Chromosome Res DOI 10.1007/s10577-013-9385-5

REVIEW

Maintenance of epigenetic information: a noncoding RNA perspective Tanmoy Mondal & Chandrasekhar Kanduri

# Springer Science+Business Media Dordrecht 2013

Abstract Along the lines of established players like chromatin modifiers and transcription factors, noncoding RNA (ncRNA) are now widely accepted as one of the key regulatory molecules in epigenetic regulation of transcription. With increasing evidence of ncRNAs in the establishment of gene silencing through their ability to interact with major chromatin modifiers, in the current review, we discuss their prospective role in the area of inheritance and maintenance of these established silenced states which can be reversible or irreversible in nature. In addition, we attempt to understand and speculate how these RNA dependent or independent maintenance mechanisms differ between each other in a developmental stage, tissue, and genespecific manner in different biological contexts by utilizing known/unknown regulatory factors. Keywords Noncoding RNA . Long noncoding RNA . Chromatin . Histone modifications . DNA methylation . Genomic imprinting . X chromosome inactivation . Kcnq1ot1 . Xist . Replication . PRC1 . PRC2 Responsible Editors: Brian P. Chadwick, Kristin C. Scott, and Beth A. Sullivan T. Mondal : C. Kanduri (*) Department of Medical Genetics, Institute of Biomedicine, The Sahlgrenska Academy, Gothenburg University, Medicinaregatan 9A, 40530 Gothenburg, Sweden e-mail: [email protected]

Abbreviations

DMR Dnmt1 Dnmt3a Dnmt3b Eed ES cells EZH2 H3K4me1 H3K4me3 H3K27Ac H3K27me3 Kb lncRNA MEFs ncRNA PcG PIGs piRNA pit-RNA PRC1 PRC2 Suz12 TrxG UIGs Xa

Differentially methylated region DNA (cytosine-5)-methyltransferase 1 DNA (cytosine-5)-methyltransferase 3 alpha DNA (cytosine-5)-methyltransferase 3 beta Embryonic ectoderm development Embryonic stem cells Enhancer of zeste homolog 2 Histone H3 monomethylated at lysine 4 Histone H3 trimethylated at lysine 4 Histone H3 acetylated at lysine 27 Histone H3 trimethylated at lysine 27 Kilobase Long noncoding RNA Mouse embryonic fibroblasts Noncoding RNA Polycomb group of proteins Placentally imprinted genes Piwi-interacting RNA piRNA-targeted long noncoding RNA Polycomb Repressive Complex 1 Polycomb Repressive Complex 2 Suppressor Of Zeste 12 Homolog Trithorax Group proteins Ubiquitously imprinted genes Active X chromosome

T. Mondal, C. Kanduri

XCI Xi Xist

X chromosome inactivation Inactive X chromosome X-inactive specific transcript

Introduction Epigenetics is the term being used for the studies that investigate extra-genetic information layered on DNA. Epigenetic information exists both on DNA and its associated histone and non-histone proteins. Cytosine methylation at CpG nucleotides is the major epigenetic modification of DNA (Cedar 1988), and histones on the other hand, subjected to several modifications, such as methylation, acetylation, phosphorylation, and ubiquitination at key lysine residues (Jenuwein and Allis 2001). DNA methylation and histone modifications constitute a layer of heritable epigenetic information or epigenetic memory. The epigenetic information from parental genomes is transferred to the offspring via mature gametes upon fertilization. This inherited epigenetic information in the offspring has been shown to program gene expression in a spatio-temporal fashion to ensure proper embryonic development (Robertson 2001). Inheritance of DNA methylation through cell divisions has been relatively well investigated, while that of chromatin marks has just begun to be unraveled. DNA (cytosine-5)-methyltransferase 3 alpha (DNMT3a)/DNA (cytosine-5)-methyltransferase 3 beta (DNMT3b) in association with a cofactor DNMT3L establishes genome-wide methylation patterns. The maintenance of these methylation patterns from one cell generation to another is carried out by DNA (cytosine-5)-methyltransferase 1 (DNMT1) (Robertson 2001). Though DNA methylation can be stably maintained through cell divisions by DNMT1, it can be erased or re-fixed depending on the external cues (Reik 2007). Histone modifications such as histone H3 dimethylated at lysine 4, trimethylated at lysine 36, and acetylated at lysine 9 invariably shown to be part of chromatin compartment enriched with actively transcribed genes, whereas inactive chromatin, which contains silent genes or genes with low expression levels has been shown to be enriched with histone H3 trimethylated at lysine 27 (H3K27me3) and lysine 9, as well as histone H4 trimethylated at lysine 20 modifications (Gardner et al. 2011). These chromatin modifications are a result of complex functional interaction between chromatin and chromatin-modifying

proteins such as G9a, SUV39H1/H2, LSD1, polycomb (PcG) and trithorax (TrxG) group of proteins. Recent investigations on PcG and TrxG group of proteins provided insights into how repressive chromatin structures are heritably maintained through multiple rounds of cell divisions (Hansen and Helin 2009; Margueron and Reinberg 2011; Petruk et al. 2012). These proteins have been shown to faithfully maintain lineage-committed gene expression patterns established by maternal and zygotic transcription factors during early embryonic development (Ringrose and Paro 2007). In Drosophila melanogaster, both PcG and TrxG proteins are recruited in a sequence-specific manner by the common cis acting sequences. However, in mammals, it is unclear how these epigenetic modifiers are targeted to specific regions across the genome. Accumulating evidence in recent years suggests that noncoding RNA (ncRNA) regulates gene transcription by acting as a cofactor in the targeted recruitment of both DNA and chromatin modifiers to specific loci (Kanduri 2011; Tsai et al. 2010; Rinn et al. 2007). Here, we review primarily the model systems where ncRNAs and the act of transcription have been implicated in both establishment as well as maintenance of epigenetic information.

Genomic imprinting and X chromosome inactivation: paradigms for understanding the functional role of ncRNA in the initiation and maintenance of epigenetic information Genomic imprinting is considered as one of the paradigms for understanding the functional role of long ncRNAs (lncRNAs) in transcriptional gene silencing in cis. It is an epigenetic phenomenon by which a subset of autosomal genes is monoallelically expressed depending on their parent of origin. So far, more than 100 imprinted genes have been identified and they are often found to coexist in gene clusters with at least one lncRNA as a partner. The expression of lncRNA is often inversely correlated with the expression of neighboring proteincoding RNAs (Royo and Cavaille 2008; Koerner et al. 2009). By using Xist, master regulator of X chromosome inactivation (XCI), and imprinted lncRNAs (Kcnq1ot1 and Airn) as model systems, we discuss how ncRNAs initiate and maintain epigenetic information at the target gene promoters and flanking differentially methylated CpG islands (differentially methylated regions (DMRs)).

Long noncoding RNA in the maintenance of epigenetic information

Kcnq1ot1 lncRNA is transcribed from within the Kcnq1/Cdkn1c imprinted domain, located at the distal end of the mouse chromosome 7. The orthologous region in humans is located on chromosome 11p15.5. It is about one mega-base in size, harbors 8–10 maternally expressed protein-coding genes and one paternally expressed lncRNA Kcnq1ot1. Expression of the lncRNA is thus inversely correlated with the expression of the protein-coding RNAs. The protein-coding imprinted genes, based on the extent of imprinted expression in embryonic and extra-embryonic lineages, are divided into two sub-classes: ubiquitously imprinted genes (UIGs) and placental-specific imprinted genes (PIGs) (Mohammad et al. 2009). Published evidence over the last few years suggests that Kcnq1ot1, upon its expression from the paternal chromosome, interacts with both DNA and chromatin-modifying enzymes and recruits them in cis to silence both UIGs and PIGs (Kanduri 2011; Pandey et al. 2008). Recent evidence highlighted differences in mechanisms by which Kcnq1ot1 RNA brings about the paternal chromosome-specific silencing of UIGs and PIGs in embryonic and extra-embryonic tissues (Kanduri 2011). UIGs, as the name implies, are imprinted in all embryonic tissues. Both DNA methylation and chromatin modifications have been implicated in the silencing of UIGs (Lewis et al. 2004; Terranova et al. 2008). Mice carrying various mutant versions of DNMT1 showed specific loss of silencing of UIGs but not PIGs on the paternal chromosome (Lewis et al. 2004; Weaver et al. 2010). In a recent investigation, it has been shown that a functional motif (silencing domain) at the 5′end of Kcnq1ot1 RNA by interacting with DNMT1 and initiate DNA methylation at the DMRs flanking some of the UIGs (Mohammad et al. 2010). By conditionally deleting Kcnq1ot1 RNA at different developmental stages during early embryonic development, it was demonstrated that Kcnq1ot1 is also required for maintaining methylation at the UIG DMRs (Mohammad et al. 2012). DNA methylation is considered a stable epigenetic mark, which, once established, can be faithfully maintained through successive cell generations via DNMT1PCNA-UHRF1 pathway (Bostick et al. 2007). Loss of DNA methylation and DNMT1 recruitment at the UIG DMRs upon conditional loss of Kcnq1ot1 RNA indicate that the UIG DMRs may additionally require Kcnq1ot1 RNA as a cofactor both in the initiation and maintenance of DNA methylation. From these observations, it appears that Kcnq1ot1 RNA acts as a scaffold for DNMT1

recruitment and Kcnq1ot1’s exclusive cis function allows DNMT1 to access the flanking sequences (Fig. 1). The paternal chromosome-specific silencing of PIGs has been shown to occur primarily by chromatin modifications but independent of DNA methylation (Pandey et al. 2008; Terranova et al. 2008; Lewis et al. 2004; Umlauf et al. 2004). Previously, the transgenic mice carrying mutations of chromatin modifiers like Eed, Ezh2, G9a, and Rnf2 were used to address the chromatin-based mechanisms underlying the paternal chromosome-specific silencing of PIGs and UIGs in the Kcnq1 domain. The first report of a link between chromatin modifiers and regulation of allele-specific gene silencing was observed in a transgenic mouse carrying a mutation in the PRC2 complex member Eed (Mager et al. 2003). Out of the 18 imprinted genes analyzed in the post-gastrulation Eed mutant embryos, 2 imprinted genes map to the Kcnq1 imprinted cluster: Ascl2-a PIG and Cdkn1c- an UIG. A functional role of chromatin modifiers in allele-specific silencing at the Kcnq1 locus was further reinforced by a recent investigation of the Ezh2 (polycomb repressive complex 2 (PRC2) complex) and Rnf2 (polycomb repressive complex 1 (PRC1) complex) mutant mice (Terranova et al. 2008). These investigations have demonstrated that both Ezh2 and Rnf2 are crucial for transcriptional silencing of UIGs (Cdkn1c) and PIGs (Tssc4 and Cd81), suggesting that the PRC2 and PRC1 complexes act in nonredundant and synergistic manner. More importantly, the recruitment of enhancer of zeste homolog 2 (EZH2) and RNF2 to the Kcnq1 locus results in the contraction of the Kcnq1 locus into a higher order repressive chromatin compartment, devoid of RNAP II and active chromatin mark histone H3 trimethylated at lysine 4 (H3K4me3). The stability of the chromatinbased silencing at the Kcnq1 locus was investigated by conditionally deleting Kcnq1ot1 at different stages (E5.5 and E8.5) of mouse embryonic development. The ChIP on chip profiles of repressive histone modification H3K27me3 and chromatin modifier EZH2 remain unchanged prior or after conditional removal of Kcnq1ot1, indicating that these modifications, once established, are maintained in a Kcnq1ot1 RNA independent manner (Mohammad et al. 2012) (Fig. 2a). Analyses of genomic imprinting and various histone modification profiles along the Kcnq1 locus upon conditional deletion of Kcnq1ot1 promoter revealed that the ncRNA is required only for the initiation of imprinted silencing of PIGs but not for their maintenance (Mohammad et al. 2012).

T. Mondal, C. Kanduri Fig. 1 Kcnq1ot1 lncRNAdependent maintenance of DNA methylation at the DMRs flanking UIGs in the Kcnq1 imprinted locus. Kcnq1ot1 interacts with and recruits DNMT1 to the DMRs flanking UIGs in the Kcnq1 imprinted domain. Conditional deletion of the Kcnq1ot1 leads to both the loss of DNMT1 recruitment and DNA methylation at the UIG promoter regions

Airn lncRNA, from the Igf2r locus on mouse chromosome 17, is another cis acting RNA being extensively investigated for understanding the epigenetic mechanisms by which lncRNA influences the gene expression of multiple genes in cis (Latos and Barlow 2009). One hundred eight kilobase (kb) Airn lncRNA has been implicated in the silencing of three genes, spanning over 400-kb region on the paternal chromosome (Koerner et al. 2009). Like Kcnq1ot1, Airn expression is restricted to the paternal chromosome due to the methylation of its promoter on the maternal chromosome. Published evidence suggests that both Airn lncRNA and the act of its transcription are functionally linked to the repression of neighboring protein-coding genes located on its overlapping and non-overlapping sides. The act of Airn transcription has been shown to control the Igf2r gene activity on the overlapping side. On the non-overlapping end, Airn lncRNA itself carries out silencing of two genes by epigenetically regulating the chromatin structure via interacting with G9a chromatin modifier (Mohammad et al. 2009). Previously, by using the mouse embryonic stem (ES) cell differentiating model system, which recapitulates the developmental onset of Igf2r imprinted expression, it has been shown that the act of Airn transcription is causally linked to Igf2r silencing via DNA methylation of a DMR, flanking the Igf2r promoter. During initial stages of ES cell differentiation, continuous expression of Airn

is required to initiate and maintain Igf2r silencing; but the functional requirement of Airn becomes dispensable for the Igf2r silencing once DNA methylation of the Igf2r DMR occurs at later stages of ES cell differentiation (Santoro et al. 2013). The Airn transcriptiondependent DNA methylation of the Igf2r DMR occurs during a specific window period between days 3 and 14 of ES cell differentiation. DNA methylation at the Igf2r DMR appears to act as a locking mechanism for silencing, which is suggested to be triggered by Airn-mediated transcriptional occlusion or transcriptional interference. Although the published data supports a functional role for noncoding transcription in initiating DNA methylation at the DMR, the role of Airn ncRNA per se in DNA methylation of the DMR cannot be ruled out (Latos and Barlow 2009). The similar noncoding transcriptionalbased mechanisms have also been proposed in the sequence-specific methylation of the DMRs that acquire methylation during germ cell development, the promoters of disease-associated genes and tumor suppressor genes (Chotalia et al. 2009; Yu et al. 2008; Morris et al. 2008; Ligtenberg et al. 2009; Tufarelli et al. 2003). Hence, it would be pertinent to understand the functional roles of noncoding transcription and its product in sequence-specific DNA methylation in normal and disease conditions. XCI is the most researched biological process to understand the functional role of lncRNAs in the

Long noncoding RNA in the maintenance of epigenetic information

initiation and maintenance of epigenetic information using the mouse as a model system. In mammals, XCI is a dosage control mechanism required for balancing the encoded X-linked gene products between males (XY) and females (XX) by inactivating one of the two X chromosomes in females. XCI is controlled by a 500kb region on the X chromosome called the X inactivation center, which primarily contains multiple lncRNAs Xist, RepA, Jpx, and Tsix (Lee 2009; Kanduri et al. 2009). The functional interplay among these lncRNAs has been shown to be critical for determining the future active (Xa) and inactive X chromosomes (Xi). Gene silencing on the Xi, once established during early embryonic development, is faithfully maintained through successive cell divisions lasting for the entire lifetime of the female mammal. Thus, XCI is one of the paradigms for understanding the mechanisms that maintain longterm epigenetic memory. It is a well-established fact that Xist plays an important role in the initiation of X-linked gene silencing on the future Xi but its role in the maintenance of gene silencing has been debated (Wutz and Jaenisch 2000). XCI occurs in two forms during early female embryonic development: nonrandom (imprinted) and random. In imprinted XCI, the paternal X (Xp) is always chosen for XCI, while in random XCI, either X is chosen. Xist dependent X-linked gene silencing on the imprinted Xi occurs between 8 and 16 cell stages of mouse preimplantation development. The Xist accumulation on the Xp has been shown to correlate with the enrichment of a repressive histone modification H3K27me3. This repressive chromatin conformation allows the imprinted Xp to undergo reactivation in the inner cell mass of implanted blastocyst embryos, and subsequently, two active X chromosomes are randomly chosen for XCI. Thus, the epigenetic information established in response to the Xist expression during imprinted X inactivation is labile and reversible in nature (Lessing and Lee 2013). During random XCI, the accumulation of Xist on the future Xi accompany several hierarchical changes in the chromatin structure comprising both DNA and histone modifications. The stability of silencing conferred by the random XCI varies with tissue and developmental stage and this has been very well investigated in mouse using ES cells and embryonic fibroblasts (MEFs) as model systems. By conditionally expressing the Xist cDNA transgene on an autosome in ES cells and the differentiating ES cell cultures, it was concluded that the

XCI is reversible and Xist dependent during an initial differentiation time window (i.e., first 72 h of differentiation), but that XCI became irreversible and Xist-independent in fully differentiated cells (Wutz and Jaenisch 2000). However, later studies provided a contrasting picture for Xist in the maintenance of X-linked gene silencing. Conditional deletion of Xist in the MEFs showed a significant decrease in the macroH2A levels, a characteristic histone variant of the Xi and increase in the levels of active histone marks H4 acetylation and H3K4me3. These MEFs also showed a marginal increase in the activation of X-linked gene Hprt and a green fluorescent protein (GFP) reporter gene, integrated on the Xi, indicating that the Xist participates in maintaining the epigenetic content of the Xi post-XCI (Csankovszki et al. 1999). A recent investigation provided further insights into lineage-specific maintenance of epigenetic information on the Xi in adult mouse. When Xist was conditionally deleted in murine hematopoetic cells 6 days after XCI has occurred, activation of 86 X-linked genes was observed from the Xi (Yildirim et al. 2013). These mutant mice developed hemalogical malignancies and showed defects in maturation of hematopoietic stem cells into differentiated cell types. Of note, these observations highlight the lineage-specific silencing mechanisms triggered by the Xist accumulation, and also that Xist RNA is absolutely required for maintenance of X-linked gene silencing (Lessing and Lee 2013). The post-XCI maintenance of epigentic information of the Xi seems to involve localization of the Xi to the perinucleolar region enriched with PRC2 complex member EZH2. When Xist was conditionally deleted in mouse skin fibroblasts, loss of H3K27me3 as well as its localization to perinucleolar region was observed, indicating that the Xist-mediated perinucleolar localization is critical for the maintenance of epigenetic information post-XCI (Zhang et al. 2007).

ncRNA-dependent initiation and maintenance of transcriptional silencing at non-imprinted loci HOTAIR and ANRIL/p15-AS are the other wellcharacterized lncRNAs that have been explored for their role in the initiation and maintenance of epigenetic information at their target loci. ANRIL/p15-AS is

T. Mondal, C. Kanduri

an antisense lncRNA that overlaps INK4b/ARF/INK4a locus (Yu et al. 2008). It mediates epigenetic silencing of the INK4b gene by targeting the PRC1 complex via interacting with one of its constituents CBX1 (Yap et al. 2010). The stability of the ANRIL-initiated epigenetic information at the INK4 locus was explored in a cell culture model. Conditional expression of the 5′ portion of ANRIL/P15-AS in HeLa cells resulted in the suppression of a GFP reporter gene, which is under the control INK4a/p15 promoter. Interestingly, this repression was maintained even after conditional turn-off of the ANRIL expression, indicating that ANRIL is required only for the initiation of transcriptional silencing but not for its maintenance (Yu et al. 2008). A follow-up investigation, however, provided contrasting data regarding the stability of epigenetic information initiated by the ANRIL RNA (Yap et al. 2010). In this investigation, H3K27me3 enrichment and the PRC1 recruitment at the INK4 locus was evaluated in PC3 cells following RNaseA treatment. It was observed that continuous expression of ANRIL was required to maintain the epigenetic information at the INK4 locus. Since the two latter experimental strategies differ quite significantly in their execution, it is difficult to draw conclusions on whether the maintenance of epigenetic information at the INK4a locus requires the continuous expression of ANRIL. Given its functional connection to the disease-associated genes, conditional deletion of ANRIL should be carried out exploiting suitable model systems to address the functional role of ANRIL in the maintenance of epigenetic information. HOTAIR is another lncRNA widely implicated in the epigenetic regulation of transcription (Rinn et al. 2007). Published evidence on HOTAIR indicates that it is required to maintain the epigenetic information on its target genes. Knockdown of HOTAIR by RNA interference in breast cancer cell line resulted in the loss of PRC2 enrichment as well as relaxation of target gene silencing in the HOXD locus, indicating that HOTAIR initiated transcriptional silencing at the HOXD locus can be reversed (Gupta et al. 2010). Braveheart (Bvht), an lncRNA with epigenetic connections has been shown to regulate differentiation of early mesodermal cells into cardiomyocytes by controlling the expression of key cardiac-specific transcription factors by interacting with PRC2 complex. The mouse ES cells lacking Bvht failed to differentiate into functional cardiomycytes and showed loss of H3K27me3 and SUZ12 from its target

genes, indicating that Bvht initiates silencing of its targets through establishing repressive chromatin during the differentiation process. Though its role in the maintenance of the repressed chromatin is not studied during differentiation, its requirement in the maintenance of fate of the neonatal cardiomyocytes suggests that Bvht may be required continuously to maintain the repression of its target genes (Klattenhoff et al. 2013). Like Bvht, Fendrr lncRNA is also an epigenetic modulator controlling heart and body wall development. Fendrr represses the expression of its key target genes involved in cardiac mesoderm differentiation by interacting with PRC2. The expression of the target genes is upregulated in mouse embryos lacking Fendr. In addition, its target gene promoters showed loss of H3K27me3 enrichment, indicating that Fendrr is required for their silencing (Grote et al. 2013). Since the latter analyses were performed in the embryos with zygotic deletion of Fendrr, it is not clear whether the Fendrr initiated repressive chromatin structures require its continuous presence post cardiomyocyte differentiation.

Mechanistic explanation for the stable inheritance of repressive chromatin structures The data from Xist, imprinted and non-imprinted lncRNAs present a complex scenario regarding their requirement for maintaining target gene silencing post initiation. Some target genes require the continuous presence of lncRNA while others do not. It is currently not clear what underlies these two lncRNA-dependent alternate chromatin conformations. Recent investigations, nevertheless, provide some clues as to how the H3K27me3 repressive histone modification is maintained through cell divisions. A positive feedback loop mechanism by polycomb proteins has been proposed in the transmission of pre-existing H3K27me3 chromatin marks through cell divisions (Fig. 2b). PRC2 component embryonic ectoderm development (EED) specifically recognizes H3K27me3 and this binding of EED leads to allosteric stimulation of PRC2 activity, thus resulting in EZH2-mediated trimethylation of neighboring H3K27 moieties (Margueron and Reinberg 2011). Along similar lines, a recent investigation suggested that EZH2 can bind directly to H3K27me3, when in complex with other PRC2 complex members (EED and SUZ12) (Hansen and Helin 2009). Supporting the latter observations on the

Long noncoding RNA in the maintenance of epigenetic information Fig. 2 a Kcnq1ot1 RNA is required only for the initiation of repressive chromatin structures at the PIGs but not for their maintenance. b Explains the plausible mechanism by which ncRNA-initiated repressive chromatin structures at the PIGs and X-linked genes are maintained through successive cell divisions. Briefly, once the PRC2/PRC1 proteins are deposited on their target genes by ncRNA, they are stably associated with the replicative chromatin over the parental strands and establish new histone marks on the chromatin of newly formed daughter strands during replication

a

PIG

Kcnq1ot1

PIG

Conditional deletion of the Kcnq1ot1 promoter

PIG

Kcnq1ot1

PIG

b

H3K27me3 repressive mark

Nucleosome

PRC1/PRC2 complex

Kcnq1ot1 promoter deletion

maintenance of H3K27me3, recent reports demonstrated that PRC1 and PRC2 complexes are associated on both parental and daughter strands during replication (Petruk et al. 2012; Lo et al. 2012), suggesting that transfer of chromatin modifiers during replication might be a way to maintain epigenetic states through cell divisions. Context and gene-dependent maintenance of transcriptional silencing by lncRNA through modifying chromatin structure raises two important questions. First, how an lncRNA-configured chromatin state in a particular developmental window is faithfully maintained in the absence of the lncRNA? Second, why do certain loci require lncRNAs continuously to maintain their silencing? Though PRC1 and PRC2 transacting factors have been the common theme in these contexts, it seems that their presence alone is not sufficient to leave an epigenetic memory that can be sustained through cell divisions in the absence of their recruiting partner like

ncRNA. Maintenance of target-gene silencing in the absence of an lncRNA can occur when epigenetic memory, established by lncRNA/PRC2 functional interaction, can be maintained in the absence of RNA by a default mechanism involving replication-dependent interaction of PRC1/PRC2, as has already been discussed in the previous paragraph. In case of loci, where ncRNA is required for both initiation and maintenance of silencing, the region may harbor binding sites for lineage and/or developmentally regulated trans-activator proteins, which can override the RNA-mediated silencing in the absence of RNA (Fig. 3). In fact, UIG seems to comply with this rule as the developmentally regulated enhancers activate some of the UIGs by overriding the Kcnq1ot1 RNA-mediated silencing in the embryonic tissues (Fig. 3a) (Korostowski et al. 2011). In addition, it has been shown that regions enriched for the enhancer-specific modifications histone H3 acetylated at lysine 27 (H3K27Ac) and histone H3 monomethylated

T. Mondal, C. Kanduri

Fig. 3 a Lineage and development-dependent activation of distal regulatory enhancers flanking some of the UIGs in the Kcnq1/ Cdkn1c imprinted locus confer resistance to the Kcnq1ot1 lncRNA-mediated transcriptional silencing. b The non-imprinted

genes, in imprinted clusters, which escape silencing effects of the imprinted lncRNAs, and X-linked genes, which escape XCI on the Xi, are enriched with the enhancer-specific histone modifications (H3K27Ac and H3K4me1)

at lysine 4 (H3K4me1), are also refractory to the RNAmediated silencing (Fig. 3b) (Mohammad et al. 2012), indicating that there is a complex interdependence between pre-existing chromatin signatures and selection of the genomic regions by lncRNA for silencing. Characterization of trans-activators which can counteract the RNA-mediated silencing can provide further understating of ncRNA-mediated initiation and maintenance of silencing.

2010; Guseva et al. 2012). Likewise, several mechanisms have been proposed to explain how CpG islands escape CpG methylation on both parental alleles in the context of housekeeping genes and on one of the parental alleles in the case of DMRs (Thomson et al. 2010; Guseva et al. 2012). Here, we discuss the functional role of ncRNAs and the act of transcription in the initiation and maintenance of methylation or undermethylation at the CpG islands. DMRs are specialized DNA sequences as only one of the alleles that undergo DNA methylation while the other allele remains unmethylated, though both parental alleles are equally amenable to DNA methylation in the same nuclear space. Recent investigations have uncovered a functional role for ncRNA in the allele-specific methylation of DMRs. The functional interaction between piwiinteracting RNA (piRNA) and lncRNA has been implicated in the allele-specific de novo methylation of a DMR at the Rasgrf1-imprinted locus. piRNAs, derived from a piRNA cluster on the mouse chromosome 7, have been suggested to act as a scaffold for piRNA interacting

Maintenance of the methylated and unmethylated state of CpG islands: a link to transcription and ncRNA In the mammalian genome, most CpG islands are largely unmethylated in somatic cells except in the context of Xlinked genes and imprinted gene clusters. ncRNA and the act of transcription have been implicated in the methylation of CpG islands in the case of DMRs associated with imprinted clusters and X-linked genes (Arnaud

Long noncoding RNA in the maintenance of epigenetic information

proteins MILI and/or MIWI2 and Dnmt3a/Dnmt3b/ Dnmt3L complex and target the Rasgrf1 locus via sequence-specific interactions with a piRNA-targeted lncRNA (pit-RNA), which is derived from direct repeats upstream of the Rasgrf1 DMR (Watanabe et al. 2011). This type of mechanism is proposed only for the Rasgrf1 DMR but not for the other paternally methylated DMRs. Other investigations have also implicated the act of transcription as the underlying mechanism in sequence-specific methylation of a subset of DMRs, which acquire epigenetic modifications during gametogenesis (Chotalia et al. 2009). Though mechanisms underlying the transcription-dependent sequencespecific methylation are unclear, the published evidence suggests that the transcription across DMRs generates RNA-DNA hybrids or triple-helical structures in a sequence-specific manner (Schmitz et al. 2010). In silico approaches predicted the presence of several such triple-helical RNA-DNA structures on a genome-wide scale at many human gene promoters (Buske et al. 2012). Given that sense and antisense transcription is widespread across several human promoters, it is tempting to speculate that noncoding transcription across tissue-specific promoters may generate triple-helical structures and thus become targets for de novo methylation. One of the unique features associated with the mammalian genome is that the CpGs in the majority of CpG islands remain unmethylated, while 80–90 % of CpGs of the non-CpG islands are prone to methylation. A recent study put forward an elegant mechanism for the maintenance of hypomethylation at CpG islands (Ginno et al. 2012). CpG islands are characterized by strand asymmetry in the distribution of guanines and cytosines downstream of the transcription start site which, upon transcription, results in the formation of R-loop structures. The R-loop structures comprising RNA-DNA hybrids have been shown to prevent the binding of DNMT3B, thereby protecting the CpGs within the R loop from DNA methylation. In addition, a similar mechanism has been shown to occur on the unmethylated allele of the DMR (Ginno et al. 2012). From this observation, it appears that the transcriptional process has a dual role in the maintenance of unmethylated and methylated states in the case of DMRs. In the case of the unmethylated allele of the DMR, the R-loop structure, in part, provides an explanation by preventing DNMT3B binding. But it remains to be determined how the transcriptional process

on the other allele of the DMR triggers sequence-specific methylation.

Future directions and conclusions Several lines of published evidence now clearly establish a functional link between lncRNA and initiation and maintenance of repressive chromatin structures at their target gene promoters. From recent evidence, it is also evident that lineage and gene-specific mechanisms cooperate with lncRNA to establish chromatin structures that can be maintained in the presence and/or absence of the lncRNA. Elucidation of these lineage and gene-specific mechanisms provides a greater understanding of epigenetic regulation of transcription by lncRNAs. Recent evidence also implicated lncRNA in the sequence-specific DNA methylation. In some instances, lncRNA plays a causal role in the initiation of DNA methylation, while in others, methylation occurs as a consequence of the lncRNA-initiated silent chromatin. Triple-helical structures, involving DNA and RNA, have been shown to be the underlying cause for sequence-specific DNA methylation. Although several such triple-helical structures have been identified genome-wide, it is unknown whether they are targets for sequence-specific de novo methylation. Addressing this issue by considering more lncRNAs with epigenetic connections would help us to understand this poorly investigated phenomenon in greater detail. There is an increasing appreciation for the role of lncRNA in human pathologies. Given that some target genes require the continuous presence of lncRNA for maintaining their silencing while the others do not, delineation of the mechanisms underlying the reversible or irreversible forms of silencing would be of greater help in devising better therapeutic strategies in disease treatment. Acknowledgments This work was supported by the grants from the Swedish Cancer Research foundation (Cancerfonden: Kontrakt no.100422), Swedish Research Council (VR-M:K2011-66X-2078104-3; VR-NT: 621-2011-4996), and Barncancerfonden (PROJ11/ 067) to CK. CK is a Senior Research Fellow supported by VR-M.

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Maintenance of epigenetic information: a noncoding RNA perspective.

Along the lines of established players like chromatin modifiers and transcription factors, noncoding RNA (ncRNA) are now widely accepted as one of the...
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