3.3 H3K4me2

H3K27ac

H3K36me3

CG

H3K4me2

H3K4me3

H3K9me2 H3K4me3

H2Bub

CHG

CG

H3.1

CHH

H3K27me3

CHG

H3K9me2 H2A.Z

H3K4me1 H3K9me3

H3K36me2

H2Bub

CHH

H3K4me2/3

H3K18/23ac

Coordinated modification pattern

Unilateral promotion pattern

Histone modification not precise or little with active transcription

Bivalent modification pattern

Unilateral inhibitory pattern

Histone modification with regressive transcription

Independent modification pattern

Mutual inhibitory pattern

DNA methylation Histone variant Figure 3. Cross-talk network of chromatin marks in chromatin. Nodes represent chromatin marks (circle for histone modification; diamond for DNA methylation; hexagon for histone variant). Nodes are colored according to connection between this mark and active transcription (red for association with transcription repression; green for association with transcriptional activation; yellow for not precise or little association with active transcription). Edge color and styles represent the forms of crosstalk. Keys for the networks are listed following the graphs. (A) Cross-talk among different histone modifications. (B) Interplay between histone modifications and DNA methylation. (C) Relationship between histone variants and DNA methylation as well as histone modifications.

An example of an ‘independent’ histone modification combination is the pattern of H3K4me1 and H3K27me3 [11]. One possible factor could be the differential distribution patterns along genes: H3K4me1 tends to be enriched at the 30 end of long genes (gene length > 4 kb), while H3K27me3 does not share similar preferences for either location within genes or gene length [11]. Moreover, H3K4me1 binds more frequently on ubiquitously expressed housekeeping genes, whereas H3K27m3 was more likely to target tissue-specific genes [11]. H3K9me2 and H3K27me3 are major silencing mechanisms in plants [15]. They are functionally interconnected through the establishment of repressive heterochromatic structures and gene silencing. However, they also appear to be independent epigenetic marks in Arabidopsis, as the influence of kyp mutant in H3K9me2 had no effect on the methylation pattern of H3K27, with changes in H3K27me3 occurring independently of the levels of H3K9me2 [60].

Interplay between histone modifications and DNA methylation There is an increasing evidence of the interplays between DNA methylation and histone modifications (Figure 3B). High-resolution genome-wide mapping and functional analysis of DNA methylation and histone modifications in Arabidopsis reveal a very high co-occurrence between H3K9me2 and DNA methylation [61]. KYP/SUVH4 is the H3K9me2-specific histone methyltransferase in Arabidopsis. Johnson and his colleagues have showed that the SRA domain of KYP/SUVH4 binds directly to methylated CG sequence contexts, suggesting a role of DNA methylation in recruiting H3K9 methyltransferases [62]. In turn, in the mutant of KYP/SUVH4, reduced H3K9me2 led to hypomethylation at CHG sites and CHH sites but not at CG sits. This kyp/ suvh4-induced genic non-CG hypomethylation is attributed primarily to the activity of CMT3, which is involved in methylation

of non-CG sites [63]. CMT3 contains a chromodomain that biochemically interacts with H3K9me2 in vitro, indicating maintenance of non-CG methylation requires di-methylation of H3K9 [64, 65]. In general, the cross-talk between H3K9me2 and DNA methylation is a mutually reinforcing partnership, a socalled ‘self-reinforcing loop’ [2]. In Arabidopsis, the mutant of H3K4me demethylase JMJ14 could increase H3K4 methylation, which causes a decrease DNA methylation at non-CG sites but not CG sites [66, 67]. Similar findings were made in rice. JMJ703 is a histone H3K4-specific demetylase in rice. In jmj703 mutant, the LINE element Karma showed significantly increased levels of H3K4me3 and dramatically reduced DNA methylation with upregulated transcription levels [68]. Zhang et al. [11] found that a negative correlation existed between DNA methylation and H3K4 methylation, with DNA methylation erased in H3K4 methylation-containing regions. Therefore, there is an inhibitory effect of H3K4me2/3 on DNA methylation. In mammalian cells, H3K27me3 may directly target DNA methylation [69], whereas there is an inverse correlation between H3K27me3 and DNA methylation in Arabidopsis [18]. Regions enriched by H3K27me3 are significantly hypomethylated on a genome-wide scale in Arabidopsis, even though they have significantly higher CG contents than either the genome average or randomly selected control regions [18]. In addition, under the met1-3 and ddm1-5, H3K27me3 is largely located in selected heterochromatic regions, such as specific 180 bp repeats [60], while the CG methylation states have no significant effect on the distribution patterns of H3K27me1 and H3K27me2, as the association of H3K27me1 and H3K27me2 with heterochromatic regions did not change in the case of the mutant of MET1 or DDM1 [60]. These findings suggest H3K27me1 and H3K27me2 are not affected by changes in the methylation of CG sites, whereas CG methylation directly restrains the tri-methylation of H3K27.

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Histone modification with active transcription

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Histone acetylation and deacetylase also have an effect on the DNA methylation pattern. IDM1, an important histone H3 acetyltransferase, could bind to methylated DNA through its MBD domain and recognize unmethylated histone H3K4 through its PHD domain, by which the lysine 18 and lysine 23 on H3 can be acetylated. In return, the acetylation of H3K18 and H3K23 may be recognized by DNA demethylation enzymes to reduce the level of DNA methylation [70]. HDA6 (Histone Deacetylase 6) is a very important histone deacetylase [71]. Studies have reported that the mutant of HDA6 can cause the loss of methylation at CG, CHG and CHH sites at a portion of promoters [72, 73], which could be owing to the physical interaction between HDA6 and MET1 [74]. These indicate that HDA6 not only plays a major role in eliminating acetylation, but also has an effect on maintaining DNA methylation.

Histone variants are an important form of epigenetic regulation. The genome-wide profiling of H3.1 and H3.3 variants in Arabidopsis and animals has shown that the patterns are broadly conserved: H3.1 is enriched in heterochromatin while H3.3 is enriched in transcriptionally active regions [25, 75]. In Arabidopsis, H3.1 is significantly marked by either H3K27me3 or H3K9me2, which is associated with the areas of silent chromatin. However, H3.3 is modified by the methylation of H3K36, H3K4 and H2Bub, which is most highly enriched in the 30 end of active genes [75]. Whole genome bisulfite sequencing data indicate that H3.1 is enriched in promoter-methylated genes, where DNA methylation of all three sequence contexts (CG, CHH and CHH) was also relatively enriched. In contrast, H3.3 was preferentially enriched within body-methylated genes and preferentially associated with CG methylation, but not with CHG and CHH methylation (Figure 3C) [75, 76]. There is a positive correlation between H3.3 and histone H2A.Z in Drosophila and humans, with both enriched near the TSS, suggesting there is a population of ‘double-variant’ nucleosomes [77]. However, genome-wide analysis has found a negative correlation between H3.3 and H2A.Z in Arabidopsis [75], as nucleosomes containing both H3.3 and H2A.Z are extraordinarily unstable. That is, the distribution patterns of these histone variants are different between animals and plants. H2A.Z and DNA methylation have a mutually antagonistic relationship in Arabidopsis [30] (Figure 3C): H2A.Z protects genes from DNA methylation, while DNA methylation excludes H2A.Z. The distribution of H2A.Z falls into three classes: low levels are found in TE regions; intermediate levels in methylated genes; and high levels in unmethylated genes. Therefore, H2A.Z has a uniquely positive correlation with transcriptional activation.

Mutual regulation between ncRNAs and chromatin marks Recently, many studies have demonstrated that chromatin marks and ncRNAs can affect each other in various ways (Table 1). Researchers have found siRNAs and lncRNA both play important roles in establishing de novo cytosine methylation in all sequence context by the RdDM process (Figure 2). Wierzbicki et al. found Pol V-dependent lncRNAs recruited AGO4 through sequence complementarity between lncRNA and AGO4-associated siRNAs (the Pol IV-mediated production of siRNAs) [87]. The possible interaction between Pol V and AGO4 can recruit DRM2 to the

Toward a network of cross-talk epigenetic patterns Several lines of evidence support the role of cross-talk of epigenetic marks in biological processes. Although each epigenetic mark seems to have its own role in determining transcriptional outcomes, there is clearly a built-in connection among them. Here, we consider the four main types of epigenetic marks, namely, histone modifications, histone variants, DNA methylation and ncRNAs. We summarize what is known about the complex relationship among them. The form of cross-talk among chromatin marks can be categorized into six classes (Figure 3). First, coordinated modification pattern may enhance the activity of expressed genes or the repression of unexpressed genes, such as the co-occurrence of H3K4me3 and histone acetylation [5, 59]. Second, a bivalent modification pattern may either weaken the activity of

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Relationship between histone variants and DNA methylation as well as histone modifications

target locus, which mediates establishment of CHH methylation [49]. CHH methylation would repress the binding of transcription factors (TFs) or other DNA-binding protein, which in turn negatively affects Pol II transcription and gene expression. Dai et al. have established the EpimiR database to collect the mutual regulation between chromatin marks and smRNAs [88]. On one hand, chromatin marks have an effect on miRNA or siRNA production, which is called Epi2miR or Epi2siR regulation pathway [88]. An example, under the control of histone acetyltransferase GCN5 (General control non-repressed protein 5), H3K14ac regulates miRNA production, such as miR-399d, miR395e, miR-165a, miR-157 and miR-172a [78]. Another example, H3K27me3 tissue specifically targets specific miRNA gene families, such as miR169, miR156/157 and miR167 [79]. On the other hand, miRNA or siRNA has a general repressive effect on the binding of chromatin marks through directly targeting modifying enzyme transcripts, which is called miR2Epi or siR2Epi regulation pathway [88]. In the EpimiR database, there are total 103 miR2Epi pathways in vertebrate, such as human, mouse and chicken [88]. Recently researchers also found several miR2Epi pathways in plants (Table 1). Baldrich et al. found miR823 can target CMT3 transcripts, whose encoding protein is involved in the RdDM process [84]. Several studies have shown that lncRNAs, such as Xist, HOTAIR, Kcnq1ot1 and Air, can target several chromatin modification complexes involved in gene silencing in animals and human [89–93]. We call this lncRNA-mediated chromatin modifications lncR2Epi regulation pathway. In plants, lncR2Epi has so far only been demonstrated in the FLOWERING LOCUS C (FLC) locus in Arabidopsis. COLDAIR, a sense ncRNA in the first exon of FLC, whose expression is induced by cold exposure, physically associates with CURLY LEAF (CLF), one of the components of PCR2 [81, 82]. COOLAIR, a group of long antisense RNAs expressed from the FLC locus, is important in the coordinated switching of chromatin states by mediating reduction in H3K36me3 at FLC [83, 94–96]. Researchers found removal of COOLAIR would disrupt cold-induced reduction of H3K36me3 levels in the nucleation region [83]. Recent research also found lncRNAs expression shows a very sensitive response to changes of chromatin epigenetic status, which is called Epi2lncR. For example, at At4g15242, a ncRNA locus, the gene is expressed in the mutant of met1-1, but not in wild type in Arabidopsis. Under the mutant of met1-1, when local DNA methylation levels were reduced by about two thirds, At4g15242 has been already activated [86].

The roles of cross-talk epigenetic patterns

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Table 1. List of regulations between ncRNAs and chromatin marks Chromatin marks

Noncoding RNA

RNA type

Chromatin remodeling complex

Category

Reference

H3K14ac

miR-399d

miRNA

GCN5

[78]

H3K14ac H3K14ac H3K14ac H3K27me3 H3K27me3 H3K27me3 H3K27me3

miR-395e miR-172a miR-157 miR169 miR156/157 miR167 tasiRNA3

miRNA miRNA miRNA miRNA miRNA miRNA ta-siRNA

GCN5 GCN5 GCN5 PCR2 and REF6 PCR2 and REF6 PCR2 and REF6 PCR2 and REF6

DNA methylation H3K27me3

siRNA854 COLDAIR

siRNA lncRNA

DDM1 and MET1 CLF

H3K36me3 DNA methylation

COOLAIR miR823

lncRNA miRNA

H3K36 demethylase CMT3

DNA methylation DNA methylation DNA methylation DNA methylation DNA methylation DNA methylation DNA methylation

siRNA415 miR-169 miR-413 miR-837-5p miR-781a miR-773a At4g15242

siRNA miRNA miRNA miRNA miRNA miRNA lncRNA

DRM2 IDN2 IDN2 IDNl1 IDNl1 DRM2 MET1

Chromatin mark regulates miRNA (Epi2miR) Epi2miR Epi2miR Epi2miR Epi2miR Epi2miR Epi2miR Chromatin mark regulates siRNA (Epi2siR) Epi2siR lncRNA-mediated chromatin modification (lncR2Epi) lncR2Epic miRNA regulates chromatin modification (miR2Epi) RdDM miR2Epi miR2Epi miR2Epi miR2Epi miR2Epi Chromatin mark regulates lncRNA (Epi2lncR)

[80] [81, 82] [83] [84] [84] [85] [85] [85] [85] [85] [86]

In this article, we have developed an integrative framework that describes known relationships among histone modifications, histone variants, DNA methylation and ncRNAs. There may be more epigenetic marks to be discovered and we will need to find the interplay between them and known marks. We believe that the formulation of the epigenetic cross-talk will be applicable to understanding the biology of organisms other than Arabidopsis. Comparisons between the specific networks for different species will open up a new era of comparative epigenetics. For example, there are many common patterns between epigenetic marks in humans and plants, but there are some pairings that act differentially between humans and plants. Understanding the origins of these differences as well as determining when they came into existence will open up a new vista on the history of life.

Key points • Different epigenetic marks interplay with each other

rather than working independently. • There are different cross-talk patterns among chroma-

tin marks. • We found a mutual regulation between chromatin

marks and noncoding RNAs.

Supplementary data Supplementary data are available online at http://bib. oxfordjournals.org/.

Acknowledgments The authors are grateful to the all members of their laboratory for helpful discussions and helpful comments. They

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expressed genes or weaken the repression of unexpressed genes, such as the co-occurrence of H3K4me3 and H3K27me3 [5]. Third, a unilateral promotion pattern exists among some histone modifications, histone variants and DNA methylation. For instance, H2Bub promotes the binding of H3K4me3 and H3K36me2/3 [58]. It is also possible for pairs of modification to mutually promote each other. For example, there is a ‘selfreinforcing loop’ between H3K9me2 and DNA methylation [2]. Fourth, a unilateral inhibitory pattern is found between DNA methylation and some histone modifications: CG methylation restrains the binding of H3K27me3 [60]; H3K4me2/3 has an inhibitory effect on DNA methylation [11]. Fifth, there is a mutual inhibitory pattern between H2A.Z and DNA methylation: H2A.Z protects genes from DNA methylation; DNA methylation excludes H2A.Z [30]. The last pattern is independence, for example, between H3K27me3 and H3K9me2 as well as between H3K27me3 and H3K4me1 [11, 60]. The form of mutual regulation between ncRNAs and chromatin marks also can be divided into six parts (Table 1). DNA methylation of promoters usually correlates with reduced transcription levels of corresponding ncRNAs, such as siRNA854 [80] and At4g15242 [86]. Histone modifications also have been discovered to have positive or negative roles in controlling ncRNAs expression, such as H3K14ac positively regulating miR-157 [78] and H3K27me3 negatively regulating miR169 [79]. NcRNA also plays an important role in control of DNA methylation or histone modification through directly targeting modifying enzymes or functional protein complexes. For instance, miR823 and miR-773a have been approved to reduce the DNA hypomethylation by targeting CMT3 and DRM2 directly, respectively [84, 85]. In addition, Zhang et al. [81] determined that COLDAIR, a sense ncRNA molecular, could upregulate H3K27me3 level through directly targeting CLF, one of the components of PCR2. Recent evidence also suggested that COOLAIR, a long antisense RNA molecular, could downregulate H3K36me level by directly binding H3K36 demethylase [83].

[78] [78] [78] [79] [79] [79] [79]

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also thank the anonymous reviewer for valuable suggestions to improve this manuscript.

Funding The work was supported by the National Natural Sciences Foundation of China (No. 31371328, 31450110068), the Henry Lester Trust, UK, Science Technology Department of Zhejiang Province, Jiangsu Collaborative Innovation Center for Modern Crop Production and Nanjing Agricultural University.

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