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Annu. Rev. Genet. 2015.49:673-695. Downloaded from www.annualreviews.org Access provided by Wayne State University on 12/03/15. For personal use only.

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Modulation of Chromatin by Noncoding RNA Victoria H. Meller,∗ Sonal S. Joshi, and Nikita Deshpande Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202; email: [email protected], [email protected], [email protected]

Annu. Rev. Genet. 2015. 49:673–95

Keywords

The Annual Review of Genetics is online at genet.annualreviews.org

long noncoding RNA, short noncoding RNA, Polycomb group, Trithorax group, heterochromatin, nuclear organization

This article’s doi: 10.1146/annurev-genet-112414-055205 c 2015 by Annual Reviews. Copyright All rights reserved ∗

Corresponding author

Abstract Noncoding RNAs (ncRNAs) are remarkably powerful, flexible, and pervasive cellular regulators. The involvement of these molecules in virtually all aspects of eukaryotic chromatin function is notable. Long and short ncRNAs play broadly complementary roles in these processes. Short ncRNAs underlie a programmable system of chromatin modification that silences mobile elements, identifies boundaries, and initiates the formation of constitutive heterochromatin in yeast. In contrast, long noncoding RNAs (lncRNAs) enforce developmentally appropriate expression and switch gene expression programs. lncRNAs accomplish this through diverse mechanisms, but often by modulating the activity or localization of chromatin regulatory complexes. Both long and short ncRNAs play key roles in organization of complex genomes of higher eukaryotes, and their coordinated actions appear to underlie some of the more dramatic examples of epigenetic regulation. This review contrasts well-studied examples of chromatin regulation by RNA and introduces examples of coordination between these systems.

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INTRODUCTION


It is appropriate to ask why RNA plays such a prominent role in organization and regulation of the genome. Most of the genome is transcribed, providing an enormous pool of raw material for natural selection to act upon. Indeed, the large noncoding Xist RNA, which plays a central role in X inactivation, arose de novo in the mammalian lineage from mobile elements and fragments of protein coding genes (33). Noncoding transcripts are unconstrained by the need to maintain an open reading frame, facilitating rapid evolution. Although interactions between long noncoding RNAs (lncRNAs) and proteins can achieve the specificity required for regulatory factors, the study of RNA-protein interactions has lagged behind that of protein-protein interactions. Finally, the rise of small RNA–dependent chromatin modification in eukaryotes enables the rapid redirection of a powerful regulatory system to virtually any region of the genome. This is taken to its logical conclusion in plants, where the primary mode of protection against mobile elements is small RNA–directed chromatin modification. The relatively late recognition of the pervasive regulatory function of RNA is due in part to the protein-centric nature of gene discovery. More recently, high-throughput analysis of transcribed regions has identified thousands of noncoding RNAs (ncRNAs). Recently developed techniques for detecting RNAs in protein complexes (RIP-Chip) (61) and identifying DNA and proteins associated with specific lncRNA domains (dChIRP) (102) have determined that many lncRNAs fold into domains, bind proteins, and localize to specific regions of the genome. This review addresses the principles of chromatin regulation by RNA, focusing on the general mechanisms by which these molecules recruit, tether, and modulate the activity of chromatin-modifying enzymes.

COTRANSCRIPTIONAL RECRUITMENT OF CHROMATIN MODIFIERS BY SHORT NONCODING RNA ncRNAs are often referred to as short if they contain fewer than 200 nucleotides, and long if they contain more. This arbitrary distinction fails to differentiate the 20- to 34-nucleotide RNAs bound by Argonaute proteins and with a mode of action distinct from that of other short RNAs. To avoid confusion we reserve the term short ncRNA for the numerous families of RNA that are united by incorporation into effector complexes that contain an Argonaute protein. Short ncRNAs were initially identified as posttranscriptional regulators but have remarkably varied roles in chromatin regulation, including heterochromatin formation, gene regulation, centromere function, and even programmed DNA elimination in Tetrahymena (reviewed in 12). A singular feature of all chromatin regulatory pathways that rely on small RNAs is that they are programmable, a property that enables them to defend against novel mobile elements. This flexibility has been capitalized on in higher eukaryotes that use endogenous small RNAs to regulate chromatin and organize large and complex genomes. A notably widespread role remains a reliance on small RNAs to silence mobile elements in the germlines of organisms as evolutionarily distant as plants, Drosophila, and mammals (reviewed in 122).

Formation of Constitutive Heterochromatin in Schizosaccharomyces pombe Small RNA–directed chromatin modification was first described in studies of heterochromatin formation in the fission yeast Schizosaccharomyces pombe, but related mechanisms in many higher eukaryotes have subsequently been discovered (reviewed in 88). Constitutive heterochromatin forms at repetitive regions surrounding the S. pombe centromeres. These regions are characterized by compaction, reduced expression, and enrichment for H3K9 methylation (H3K9me) and Swi6, 674

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a heterochromatin protein 1 (HP1) homolog that binds H3K9me. Heterochromatin formation is initiated by bidirectional transcription to produce double-stranded RNA (dsRNA), which is processed by a Dicer exonuclease (Dcr1) into 21–base pair short interfering RNAs (siRNAs). A single strand of the siRNA is loaded onto the Argonaute1 (Ago1) protein, part of the RNAinduced transcriptional silencing (RITS) effector complex (Figure 1a). This guide strand enables RITS to bind complementary nascent transcripts and thus associate with transcribed regions of the genome that produce small RNA. RITS contains the H3K9me binding protein Chp1 and the RNA binding protein Tas3, which contribute to stable association with chromatin (113). RITS also interacts with two critical complexes: One contains an RNA-dependent RNA polymerase (RdRP), which converts primary transcripts into dsRNA, and the other contains Clr4, an H3K9 methyltransferase (90). Clr4 methylates histone H3 at sites of RITS recruitment, and this enables Swi6 binding (reviewed in 53). As Chp1 and Swi6 recruit activities that promote introduction of H3K9me marks, association of these proteins with H3K9me reinforces silencing (18, 93). For example, synthesis of dsRNA by RdRP serves to amplify the signal produced by weak transcription of heterochromatic regions (88). The enzymatic activities necessary for amplification are tethered to chromatin, producing targeted and local signals. Transcription of S. pombe heterochromatic regions is limited to S phase, and thus small RNA synthesis occurs during assembly of replicated chromatin. Because low levels of transcription are necessary to generate nascent transcripts and initiate production of small RNA, this process is often referred to as cotranscriptional silencing.

Germline Silencing by Short Noncoding RNA Effector Complexes Small RNA–dependent epigenetic regulation is also found in plants, mammals, flies, and worms. One of the most notable adaptations is the system to silence transposons in the germlines of these organisms (reviewed in 55). In animals, these systems rely on a subfamily of Argonaute family proteins that includes Piwi (P-element induced wimpy testes) in flies and the MILI and MIWI proteins in mice. The short Piwi RNAs (piRNAs) are distinct, being 24–34 nucleotides in length and generated from long transcripts encoded by the host genome, or from active transposons, by a Dicer-independent mechanism (55, 142). Clusters of transposon fragments that are embedded in heterochromatin encode Drosophila piRNAs. The heterochromatic environment and epigenetic properties of the piRNA loci are necessary for correct processing of primary transcripts (142). Processing of piRNA is completed in the cytoplasm, where propagation of the signal occurs by ping-pong amplification (Figure 1b, inset). Amplification relies on the slicer activity of Argonaute family proteins to cut the 5 end of piRNAs, followed by exonuclease trimming. This process necessarily destroys transcripts with identity, generating piRNAs that mediate posttranscriptional gene silencing (PTGS) of mobile elements. piRNAs bound to the Piwi protein are also imported into the nucleus, where they associate with chromatin and direct transcriptional gene silencing (TGS), as illustrated in Figure 1b (72). Piwi localization on chromatin is RNA dependent, suggesting association with nascent transcripts that is directed by hybridization of the bound piRNA (Figure 1b) (69). The Su(var)3-9 H3K9 methyltransferase is necessary for Piwi-dependent TGS in flies, and H3K9me is enriched at transposons that are silenced (110). Piwi itself interacts with HP1, which binds H3K9me marks (11). It is thus possible that modification of fly chromatin by Piwi involves interactions similar to those in the RITS complex of S. pombe. The mouse genome also encodes piRNA precursors in clusters. Whereas Drosophila piRNAs act in the germlines of both sexes, the mouse piRNA pathway is necessary only in the male germline, where it acts to direct DNA methylation of transposons (5, 14). Mouse piRNAs are also amplified by the ping-pong mechanism, and, oddly, different classes of mouse piRNAs are produced sequentially during spermatogenesis. Plants similarly rely on small RNAs to control www.annualreviews.org • Modulation of Chromatin by Noncoding RNA

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mobile elements in germ cells. In this instance, the small RNAs are not produced from the germ cells themselves, but by derepression of transposons in companion cells. Transcripts from active mobile elements in the companion cells are processed into small RNAs that are transmitted to the germ cells, where they direct silencing of homologous sequences (reviewed in 15, 123). This strategy enables transmission of information about active mobile elements without the risk of transposition damage to the germ cells.

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Silencing Pathways in the Soma The silencing role of small RNAs in higher eukaryotes is not limited to the germline. Considerable evidence from multiple species points to a role in repression of mobile elements and heterochromatin formation in somatic cells (reviewed in 72). Dicer and Argonaute proteins are required for methylation of the promoters of L1 (long-interspersed nuclear element 1) elements, reducing L1 mRNA in human and mouse embryonic stem (ES) cells (16, 21). In addition, heterochromatic silencing in adult flies is reduced when Piwi protein is knocked down in early embryos (43). In contrast, knockdown in later stages is without effect, consistent with a role in initiation, not maintenance, of silencing. Although it is clear that small RNA pathways play a role outside the germline, the extent of their contribution to the complex epigenetic landscapes of somatic cells remains to be fully elucidated.


RNA-Directed DNA Methylation in Plants Plant genomes are remarkably rich in mobile elements of all sorts. RNA-directed DNA methylation (RdDM) in plants was discovered through its role in controlling the propagation of viruses and transposable elements (reviewed in 79). In contrast to S. pombe and Drosophila, neither of which displays appreciable DNA methylation, cytosine methylation is a primary mode of genome defense in plants. Transposon DNA is heavily methylated, and disruption of methylation derepresses transposons and increases spontaneous mutation by new insertions (reviewed in 56). Plants have devoted extensive genetic resources to RdDM, including two dedicated RNA polymerases, Pol IV and Pol V, as well as 10 Argonaute proteins, many with yet unresolved roles (99). Pol IV is necessary for the production of small RNA and localizes to sites undergoing RdDM, where it also acts to recruit additional factors (Figure 1c). Pol IV transcripts serve as a template for amplification of signal by the RNA-directed RNA polymerase RDR2, which associates with Pol IV. Resulting dsRNAs are cleaved by Dicer-like 3 (DCL3) to generate 24-nucleotide small interfering RNAs (siRNAs) that are loaded onto Argonaute proteins, such as AGO4, enabling recruitment to nascent Pol V transcripts (reviewed in 79). Pol V is enriched at transposons and ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 1 Small RNA–directed chromatin silencing in yeast, flies, and plants. (a) Silencing of Schizosaccharomyces pombe heterochromatin relies on an amplified double-stranded RNA (dsRNA) signal and chromatin-tethered enzymes. Bidirectional transcription of pericentromeric repeats in S. pombe produces dsRNA that is processed by Dcr1 to produce small interfering RNA (siRNA). A guide strand (orange) bound to Ago1, a member of the RITS (RNA-induced transcription silencing) complex ( green), enables association with nascent, complementary transcripts. Chp1, a RITS subunit, binds H3K9me. A complex containing the H3K9 methyltransferase Clr4 is recruited by RITS and modifies chromatin. The small RNA signal is amplified by an RNA-dependent RNA polymerase (RdRP) that interacts with RITS, Swi6, and Dcr1. (b) Piwi RNAs defend against transposable elements in Drosophila melanogaster. Piwi binds germline-specific piRNAs (short Piwi RNAs), enabling localization to complementary, nascent transcripts. Piwi also interacts with HP1 and recruits the H3K9 methyltransferase Su(var)3-9, leading to enrichment of H3K9me. Secondary piRNAs are generated by ping-pong amplification (inset). The proteins Piwi, Aub, and Ago3 cut the 5 ends of piRNAs. Exonucleases trim the 3 ends. (c) Small RNA–directed DNA methylation (RdDM) silences transposable elements in plants. The RNA polymerase Pol IV generates templates for the RdRP RDR2. dsRNA is processed by DCL3, as well as other enzymes not depicted. The resulting small RNA binds AGO4, enabling association with nascent transcripts produced by Pol V. AGO4 recruits a complex containing the DNA methyltransferase DRM2, leading to methylation of DNA. Pol V is enriched in RdDM-silenced regions by interactions with specific proteins, including AGO4. The H3K9 methyltransferases SUVH2/9 are recruited to methylated DNA. Additional chromatin-modifying activities, such as histone deacetylation and ATP-dependent chromatin remodeling, are recruited to repressed regions and contribute to silencing. www.annualreviews.org • Modulation of Chromatin by Noncoding RNA

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interacts with AGO4. The siRNA signal maintains methylation of daughter strands by DRM2 (domains rearranged methyltransferase 2), present in a complex that binds AGO4 (37). Enrichment of H3K9me at plant transposons also depends on the production of small RNAs, and there is considerable interdependence of DNA and histone methylation marks (reviewed in 15). The SUVH2 and SUVH9 enzymes that introduce the H3K9me mark are recruited by DNA methylation. Likewise, DNA methylation is at least partially dependent on H3K9me marks (15). Formation of heterochromatin also involves removal of activating marks by histone deacetylase and demethylase activities that are recruited to regions of RdDM (79). Although far from exhaustive, the preceding examples illustrate common features of small RNA–directed chromatin regulation, including amplification of signal, targeting of chromatinmodifying complexes to nascent transcripts, and multiple modes of tethering effector proteins to chromatin. These properties make small RNA remarkably effective at limiting the spread of mobile elements and well-adapted to stable regulation of epigenetic marks by the cell.

LONG NONCODING RNAs RECRUIT AND REGULATE CHROMATIN-MODIFYING COMPLEXES Chromatin-modifying complexes are also controlled by lncRNAs. These typically function in a complementary mode to chromatin regulators directed by short ncRNAs. For example, although many lncRNA regulators act close to their sites of transcription (action in cis), their function rarely depends on sequence complementarity to the regulated region. lncRNAs act by transcriptional interference, as structural or scaffolding components or as regulators of complexes that modify chromatin. These complexes are remarkably varied, but because the classical developmental regulators in the Polycomb and Trithorax groups (PcG and TrxG) are frequently controlled by lncRNA, this section begins with a brief introduction to the complexes formed by the proteins in these two groups.

The Polycomb and Trithorax Group Genes Regulate Pluripotency and Development The PcG and TrxG genes were initially identified by mutations that produce homeotic transformations in Drosophila (reviewed in 63). Members of these groups generally act in opposition to each other, with PcG repressing and TrxG activating the expression of target genes. But rather than acting as conventional transcription factors, the proteins encoded by PcG and TrxG genes work together to maintain activated and repressed states that have been established by other factors. Genes controlled by PcG and TrxG are often developmentally important regulators of differentiation or patterning. In flies, loss of PcG or TrxG activities produces aberrant patterns of Hox gene expression, leading to patterning abnormalities. The mammalian homologs of PcG and TrxG control development and are notable regulators of pluripotency and differentiation (76, 94; reviewed in 52). Consistent with this, loss-of-function PcG and gain-of-function TrxG mutations are found in many cancers (reviewed in 85). In mammals, the PcG and TrxG regulatory families are major points of lncRNA-mediated epigenetic regulation.

Polycomb Group Proteins Form Repressive Complexes The PcG proteins are grouped into multiple complexes, most prominently Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). PRC1 contains a ubiquitin ligase (RING1 or PCGF in mammals, Sce in flies), and one of several mammalian Chromobox (CBX) subunits (homologous to 678

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fly Pc) was found to have SUMO ligase activity (137). CBX subunits may interact with H3K9me or H3K27me marks, enabling recruitment to repressed chromatin (36, 86). PRC2 complexes contain an H3K27 methyltransferase [E(Z) in flies, EZH1 or EZH2 in mammals, CLF and SWN in plants], as well as subunits that interact with nucleosomes and regulate methylation activity. One of these, EED in mammals and Esc in flies, binds H3K27me and increases the methylation activity of the complex. A central member of PRC2, SUZ12 in mammals and Su(z)12 in flies, is essential for enzymatic activity. An excellent discussion of the compositional complexity of PRC1 and PRC2 in flies and mammals can be found in a review by Schwartz & Pirrotta (118). PRC1 and PRC2 frequently colocalize at developmentally repressed genes, where they stably associate through the life of the animal. For example, silencing of the Drosophila Hox genes is enforced by the binding of both complexes within a broad region of H3K27me3 enrichment (10, 134).

Trithorax Group Genes Work in Opposition to Polycomb Group Repressors TrxG activities are diverse. Included in this group are complexes containing the activating H3K4 methyltransferase Trx (fly) or MLL (mixed lineage leukemia; hereafter Trx/MLL) of mammals. Translocations that produce gain-of-function Trx/MLL fusion proteins frequently contribute to the development of leukemia (91). These mutations elevate expression of genes normally regulated by Trx/MLL, including the Hox genes. Other histone-modifying activities among TrxG genes are an H3K27 demethylase and H3K27 acetyltransferase (125, 133). The molecular activities of TrxG-encoded proteins thus oppose the methylation of H3K27 by PRC2. In accord with this, TrxG mutations have been isolated as genetic suppressors of PcG mutants (64). TrxG also contains chromatin remodelers, as well as a few proteins that serve as general transcription factors. An excellent introduction to the TrxG family can be found in Reference 117.

Recruitment of Polycomb and Trithorax Complexes by DNA Binding Proteins The molecular activities of PcG proteins appear broadly similar across a range of organisms, but there are notable differences in the preferred mode of recruitment to chromatin. PRC1 and PRC2 core complexes lack DNA binding activity but interact with DNA binding proteins, histone marks, and lncRNAs that direct recruitment. The fly genome contains regulatory regions called Polycomb Response Elements (PREs), which are made up of clustered binding sites for proteins that recruit PcG and TrxG complexes, including the zinc finger protein Pho (reviewed in 60). Drosophila genes regulated by PREs are strikingly enriched for transcription factors that control development, including the Hox genes (116). Fly PREs function within the context of higher-order nuclear architecture. Some PREs confer pairing-dependent silencing and participate in long-range interactions that result in clustering in the nucleus. These interactions contribute to silencing (7). One consequence of the well-defined PREs of flies is that transgenes carrying these regions display many of the regulatory properties, and long-range interactions, of the element at its endogenous locus (60). DNA binding proteins also recruit PRC2 in mammals. For example, YY1, homologous to Drosophila Pho, often recruits PRC2 complexes in vertebrates (139). However, the regions that recruit PcG and TrxG are poorly defined in mammals, with little similarity to the fly PREs. In contrast, lncRNAs have an outsized role in recruiting and regulating PcG and Trx/MLL complexes in mammals. The following sections introduce examples of lncRNAs that recruit and regulate chromatin modifiers. www.annualreviews.org • Modulation of Chromatin by Noncoding RNA

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Long Noncoding RNAs Are Bound by Chromatin-Modifying Complexes


Pull-down of mammalian PcG and Trx/MLL complex members and analysis of associated RNA have yielded an abundance of lncRNAs. For example, immunoprecipitation of EZH2 from mouse ES cells and RNA sequencing (RIP-seq) identified thousands of lncRNAs associated with the PRC2 complex (147). In agreement with the large number of RNAs identified, nonspecific recruitment to RNA has been suggested as a general mechanism for localizing PRC2 to transcribed regions (28, 59). Specificity of modification could be achieved by binding of the PRC2 subunit EED to H3K27me, which increases the H3K27 methylation activity of the complex (77, 141). Even promiscuously bound PRC2 would thus have activity appropriate to the site of recruitment (Figure 2a). This model accounts for the enormous number of lncRNAs bound to EZH2, explains localization of PRC2 at sites not enriched for H3K27me, and provides a mechanism for epigenetic stability. However, many lncRNAs have been found that are very specific regulators of PRC2 localization and activity, including some that have been functionally validated by knockdown or knockout (22, 143; reviewed in 107). These act by recruiting PRC2 to chromatin adjacent to the site of lncRNA transcription (in cis), or by modulating PRC2 activity at distant sites, even on other chromosomes (activity in trans). The lncRNAs display multiple modes of control of target genes, and a single lncRNA may employ different mechanisms at different genes.

LONG NONCODING RNAs REGULATE DEVELOPMENT HOTAIR Represses Mammalian HOX Genes in Trans The mammalian HOX genes are arranged in four clusters (A–D) on different chromosomes (80). Most transcripts from these regions are noncoding but display expression coordinated with that of nearby HOX genes (106, 108). One of these is HOTAIR (HOX transcript antisense intergenic RNA), a highly conserved 2-kb transcript from HOXC on chromosome 12 (Figure 2b). RNAi depletion of HOTAIR does not affect transcription from HOXC but increases expression of protein coding genes and lncRNA from the HOXD locus on chromosome 2, as well as in numerous other regions throughout the genome (70, 108). Loss of HOTAIR reduces PRC2 recruitment and H3K27me3 accumulation over the affected regions of HOXD. In accord with these findings, HOTAIR could be immunoprecipitated with PRC2. In addition to PRC2, HOTAIR also recruits a second repressor complex, coREST/REST, containing the histone demethylase LSD1, which removes the activating H3K4me2 mark (135). The idea that HOTAIR forms the link between PRC2 and coREST/REST is supported by an association between these complexes that is lost upon HOTAIR knockdown (135). HOTAIR thus acts as a modular scaffold that coordinates recruitment of a histone methyltransferase and a demethylase that act together to maintain transcriptional silencing.

Recruitment of Repressors by Long Noncoding RNAs Regulates Plant Flowering Arabidopsis requires a period of cold before flowering, a process called vernalization (reviewed in 126). Vernalization induces stable silencing of the FLOWERING LOCUS C (FLC) gene, encoding a transcription factor that prevents flowering. Two lncRNAs from the FLC locus, the antisense COOLAIR and intronic-sense COLDAIR, regulate FLC expression (50, 131). Cold treatment induces accumulation of both transcripts. COLDAIR interacts with CURLY LEAF (CLF) and SWINGER (SWN), PRC2 complex members homologous to the mammalian EZH 680

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Figure 2 Modes of chromatin control by long noncoding RNAs (lncRNAs). (a) Promiscuous PRC2 recruitment and context-dependent histone methylation. PRC2 (turquoise) binds nonspecifically to nascent RNA. The H3K27 methyltransferase EZH2 is inactive in the context of active chromatin (right), but the silencing mark H3K27me is bound by EED, which then activates EZH2 enzymatic activity. (b) The HOTAIR lncRNA of mammals acts in trans to repress expression. HOTAIR is transcribed from the HOXC locus on chromosome 12 but recruits PRC2 and the LSD1-containing coREST/REST complex to HOXD on chromosome 2. Recruitment leads to deposition of H3K27me by PRC2 and removal of activating H3K4me marks by LSD1. (c) The COLDAIR and COOLAIR transcripts of Arabidopsis act in cis to repress expression. COLDAIR recruits plant PRC2 complexes containing CLF and SWN. This results in enrichment of H3K27me at the 5 and 3 ends of FLC. The antisense COOLAIR transcript spans the FLC gene and promotes removal of the activating H3K4me and H3K36me marks through unknown mechanisms. www.annualreviews.org • Modulation of Chromatin by Noncoding RNA

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proteins (Figure 2c). Cold exposure and PRC2 recruitment lead to deposition of H3K27me3. Removal of the activating H3K4me2 and H3K36me marks is promoted by COOLAIR, acting through an unknown mechanism (50, 71). COOLAIR and COLDAIR thus have roles analogous to HOTAIR, causing silencing by recruitment of repressive marks and removal of activating marks. Consistent with association of PCR2-bound regions in other organisms, silencing during vernalization induces clustering of transgenes carrying the FLC locus (109).

Imprinted Long Noncoding RNAs Control Gene Expression Through Diverse Mechanisms


Mammalian genomes contain approximately 150 imprinted genes that display monoallelic, parentally determined expression (reviewed in 8). These are arranged in clusters, most of which also contain lncRNAs that are monoallelic in expression, usually produced from the paternal chromosome. The promoters of these imprinted lncRNAs are regulated by differential DNA methylation that is established in the parental germlines (8). Some imprinted lncRNAs have been shown to silence genes within the imprinted cluster, but their modes of action are diverse. For example, the paternally expressed Airn lncRNA silences protein-coding genes in the Igf2r cluster. Airn, which is antisense to Igf2r, silences paternal Igf2r by transcriptional interference, but also recruits the G9a methyltransferase, which deposits the repressive H3K9me mark at another silenced gene in the cluster (67, 92). A similar situation is observed at the imprinted Kcnq1 cluster, containing the paternally expressed Kcnq1ot1 lncRNA. Kcnq1ot1 is antisense to Kcnq1 and necessary for silencing Kcnq1, as well as several other protein coding genes across nearly 1 Mb of the paternal chromosome. Similar to other imprinted gene clusters, the precise pattern of repression by Kcnq1ot1 is tissue specific. In this instance, silencing of more distant genes in the placenta is accompanied by recruitment of PRC2, G9a, and the Kcnq1ot1 RNA (95). Once again, silencing is associated with a change in the localization of the silenced chromosome, which is recruited to the nucleolus. In contrast, in the embryo proper only imprinted genes close to Kcnq1ot1 are silenced, and this involves recruitment of the DNA methyltransferase Dnmt1, but not PRC2 or G9a (89, 95).

Xist Illustrates the Power and Versatility of Long Noncoding RNAs One of the best-studied examples of a lncRNA that regulates chromatin is the X inactive specific transcript of eutherian mammals (Xist/XIST of mouse and humans). Although the mouse and human homologs are similarly required for X inactivation, details of this process differ in the two species (reviewed in 29). The Xist gene is located in the X inactivation center (Xic), a region genetically identified through its ability to silence autosomal chromatin in X:autosome translocations (104). Xist is expressed from the inactive X of females (Xi) and is essential for initiation of silencing (98). Xist is controlled by several nearby genes, including the adjacent Jpx, which produces a lncRNA that activates Xist transcription, and the antisense Tsix lncRNA, which inhibits Xist expression (reviewed in 74). During early differentiation a transient pairing of Xic homologs occurs, and this is believed to underlie the process of X-chromosome counting (6). In cells with two or more X chromosomes, Xist is silenced on the future active X (Xa), but upregulated and stabilized on the Xi (96, 120).

Initiation of X Inactivation A remarkable feature of X inactivation is that it is restricted to chromatin in cis to the active Xist allele, a property that relies on a nucleation site within the Xist gene. A strong candidate for the 682

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nucleation site is a cluster of YY1 binding sites (57). In accord with this, knockdown of YY1 blocks recruitment of Xist to X chromatin. YY1 binds relatively specifically to several regions of the Xist transcript and also interacts with PRC2 (139, 148). What prevents Xist localization to numerous other sites of YY1 binding throughout the genome remains an open question, but the proximity of nascent Xist transcripts to the nucleation site may create concentrations of Xist, YY1, and PRC2 that are not replicated at other positions. Remarkably, YY1 binds to only one of the two Xist alleles in female cells, the future Xi (148).


Spreading of X Inactivation in Stages Upregulation of Xist on the Xi is followed by the exclusion of the transcriptional machinery and spreading of silencing along the chromosome by a sequential series of modifications (reviewed in 49, 140). Xist takes advantage of interphase chromosome architecture to spread throughout the X, initially associating with gene-rich regions in close spatial proximity to the Xic (Figure 3a) (34, 121). The role of chromosome organization in X inactivation is highlighted by Xist interactions with architectural proteins, including cohesin subunits, CTCF, SAF-A (scaffold attachment factorA/hnRNP U), and topoisomerases (20, 48, 81, 87). In accord with these findings, organization of the Xi is distinct from the Xa (47, 87). The Xi is also enriched for PRC2, H3K27me3, and PRC1, and PcG proteins are pulled down with Xist (20). However, PRC2 appears important for the stability, rather than the initiation, of silencing (58). This highlights the sequential nature of the modifications that inactivate the Xi (100, 140). Numerous additional epigenetic marks characterize the Xi, including depletion of acetylated histones, enrichment for satellite binding proteins and a histone variant (1, 26, 101, 103). These modifications, as well as DNA methylation at promoter CpG islands, contribute to stable silencing that lasts throughout the life of the organism (27).

Chromosome-Specific Long Noncoding RNAs Associate with the Xa and Autosomes in Human Cells Another lncRNA that may play a role in human dosage compensation is XACT, an extraordinarily large transcript that is produced from and coats the active X chromosome of undifferentiated human cells (136). XACT is limited to higher primates, likely originating from a relatively recent retrotransposon insertion. Although functional studies have not yet been published, XACT expression is transient, consistent with a role in protection of the Xa or regulation of XIST during the onset of X inactivation. But lncRNAs that associate with a single chromosome may be more widespread than once thought. Recent studies have identified autosomal lncRNAs with properties strikingly similar to those of Xist. The ASAR6 and ASAR15 transcripts, produced from human chromosomes 6 and 15, are monoallelic in expression and associate with their chromosome of origin in a manner reminiscent of Xist (31). Intriguingly, the ASAR6 and ASAR15 lncRNAs regulate replication timing of the chromosomes that they associate with. Mutation of either gene delays replication of a single chromosome and leads to mitotic instability. The discovery of XACT, and autosome-specific lncRNAs that exert global effects, suggests that regulation of individual chromosomes by lncRNAs may be quite common.

Long Noncoding RNAs Activate in Cis and in Trans HOTTIP (HOXA transcript at the distal tip) is transcribed from the HOXA locus in distal anatomic tissues and binds to WDR5, a subunit of the Trx/MLL complex. HOTTIP recruitment of Trx/MLL leads to deposition of the activating H3K4me3 mark on genes in the HOXA cluster (138). Knockdown of HOTTIP reduces Trx/MLL and H3K4me3 at promoters and www.annualreviews.org • Modulation of Chromatin by Noncoding RNA

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a

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Figure 3 Long noncoding RNAs function in the context of nuclear organization. (a) X inactivation silences chromatin close to the XIC. Xist recruits PRC2to X chromatin close to the XIC in the interphase nucleus (solid arrows). Silencing progressively spreads to more distant regions (dashed arrows). (b) The HOTTIP lncRNA of mammals utilizes chromosome looping. HOTTIP recruits the Trx/MLL (mixed lineage leukemia) complex (orange) to genes within the HOXA locus. HOTTIP takes advantage of preexisting chromosome loops to spread from its site of transcription to regulated genes. (c) Generation of small RNA from L1 elements precedes X-chromosome compaction in mammals. Active L1 elements (blue arrow) are expressed, producing small RNA that may recruit silencing to intact and fragmentary L1s on the X chromosome. Small RNA–directed silencing is hypothesized to contribute to the formation of the silent, compact domain occupied by the inactive X (right). (d ) Generation of small RNA from X-linked repeats contributes to X recognition in Drosophila. Small interfering RNAs are produced by repeat sequences enriched on the Drosophila X chromosome. This may direct modification of chromatin at the repeats, enabling the X chromosome to assume a conformation that facilitates recognition or spreading of the MSL (male-specific lethal) complex from sites of initial recruitment.

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lowers expression of nearby HOXA genes, but homologous HOX genes in clusters on other chromosomes are unaffected. The genes regulated by HOTTIP interact by looping, but because knockdown of HOTTIP does not release the loops, preexisting long-range contacts are likely involved (Figure 3b). Another Trx/MLL recruiter is the lncRNA NeST, identified by mapping a disease susceptibility variant within the interferon locus (39). NeST, convergently transcribed with INF-γ , is coimmunoprecipitated with WDR5 (39). Consistent with this, elevated NeST leads to H3K4me3 accumulation and increased expression of INF-γ . In spite of the adjacent location of NeST and INF-γ on the chromosome, NeST from a transgene can restore INF-γ production in cells deficient in NeST, and thus the NeST lncRNA can act in trans.


Activation by Long Noncoding RNAs Maintains Pluripotency in Embryonic Stem Cells One of the most striking features of lncRNAs in mammals is their recurrent appearance as regulators of developmental processes. Pull-down of WDR5 from ES cells identified more than a thousand RNAs that interacted specifically with this protein, some with demonstrated roles in pluripotency (45, 144). In accord with this, RNA binding by WDR5 is required for the maintenance of pluripotency (144). As accumulation of Trx/MLL on chromatin is decreased in WDR5 mutants that are unable to interact with RNA, a considerable amount of recruitment appears to rely on this process. Furthermore, the roles of lncRNAs in ES cells are not limited to the PcG and Trx/MLL complexes, as approximately one-third of the protein complexes examined in a systematic pull-down from ES cells yielded associated lncRNAs (45). These complexes participate in deposition, removal, and reading of activating and repressive marks, implicating lncRNAs as integral components of the network of transcription factors and chromatin regulators that stabilize the pluripotent state. As would be expected by their central role in pluripotency, misexpression of lncRNAs is linked to several types of cancer, and the association between misregulation of particular lncRNAs and poor prognosis has led to the proposal that these be used diagnostically (105).

The roX1 and roX2 RNAs of Drosophila Recruit Activators to an Entire Chromosome Flies also use lncRNAs to modulate an entire chromosome during the process of sex chromosome dosage compensation. However, rather than silencing a chromosome, male flies increase expression from nearly all X-linked genes, thus achieving expression equivalent to that in females with two X chromosomes (130). The male-specific lethal (MSL) complex of flies is selectively recruited to transcribed genes on the male X chromosome, where it alters chromatin to increase expression (3). A key subunit of the complex is repressed in females, limiting this process to males. One subunit of the MSL complex is the protein MOF (males absent on the first), which acetylates H4 on lysine 16, a mark found in active genes and decondensed chromatin (2, 124). Although the strategy of compensation is fundamentally different in flies and mammals, both use lncRNAs to identify X chromatin. roX1 and roX2 (RNA on the X 1 and RNA on the X 2) are essential but redundant components of the MSL complex (82). Simultaneous mutation of both roX genes is male lethal, producing mislocalization of the MSL proteins to autosomes and reducing expression of X-linked genes (30). Interestingly, both roX genes are X-linked, and both are able to recruit the MSL complex to chromatin adjacent to sites of roX transcription, including autosomal chromatin flanking a transgene insertion (62). The MSL complex is initially recruited to dozens of X-linked chromatin entry sites (CESs), followed by spreading into nearby transcribed genes (reviewed in 35). The CES contain a 21-bp MSL recognition element (MRE) that binds CLAMP, a zinc finger www.annualreviews.org • Modulation of Chromatin by Noncoding RNA

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protein (4, 127). Knockdown of CLAMP prevents X-chromosome binding by the MSL complex, demonstrating its importance. However, CLAMP binding is not limited to the X chromosome, and CLAMP is essential in females as well as males, indicating additional roles outside of dosage compensation. roX1 and roX2 are dissimilar in size and sequence, complicating the search for functional domains. Elegant molecular analysis revealed that two proteins in the MSL complex, the maleless (MLE) helicase and male-specific lethal 2 (MSL2), a ubiquitin ligase, bind to the same regions of roX (54, 75). The discovery that remodeling of a stem loop by MLE is necessary for subsequent MSL2 binding explains this overlap. The roX regions that interact with proteins comprise separate, folded domains (102). The discovery that ectopic expression of individual domains could, by themselves, achieve partial to full rescue of roX mutants confirms the value of this approach for identifying functional domains of lncRNAs.

LONG NONCODING RNAs AS DEVELOPMENTAL SWITCHES A Switch Between Repression and Activation Controls Proliferation A remarkable example of regulatory lncRNA function is provided by growth factor signaling through the PRC1 complex. One member of the mammalian PRC1 complex, Pc2/CBX4, is an E3 SUMO ligase that binds histones (143). Although Suv39h1 is better known as an H3K9 methyltransferase, it also methylates Pc2/CBX4 and alters the function of this protein. In the absence of growth factor, Pc2/CBX4 is methylated and localizes to Polycomb bodies, where it silences genes that are necessary for growth and proliferation. Upon addition of growth factor, Pc2/CBX4 is demethylated and moves to interchromatin granules (ICGs), sites of active transcription (143). Interestingly, methylated Pc2/CBX4 binds the TUG1 lncRNA, a marker for Polycomb bodies, but demethylated Pc2/CBX4 binds MALAT1/NEAT2, a lncRNA associated with ICGs (143). Knockdown of TUG1 releases growth control genes from Polycomb bodies, revealing a role in nuclear organization. RNA binding switches the histone binding preference of Pc2/CBX4, which favors repressive histone modifications when bound to TUG1 but prefers active marks when associated with NEAT2. Insight into the mechanism by which RNA modulates histone binding comes from structural studies of CBX7, a homolog of Pc2/CBX4 that binds the lncRNA ANRIL, an antisense repressor of the INK4A tumor suppressor (145). ANRIL binds both PRC1 and PRC2 and thus may recruit or coordinate the activity of both complexes. Interestingly, the CBX chromodomain (a motif found in histone and RNA binding proteins) interacts with both ANRIL and H3K27me (145). In this instance, RNA and histone binding sites are distinct but partially overlapping. As would be anticipated by this arrangement, RNA binding modulates affinity of CBX7 for H3K27me.

Long Noncoding RNAs Interact with Repressing and Activating Complexes The lncRNA Fendrr is divergently transcribed from the Foxf1 gene and expressed quite specifically in developing lateral mesoderm of the mouse (42). Premature termination of Fendrr transcription causes heart defects and lethality, as well as reduction of H3K27me3 at target promoters, including Foxf1 itself. Interestingly, Fendrr binds both the PRC2 and Trx/MLL complexes and thus could facilitate switching between repressed and active states (42). Remarkably, these authors present evidence that some Fendrr binding sites may be recognized by triplex formation with genomic DNA.

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Transcripts from the Fly Hox Clusters Act as Switches Like the mammalian HOX loci, Drosophila Hox clusters are rich in noncoding transcripts that are expressed in segmental patterns similar to those of nearby protein coding genes (68). Disruption of these transcripts, or ectopic expression, can lead to homeotic transformation, supporting the idea that the RNAs produced contribute to regulation of the Hox genes (9). However, the functions of these transcripts have defied simple categorization. The lncRNA iab-8, from the bithorax complex (BX-C), represses nearby abd-A by production of miRNA and transcriptional interference (44). One study of another BX-C lncRNA, bxd, concluded that it repressed nearby Ubx by transcriptional interference, but an inversion of the 5 end of bxd, directing transcription away from Ubx, produced negligible alteration in Ubx expression and no discernable phenotype, suggesting that bxd may in fact play no significant role (97). In addition to these lncRNAs, the PREs are themselves transcribed. Transcription through PREs has been proposed to allow remodeling and has been linked to activation of nearby genes. In accord with this idea, transcription through the well-studied Fab-7 PRE of BX-C relieved silencing of a closely linked reporter (114). A recent study of a PRE adjacent to the vestigial (vg) gene supports the idea that lncRNAs from the PRE act to modulate PRC2 (51). This study found an inverse correlation between expression of vg and the forward strand of the PRE. In contrast, lncRNA from the reverse strand of the PRE specifically bound E(Z) and displaced PRC2, relieving repression.

LONG AND SHORT NONCODING RNAs ORGANIZE THE NUCLEUS RNA Organizes Chromosomes and the Nuclear Matrix Evidence from numerous organisms reveals that long and short noncoding RNAs collaborate in nuclear organization, including establishment of insulators and boundaries, formation of Polycomb bodies, and identification of X chromatin during dosage compensation. One of the most fundamental roles of RNA is as a component of chromatin itself. Although lncRNAs have long been recognized as contributing to chromosomes and the nuclear matrix, the nature of these RNAs has remained unknown until recently. Surprisingly, one matrix-associated lncRNA appears to be Xist itself. After extraction of histones and digestion of chromosomal DNA, Xist remains bound to the X-chromosomal territory, indicating an association with the nuclear scaffold (23). This suggests that one of the roles of Xist may be to anchor chromatin to the matrix.

RNA Associates with Accessible Chromatin Xist is one of many lncRNAs that associate with the nuclear matrix fraction. The fast annealing Co t-1 fraction of the genome is composed of repetitive elements, including LINEs and SINEs (long- and short-interspersed repetitive elements). Labeled Co t-1 probes reveal abundant RNA throughout the nucleoplasm, excepting nucleoli and heterochromatic regions (47). The Co t-1 RNA signal is also absent from the territory occupied by the Xi, reflecting the silencing of repetitive elements on this chromosome. In contrast to the idea that Co t-1 RNA freely diffuses, studies of hybrid rodent cells with a single human chromosome reveal that the human Co t-1 signal is tightly restricted to the nuclear territory of the parent chromosome (46). Reminiscent of Xist, this Co t-1 localization depends on SAF-A protein. The Co t-1 signal dissociates prior to mitosis and is resynthesized in early G1. Interestingly, when resynthesis of Co t-1 RNA is blocked, chromosomal condensation occurs. Similarly, studies in Drosophila and human cells found that small nucleolar RNAs

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(snoRNAs) were associated with accessible chromatin (115). A decondensation factor, Df31, that is enriched in active regions of the genome binds both H3 and snoRNAs and thus may modulate nucleosome packing (115). Taken together, these studies suggest that matrix-associated RNAs, as well as RNAs that are an integral component of chromatin, contribute to the organization and maintenance of open chromatin.

RNA Organizes Chromosome Interactions at Insulators and Polycomb Bodies


Several specialized structures organize the nucleus and mediate chromosome interactions. Some of the most important of these are the insulators that demarcate chromatin domains with differing properties and separate regulatory elements from promoters (reviewed in 128). Insulation also defines domains of transcriptional activity and replication timing and is thus a critical organizing principle of the eukaryotic genome (111). Insulators loop chromatin or anchor it to the nuclear matrix, nucleolus, or nuclear pore, properties that may be essential for their function (38, 129). Insulators also associate to form insulator bodies (32, 38, 146). Drosophila PREs, which also loop and pair, may act as insulators (60). Polycomb bodies result from the propensity of PcG-bound regions to associate in the nucleus. Long and short noncoding RNAs have been implicated in the formation and function of both insulators and Polycomb bodies. Colocalization of PcG proteins and RNAi components has been observed in mammalian cells (65). Both RNAi and Polycomb proteins are necessary for insulation in a transgene carrying the Fab-7 PRE from flies (40). However, interactions between native PREs are not disrupted by loss of RNAi, suggesting that redundant functions may be lost when PREs are removed from their natural context (17). Immunoprecipitation of fly insulator proteins has identified specific associated mRNAs that have been experimentally linked to insulator function, but the molecular details of RNA function have yet to be resolved (78). Taken together, these observations suggest recurrent roles for long and short noncoding RNAs in the formation of insulators and Polycomb bodies.

Short Noncoding RNAs Mark Edges and Boundaries A notable feature of short ncRNAs is their utility in marking the boundaries that separate chromatin domains with epigenetic differences. For example, short ncRNA is found throughout silenced regions of S. pombe but is more abundant at euchromatin-heterochromatin boundaries, and silencing in these regions is particularly sensitive to the loss of small RNA pathways (13, 25). Certain parts of Drosophila mobile elements, particularly the ends, generate abundant small RNA, and deletion of these sequences prevents epigenetic silencing of insertions (112, 119). Plant RdDM is reduced in pericentric heterochromatin, perhaps because RdDM most avidly targets the ends of certain transposons, and the types of mobile elements in pericentromeric heterochromatin differ from those on chromosome arms (56). Taken together, these observations suggest that the flexibility of small RNA pathways contributes to the complex mosaic of epigenetic regulation that is characteristic of eukaryotic genomes.

Whole-Chromosome Recognition in Mammals Although the lncRNAs Xist and roX serve central roles in recognition of X chromatin, short ncRNA has been implicated in X recognition in both mammals and flies. The precise role of short ncRNA is not clear in either system, but intriguing parallels can be detected. The mammalian X chromosome is enriched for relatively young L1 elements that are postulated to play a role 688

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in X-chromosome inactivation (73). In agreement with this idea, autosomal integrations of Xist achieve better silencing in L1-dense regions (132). Silencing takes several days to spread over the Xi, and during this process inactivated genes are recruited into the silent domain coated by Xist (24). Early in this process, L1s at the periphery of the Xist domain are actively expressed, producing short ncRNA corresponding to the elements and their promoters (19). The active L1s are subsequently recruited into the inactive domain covered by Xist. It is possible that the activation, and subsequent silencing, of the L1 elements contributes in some manner to creation of the nuclear domain occupied by the Xi (Figure 3c).


Long and Short Noncoding RNAs Collaborate to Identify the Drosophila X Chromosome X recognition in Drosophila provides a compelling argument for coordination between long and short ncRNAs. Partial loss-of-function roX1 roX2 mutant males have low male survival and reduced recruitment of the MSL complex to the X chromosome (30). In fact, failure to direct the MSL complex to X chromatin is the signature defect of these mutations (82). Mutations in the siRNA pathway are potent enhancers of roX1 roX2 mutants, increasing male lethality and further reducing X-chromosome recognition (84). Interestingly, ectopic production of siRNA from a family of satellite repeats that is nearly exclusive to the X chromosome partially rescues MSL localization and male viability in roX1 roX2 mutants (83). On the surface this appears counterintuitive, as dosage compensation increases expression of X-linked genes and the siRNA pathway is linked to chromatin-based silencing. However, no physical interactions between MSL complex members and small RNA effectors have been found, suggesting independent modes of action (83). One possibility is that the X-linked repeats underlie a chromosome-specific organization that facilitates the spread of the MSL complex over the entire chromosome (Figure 3d ). This idea is supported by the similarity of the X-linked repeats to matrix attachment sites, as well as reports that the male X chromosome exists in an interphase organization that is different from that of the female X chromosomes (41). Reminiscent of the spread of Xist during X inactivation, regions of the fly X chromosome that recruit the compensation machinery cluster in three-dimensional space in the nucleus (102).

CONCLUSIONS AND FUTURE PROSPECTS Principles of chromatin regulation by lncRNA are increasingly well established in higher eukaryotes, and the identification of thousands of lncRNAs has provided researchers with a remarkably rich resource. Functional studies are needed to determine which of these lncRNAs are true regulatory molecules. A more difficult issue is understanding the nature of the interactions between RNA and other molecules. This is no simple task as the lncRNAs involved can be enormous. For example, Kcnq1ot1 and Airn are approximately 100 kb. Furthermore, analysis of lncRNAs with easily assayed phenotypes, such as Xist and the roX RNAs, reveals rampant internal redundancy as well as large regions with no apparent function. Although this complicates mechanistic studies, more widespread application of techniques that use antisense probes to pull-down proteins and chromatin cross-linked to RNA may provide a shortcut to identifying functional domains. The reliance of small ncRNA on a limited number of Argonaute effector proteins simplifies some aspects of the study of these regulators. Nevertheless, the scope of chromatin regulation by small RNA in higher eukaryotes remains unresolved, and a topic of great interest. Perhaps most interesting is the molecular logic that underlies the considerable collaboration between long and short ncRNAs. The examples provided by dosage compensation suggest that the www.annualreviews.org • Modulation of Chromatin by Noncoding RNA

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challenge of recognizing an entire chromosome requires a suite of epigenetic processes that act in a complementary fashion. It is also intriguing that organizing features of the nucleus, such as boundaries, insulators, and Polycomb bodies, may similarly employ both pathways. Understanding the contributions of regulatory RNAs will advance our understanding of how complex genomes are organized, partitioned, and regulated.

DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. LITERATURE CITED Annu. Rev. Genet. 2015.49:673-695. Downloaded from www.annualreviews.org Access provided by Wayne State University on 12/03/15. For personal use only.

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