Confining euchromatin/heterochromatin territory: jumonji crosses the line

Keywords: JmjC, 5mC, H3K9me, euchromatin/heterochromatin, transcription

RIP generates signals for heterochromatin formation in N. crassa

Heterochromatin formation in N. crassa involves efficient targeting of H3K9me3, HP1, and 5mC to chromosome regions containing RIP-mutated (RIP'd) DNA, including centromeres, telomeres, and transposon relics (Selker et al. 2003; Lewis et al. 2009). RIP detects duplicated DNA fragments—regardless of their transcriptional state or foreign or native source—in a pairwise manner, and mutates them with numerous G:C to A:T transition mutations in the sexual phase of the life cycle (Cambareri et al. 1989; Selker 1990). RIP preferentially mutates CpA to TpA dinucleotides (Singer et al. 1995). As a consequence, RIP'd DNA sequences are both TpA- and A:T-rich. Hence, analyses of the “RIP product index” (TpA/ApT) and the “RIP substrate index” (CpA + TpG/ApC + GpT) can identify sequences that have undergone RIP in the Neurospora genome (Margolin et al. 1998; Selker et al. 2003). Consistent with the hypothesis that RIP serves to control selfish DNA, such as mobile elements, analyses of the sequences of the N. crassa genome revealed a complete absence of intact mobile elements (Galagan et al. 2003; Galagan and Selker 2004). On the other hand, because RIP is unable to distinguish between duplications of its own and parasitic foreign DNA, RIP has undoubtedly impacted the evolution of the Neurospora genome, since gene duplications are considered to be important for the evolution of new gene/protein functions and regulation (Galagan and Selker 2004). As an obvious consequence of RIP, the Neurospora genome has only a few highly similar duplicate genes. Most, if not all, N. crassa paralogs duplicated and diverged before RIP evolved (Galagan et al. 2003).

RIP'd DNA fragments typically function as portable de novo DNA methylation “signals”; i.e., when introduced in an unmethylated single-copy form into the genome by transformation, they trigger methylation of themselves and nearby sequences in all sequence contexts—CG, CHG, and CHH—in vegetative cells (Selker et al. 1993), and can potentially inhibit transcription elongation (Rountree and Selker 1997). As is typical of heterochromatin, RIP'd chromatin enriched in H3K9me, HP1, and 5mC can cause transcriptional silencing of associated genes (Selker et al. 1993; Cambareri et al. 1996; Irelan and Selker 1997). Profiling the distribution of H3K9me3, HP1, and 5mC across an entire chromosome (LGVII) of wild-type N. crassa strains by chromatin immunoprecipitation (ChIP)–chip demonstrated that H3K9me3, HP1, and 5mC colocalize extensively to form discrete heterochromatin domains in naturally RIP'd DNA sequences (Lewis et al. 2009). In contrast to the situation in animals and plants, coding sequences and virtually all promoters of genes were free of H3K9me3, HP1, and 5mC.

To examine the extent to which methylation of H3K9 and DNA depend on de novo or pre-existing methylation at naturally occurring RIP'd heterochromatic regions in the Neurospora genome, Selker and colleagues (Lewis et al. 2009) crossed dim-5 and dim-8 strains, both of which show complete loss of H3K9me3 and 5mC; isolated a dim⁺ recombinant; and tested which genomic regions were subjected to de novo methylation of H3K9me3 and DNA. Strikingly, H3K9me3 and DNA methylation were restored with 100% fidelity at all of the heterochromatic regions. This demonstrates that H3K9me3 and DNA methylation are efficiently established de novo and maintained, and that they do not depend on pre-existing methylation of H3K9 or DNA.

Interrelationship between H3K9me, HP1, and DNA methylation at heterochromatin in N. crassa

Null mutations in either DIM-5 or HPO, which encode a H3K9 HMT (Tamaru and Selker 2001) and a HP1 (Freitag et al. 2004a), respectively, eliminate all detectable DNA methylation, as does mutation of the DNA methyltransferase (DMT) gene dim-2 (Foss et al. 1993; Kouzminova and Selker 2001). DIM-5 specifically trimethylates H3K9, and trimethyl-H3K9 (H3K9me3) is associated specifically with methylated DNA (Tamaru et al. 2003). A role of H3K9me in the control of DNA methylation has been found also in plants and animals (Jackson et al. 2002; Lehnertz et al. 2003). H3K9me3 provides a binding site for the HP1 chromodomain (Freitag et al. 2004a). HP1 interacts directly with DIM-2, independently of H3K9me3, thereby acting as an adapter between methylation of histones and DNA. The interaction between DIM-2 and HP1 involves a pair of PXVXL-related motifs in the N-terminal region of DIM-2 and the chromo shadow domain of HP1, and is essential for DNA methylation (Honda and Selker 2008). The DIM-2/HP1 complex does not include DIM-5. The findings led to the conclusion that DNA methylation in N. crassa is a stepwise process in which DIM-5 methylates histone H3K9, and then the DIM-2/HP1 complex recognizes the resulting H3K9me3 mark via the chromo domain of HP1, allowing DIM-2 to methylate nearby nucleosomal DNA. Consistent with this model, genome-wide mapping of H3K9me3, HP1, and 5mC in dim-5, hpo, and dim-2 mutant strains indicated that DIM-5 is required for HP1 and 5mC localization, whereas targeting H3K9me3 does not depend on DIM-2 or HPO at the vast majority of heterochromatic regions (Lewis et al. 2009). An unsolved mystery is how RIP'd DNA-containing nucleosomes can be specifically targeted by DIM-5 for the H3K9me3 mark to establish DNA methylation and subsequent heterochromatization in N. crassa.

Does a DNA-based mechanism target heterochromatin formation in N. crassa?

It is tempting to speculate that heterochromatization in N. crassa is initiated by the recognition of features of RIP'd nucleosomal DNA by unidentified DIM-5-associated factor(s). Previous tests of nested sets of DNA fragments from a relic of RIP, the 1.6-kb zeta–eta region, or its mutated versions, for their capacity to induce de novo methylation in single copy at a specific locus revealed multiple positive signals that trigger methylation of its own and flanking non-RIP'd DNA sequences. Neither A:T richness nor a high content of TpA dinucleotides is an essential feature of methylation signals, but both promote de novo methylation (Miao et al. 2000). Thus, the A:T-rich nature of the product of RIP, rather than any specific DNA sequences, appears to be the trigger and the target for heterochromatin formation.

Subsequently, in vivo tests of various simple short synthetic DNA sequences for their potential to trigger de novo methylation of nearby cytosines at a synthetic locus “poised for methylation” revealed features of the methylation signals common with DNA binding by the AT-hook motif found in proteins such as mammalian HMG-1 (Reeves 2001), which binds to the minor groove of A:T base pairs (Tamaru and Selker 2003): (1) G:C base pairs inhibit methylation. (2) Both T and A residues are required on a given strand to induce appreciable methylation. (3) DNA lengths shorter than one nucleosome-wrapping DNA (146- bp) of the most potent signals can induce de novo methylation in sequences without RIP. (4) Extending signal lengths strengthen methylation. (5) A:T tracts as short as 2 bp are able to cooperate to induce methylation. Consistent with the hypothesis that A:T-hook proteins play a role in signaling DNA methylation, Distamycin A, an analog of the AT-hook motif, suppressed DNA methylation and transcriptional gene silencing in N. crassa (Tamaru and Selker 2003). Finally, a correlation between the strength of methylation signals and their binding to AT-hook proteins in vitro was observed. These collective findings prompted a proposal that de novo DNA methylation in Neurospora is triggered by cooperative recognition of the minor groove of multiple short A:T tracts, presumably by one or more AT-hook proteins that interact with DIM-5 (Tamaru and Selker 2003).

In a recent Genes & Development paper, Selker and colleagues (Honda et al. 2010) explored this possibility by searching for the A:T-hook proteins that read signals for initiating heterochromatin formation in N. crassa.

Identification of DMM-1 and its associated protein, DMM-2

The N. crassa genome has at least 30 genes that encode proteins containing A:T-hook motifs. Honda et al. (2010) first focused on four predicted proteins with two or more A:T-hooks, disrupted the corresponding genes by homologous recombination, and tested DNA methylation. None of the null mutations resulted in global methylation defects comparable with those of dim-5, hpo, and dim-2 strains. Interestingly, however, one of them, which Honda et al. (2010) named dmm-1, was found to cause both DNA hypomethylation and, more frequently, hypermethylation at several relics of RIP that are normally methylated in N. crassa. The dmm-1 gene encodes a protein that contains three A:T-hook motifs, a JmjC domain, and two cysteine-rich regions. Phylogenic analyses of JmjC domains from various species revealed that the JmjC domain of DMM-1 does not have any close homologs in humans, flies, worms, or yeasts. More detailed analysis of DNA methylation in the dmm-1 mutant strain revealed that the dmm-1-induced DNA hypermethylation resulted from spreading of methylation both upstream of and downstream from the methylated regions. This suggests a role for DMM-1 in confining 5mC within heterochromatic regions. Complementation tests with a series of DMM-1 constructs with mutated versions of the JmjC domain, cysteine-rich regions, or A:T-hook motifs in a dmm-1 mutant indicated that the JmjC domain and two cysteine-rich regions, but not the AT-hook motifs, ironically, are essential for the DMM-1 function.

In vivo copurification and coimmunoprecipitation of DMM-1-associated proteins identified a protein that Honda et al. (2010) named DMM-2. DMM-2 contains a fungal-specific Zn(II)₂Cys₆ binuclear cluster DNA-binding domain (MacPherson et al. 2006). In other proteins, this domain binds to simple DNA sequences such as CGG and CCG trinucleotides. Phylogenic analysis of DMM-2 revealed that it is conserved only in the filamentous fungi that possess a DMT DIM-2 homolog. Supporting the idea that DMM-2 collaborates with DMM-1 in modulating DNA methylation, a dmm-2 mutation also caused DNA hypermethylation defects similar to those in dmm-1 mutant strains, but to a lesser extent.

DMM-1 plays a role in preventing heterochromatin from spreading into genes

Interestingly, whereas dmm-2 strains grow nearly normally, dmm-1 strains show severe growth defects. This finding led Honda et al. (2010) to hypothesize that further spreading of DNA methylation across RIP'd transposon relics in dmm-1, compared with dmm-2, strains might silence neighboring housekeeping genes, resulting in growth inhibition. Consistent with the idea, a null mutation in the dim-2 DMT gene fully rescued the growth defects of dmm-1. To further test this hypothesis, Honda et al. (2010) conducted ChIP-chip analysis of LGVII with anti-5mC antibodies on DNA from wild-type, dmm-1, and dmm-2 strains. Almost all of the peaks of 5mC in dmm-1 and dmm-2 strains colocalized, but close inspection revealed that dmm-2 strains displayed less extensive spreading of 5mC, and at a smaller number of the regions, compared with dmm-1 strains. Strikingly, in dmm-1, but not in dmm-2, strains, DNA methylation spread into 10 genes, and, of these, a putative essential gene encoding a transcription factor, whose expression is reduced in dmm-1 strains, was found. Similar ChIP–chip analysis with anti-H3K9me3 antibodies on chromatin from wild-type and dmm-1 strains demonstrated that H3K9me spreads beyond RIP'd heterochromatic regions, concomitantly with 5mC, in dmm-1 strains.

To gain insights into the mechanisms by which DMM-1 prevents spreading of H3K9me3 and DNA methylation beyond heterochromatic regions, Honda et al. (2010) investigated the distribution of DMM-1 on LGVII in wild-type N. crassa strains by ChIP–chip analyses using antibodies against a DMM-1-HA fusion protein. Remarkably, peaks of DMM-1 localization were found to coincide with the edges of RIP'd heterochromatic regions, resembling the localization pattern of HP1 observed previously (Lewis et al. 2009). ChIP analyses using an N. crassa strain expressing DMM-1, DMM-2, and HP1, each tagged with a different epitope, revealed that all three proteins were preferentially localized at the edges of methylated regions. In contrast, H3K9me3 was distributed uniformly throughout the regions. Enrichment of DMM-1 and DMM-2 at RIP'd regions was greatly reduced in dim-5 or hpo, but not dim-2, backgrounds, indicating that chromatin binding by the DMM-1/DMM-2 complex depends on the presence of H3K9me3 and HP1, but not 5mC. Association of DMM-2 with chromatin depends on DMM-1, as suggested by the loss of DMM-2 enrichment throughout the heterochromatic regions in the dmm-1 background. Careful biochemical analyses detected a weak interaction between DMM-1 and HP1, which depends on the salt concentration in the extraction buffer. Coimmunoprecipitation assays in various mutant backgrounds revealed that the HP1/DMM-1 interaction is independent of DIM-2, DIM-5, and DMM-2. Collectively, the findings support the idea that HP1 recruits two opposing activities of DIM-2 and DMM-1/DMM-2, which establishes and antagonizes DNA methylation, respectively, to maintain and restrict heterochromatin within RIP'd DNA sequences in N. crassa.

A role for DMM-2 in anchoring DMM-1 to the euchromatin/heterochromatin boundary

Interestingly, a null mutation of dmm-2 led to the spreading of HP1 and DMM-1 into adjacent non-RIP'd euchromatic sequences. This suggested that DMM-2 is required for docking HP1/DMM-1 at euchromatin/heterochromatin boundaries, perhaps by binding to DNA near the edges of RIP'd DNA sequences through its Zn(II)₂Cys₆ DNA-binding domain. Honda et al. (2010) propose that the HP1/DIM-2 complex binds transiently to H3K9me3 at RIP'd chromatin, nucleates and spreads to allow DIM-2 DMT to introduce 5mC throughout the RIP'd sequences, and then recruits the DMM-1/DMM-2 complex to stop HP1/DIM-2 at the euchromatin/heterochromatin borders. In the absence of DMM-1 or DMM-2, HP1/DIM-2 continues to nucleate and spread across the adjacent nucleosomes that do not contain H3K9me3, allowing DIM-2 to methylate the neighboring non-RIP'd DNA sequences. Although it is not clear whether or not a DIM-2/HP1/DMM-1/DMM-2 complex exists, a simpler explanation could be that such a complex would progressively methylate RIP'd regions but stops when it reaches its edges.

How does DMM-1/DMM-2 recognize the euchromatin/heterochromatin boundary?

Interestingly, DMM1 has cysteine-rich domains that resemble that of mammalian DNMT3L, which binds to unmethylated H3K4 (Ooi et al. 2007). Given that, as in animals and plants, silent chromatin in N. crassa is typically deprived of the H3K4me mark, this observation raised the possibility that DMM-1 binds to unmethylated H3K4 on RIP'd heterochromatin. However, no binding of the DMM-1 cysteine-rich domains to any unmethylated or methylated H3 and H4 tails was detectable (Honda et al. 2010). It still remains possible that the binding requires one or more unidentified factors in vivo. Loss of DMM-2 association with RIP'd chromatin in dmm-1 strains suggests that the DNA-binding domain of DMM-2 is not sufficient for the association. In the absence of specific DNA boundary elements, recognition of both euchromatic and heterochromatic features would facilitate the preferential localization of the DMM-1/DMM-2 complex to euchromatin/heterochromatin borders. It is interesting to consider that DMM-1/DMM-2 may bind to both RIP'd and non-RIP'd sides of euchromatin/heterochromatin boundaries through the DMM-1 cysteine-rich domains and the DMM-2 Zn(II)₂Cys₆ DNA-binding motif, respectively, resulting in a high concentration of the complex at the boundaries (Fig. 1).

Does DNA methylation take the lead when heterochromatin spreads into euchromatin in N. crassa?

Positive feedback loops have been implicated in the propagation of heterochromatin in several eukaryotic systems. For example, in S. pombe, the Clr4 H3K9 lysine methyltransferase (KMT) binds to methylated H3K9 via its chromodomain, thereby facilitating the spreading of H3K9me beyond RNAi-dependent nucleation sites in cooperation with the methyl H3K9-binding protein Swi6 (homolog of HP1), which also has a chromodomain (Hall et al. 2002; Zhang et al. 2008). Similarly, Drosophila and mammalian SU(VAR)3–9 H3K9 KMTs interact with HP1 (Aagaard et al. 1999; Schotta et al. 2002). In A. thaliana, the chromodomain of the CHROMOMETHYLASE 3 (CMT3) DMT interacts with histone H3 methylated at K9 and K27 to target 5mC, and the KRYPTONITE H3K9 KMT binds to 5mC via its SRA domain to target histone methylation (Lindroth et al. 2004; Johnson et al. 2007). In mammals, H3K9 KMTs interact with DNA methyl-binding domain (MBD) proteins (Fujita et al. 2003; Sarraf and Stancheva 2004). The lack of putative methyl H3K9 binding, methyl DNA binding, and HP1-interacting domains in the DIM-5 KMT suggested limited positive feedback from the HP1/DIM-2 DMT complex in the DNA methylation pathway of N. crassa. Consistent with this view, HP1 has only a modest impact on the distribution of H3K9me3 (Lewis et al. 2009). Similarly, while a null mutation in the H3K9 HMT gene dim-5 eliminates all known DNA methylation in Neurospora, loss of the DMT gene dim-2 does not influence H3K9me3 and HP1 binding to chromatin at most, if not all, natural relics of RIP (Lewis et al. 2009). This indicates that DNA methylation depends on H3K9me, but not vice versa.

Unexpectedly, ChIP experiments with antibodies against H3K9me3 revealed that spreading of methylation beyond some RIP'd heterochromatic regions in dmm-1 strains was suppressed by a dim-2-null mutation, suggesting that this ectopic H3K9me depends on DNA methylation. How does this transition in the “methylation leadership” occur? It seems likely that, in the wild-type situation, the DIM-5/X complex (in which X is a hypothetical RIP'd DNA-binding protein) specifically recognizes RIP'd nucleosomal DNA through X, and DIM-5 trimethylates nearby H3K9 (Fig. 1). The HP1/DIM-2 complex then binds to H3K9me3 via the chromodomain of HP1, and DIM-2 methylates the associated DNA. The HP1/DMM-1/DMM-2 complex enriched at non-RIP'd/RIP'd euchromatin/heterochromatin boundaries presumably restricts the localization of the HP1/DIM-2 complex and DNA methylation within RIP'd regions. In dmm-1 or dmm-2 strains, HP1/DIM-2 and subsequent DNA methylation spread into the adjacent non-RIP'd nucleosomal DNA, and then the DIM-5/Y complex (in which Y is a hypothetical methyl DNA-binding protein) binds to 5mC through Y, and DIM-5 trimethylates H3K9 (Fig. 1).

Notably, unlike the situation of natural relics of RIP, experimentally RIP'd DNA fragments, when introduced into the wild-type N. crassa genome by transformation, frequently cause spreading of DNA methylation far across non-RIP'd flanking sequences (Miao et al. 2000; Tamaru and Selker 2003). Assuming that DIM-5 HMT loses its control of DNA methylation in non-RIP'd euchromatic sequences, the spreading of DNA methylation over non-RIP'd DNA might direct H3K9me also in the wild-type background. Moreover, there are indications that DNA methylation is required for H3K9me of transcriptionally competent sequences. For example, H3K9me3 is lost in an experimentally RIP'd allele of the N. crassa am gene (am^RIP8) in a dim-2 DMT mutant (Supplemental Fig. 2; Tamaru et al. 2003). A methyl DNA-binding activity has been identified in N. crassa cell extracts (Selker et al. 2002). Thus, it is reasonable to assume that, despite a limited feedback loop, factors that read DNA methylation are required to reinforce the establishment of H3K9me3 by DIM-5 HMT under certain conditions also in Neurospora. It would be interesting to investigate how the DMM-1/DMM-2 complex would work under such conditions.

Epe1 has a role in preventing spreading of heterochromatin into euchromatin

The JmjC domain protein Epe1was identified originally in a genetic screen for mutations that promote gene silencing beyond the natural borders of heterochromatin domains in S. pombe (Ayoub et al. 2003). Later, Epe1 was also identified as a Swi6-interacting protein (Zofall and Grewal 2006; Trewick et al. 2007). Loss of Epe1 causes spreading of H3K9me deposited by Clr4 HMT and its binder, Swi6, across heterochromatic barriers, otr at centromeres, and IR-L/R at the mating type (mat) locus, resulting in silencing of reporter genes inserted at adjacent euchromatin (Ayoub et al. 2003; Zofall and Grewal 2006). Of note, whereas an epe1 mutation predominantly causes enhanced silencing in the majority of cells, a smaller population of Δepe1 cells exhibits variegated expression of marker genes located within the heterochromatic regions that are normally silenced (Trewick et al. 2007). Given that loss of Epe1 causes reduction in siRNAs derived from centromeric repeat transcripts (Trewick et al. 2007), the simplest account for the reverse epe1 effect is that, in a small population of cells, Δepe1 leads to complete loss of the repeat transcripts, which in turn affects the siRNA production–heterochromatin assembly loop, ultimately resulting in variegated derepression. Δepe1 still results in the formation of extended heterochromatin domains in RNAi pathway mutant backgrounds (e.g., dcr1 and rpb2-m203), suggesting that Epe1 is not a component of the RNAi machinery (Trewick et al. 2007).

Swi6/HP1 recruits Epe1 and Clr3 to balance transcription and silencing of heterochromatin

The emerging concept of heterochromatin is that it needs to be transcribed to maintain its assembly and silencing (Grewal and Elgin 2007; Kloc and Martienssen 2008). Paradoxically, maintenance of heterochromatin formally requires transcription of the target tandem repeats to generate precursor RNAs (pre-siRNAs) to allow continuous production of siRNAs (Martienssen 2003). Accordingly, it has been found that centromeric repeats are transcribed by RNA polymerase II (Pol II), even in the presence of repressive heterochromatin complexes (Cam et al. 2005; Djupedal et al. 2005; Kato et al. 2005; May et al. 2005). How Pol II transcribes repeat sequences within a highly condensed heterochromatin environment remains enigmatic. Recent studies have revealed that Epe1 facilitates transcription of heterochromatic repeats (Zofall and Grewal 2006). The HDAC Clr3 plays an opposing role in limiting Pol II accessibility, thereby promoting transcriptional silencing of heterochromatic domains (Yamada et al. 2005). Transcripts from the cenH repeat elements at the silent mat locus are not detectable in wild-type S. pombe strains, since they are processed into siRNAs. Loss of Clr3 results in increased Pol II occupancy at cenH and higher levels of cenH transcripts. Mutation of epe1 reduces Pol II and transcription at cenH in a clr3 background, suggesting a role for Epe1 in promoting Pol II accessibility and transcription at heterochromatic repeats. Given that Swi6 recruits Epe1 (Zofall and Grewal 2006; Isaac et al. 2007; Trewick et al. 2007), and that Swi6 and another HP1 protein, Chp2, recruit Clr3 (Sugiyama et al. 2007) to heterochromatin, a newly arising aspect of heterochromatin is that it recruits functionally antagonizing factors via Swi6 to tune up the balance between transcription and silencing of heterochromatic sequences, which is required for proper functioning of heterochromatin (Fig. 2; Zofall and Grewal 2006).

Epe1 appears to facilitate transcription of repeats in association with Swi6 in the context of a repressive chromatin environment (Zofall and Grewal 2006; Isaac et al. 2007). It remains to be established whether Epe1 directly recruits transcriptional machinery, which in turn destabilizes heterochromatin, or Epe1 first destabilizes heterochromatin components such as Swi6, thereby indirectly promoting Pol II transcription. More recent studies have revealed that S phase of the cell cycle provides a short period of time during which heterochromatin becomes relatively accessible to Pol II for transcribing the underlying repeat sequences (Chen et al. 2008; Kloc et al. 2008). Taken together, the findings imply the existence of multiple mechanisms to transcribe heterochromatin.

Epe1 promotes transcription of DNA barrier elements and prevents spreading of heterochromatin across euchromatin

Supporting the previous cytological observation that Epe1 and Swi6 colocalize, ChIP–chip analyses of the distribution of Epe1 using antibodies against Epe1 and a microarray that covered the entire S. pombe genome revealed Epe1 at all the major heterochromatic loci, including centromeres, telomeres, the mat locus, and certain meiotic genes (Zofall and Grewal 2006). Epe1 association with heterochromatic loci depends on H3K9me and Swi6. Interestingly, close inspection of the enrichment of Epe1at centromeric regions revealed prominent peaks at the inverted repeat (IRC) elements located at boundaries of the heterochromatin domain. The Epe1 peaks coincide with a sharp decrease in heterochromatin marks such as H3K9me and Swi6. IRCs are transcribed by Pol II within the context of heterochromatin, correspond to hot spots of siRNA production (Cam et al. 2005), and have been shown to serve as heterochromatin barrier (or boundary) elements (Noma et al. 2006). Loss of Epe1, like deletion of IRCs, results in spreading of H3K9me and Swi6 across centromeric sequences, indicating that Epe1 is required for barrier function of IRCs. An epe1 mutation also leads to reduced Pol II occupancy at IRC1 and IRC3, and to reduced levels of their transcripts.

In view of these results, it seems likely that high concentrations of Epe1 and the resulting high levels of transcription at IRCs provide an “open” chromatin environment that counteracts the repressive effects of heterochromatin and prevent heterochromatin spreading across euchromatic sequences (Fig. 2; Grewal and Moazed 2003; Zofall and Grewal 2006). Remarkably, Epe1 localization within cen1 and cen3 but not at IRC1 and IRC3 barrier elements fully depends on Swi6. Epe1 is still enriched at IRCs in the swi6 mutatnt (Zofall and Grewal 2006), raising the possibility of an unidentified factor that recruits Epe1 to IRCs, and suggesting a specific Swi6-independent role for Epe1 at boundaries of heterochromatin. An intriguing possibility is that Epe1 interacts with proteins with similar function as DMM2 in N. crassa that recognize DNA barrier elements in S. pombe.

Three DNA methylation pathways cooperate to establish and maintain 5mC in the genome of A. thaliana

Unlike in mammals, 5mC in the flowering plant A. thaliana is found in all sequence contexts: CG, CHG, and CHH (in which H is A, T, or C) (Chan et al. 2005). MET1, a homolog of the mammalian DNMT1, is responsible for the maintenance of CG methylation (Kankel et al. 2003). CMT3 is responsible for the majority of CHG and a small fraction of CHH methylation (Bartee et al. 2001; Lindroth et al. 2001). The KRYPTONITE (KYP) H3K9 HMT is required for cytosine methylation by CMT3 at CHG sites (Jackson et al. 2002; Malagnac et al. 2002). DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is homologous to the mammalian DNMT3, is required for RNA-directed DNA methylation (RdDM) in all sequence contexts (Cao and Jacobsen 2002a,b). RdDM employs RNAi components—including RdRP (RDR2) (Xie et al. 2004), DICERLIKE3 (DCL3) (Xie et al. 2004; Daxinger et al. 2009), AGO4 (Zilberman et al. 2003, 2004; Pontes et al. 2006), and plant-specific RNA polymerases Pol IV and Pol V (Herr et al. 2005; Kanno et al. 2005; Onodera et al. 2005)—to guide de novo methylation of DNA sequences homologous to 24-nucleotide (nt) siRNAs, and leads to transcriptional gene silencing (Matzke and Birchler 2005). Previous genome-wide mapping of DNA methylation in the Arabidopsis genome using methylated DNA immunoprecipitation (MeDIP) coupled with microarray analysis (Zhang et al. 2006; Zilberman et al. 2007), and, more recently, whole-genome shotgun sequencing of bisulphate-treated DNA (Cokus et al. 2008; Lister et al. 2008), revealed dense CG and non-CG methylation of repeated sequences and transposable elements (TEs) that are concentrated within pericentromeric heterochromatin domains. Unexpectedly, >30% of expressed genes were also found methylated at relatively lower levels, exclusively in CG context in their coding, but not promoter, regions. The function of the gene body methylation is not yet known.

A ddm1-induced syndrome, bonsai (bns), caused by ectopic DNA methylation in A. thaliana

The SWI2/SNF2-like chromatin remodeling ATPase DECREASE IN DNA METHYLATION (DDM1) was originally identified through a genetic screen for mutants defective in DNA methylation in A. thaliana. DDM1 plays a vital role in controlling pericentromeric heterochromatin and TEs (Jeddeloh et al. 1999). DDM1 is involved in maintenance of both CG and non-CG methylation (Vongs et al. 1993; Jeddeloh et al. 1999). In ddm1 plants, 180-bp centromeric (180CEN) repeats and most TEs lose DNA methylation, H3K9me2, and 24-nt siRNAs corresponding to TE sequences (Gendrel et al. 2002; Lippman et al. 2004). This results in transcriptional reactivation and transposition of TEs (Miura et al. 2001; Singer et al. 2001; Gendrel et al. 2002; Tsukahara et al. 2009). Repeated self-pollination of ddm1 plants generates a variety of developmental abnormalities by inducing both genetic and epigenetic heritable alterations in loci other than DDM1 (Kakutani et al. 1996). Typically, phenotypes of ddm1-induced epigenetic alleles (epi-alleles) are associated with loss of DNA methylation at normally methylated loci. For instance, ddm1 induces a late-flowering phenotype through hypomethylation and ectopic expression of the imprinted homeobox gene FWA, which is normally silenced in embryonic plant tissues (Kakutani 1997; Soppe et al. 2000; Kinoshita et al. 2004).

Paradoxically, ddm1 occasionally induces de novo DNA methylation and silencing of a normally unmethylated locus, SUPERMAN (SUP), resulting in morphological defects (Jacobsen et al. 2000). The bns phenotype, which is characterized by miniature plant morphologies with compact leaves and short shoots (Saze and Kakutani 2007), represents another example of a ddm1-induced syndrome that is associated with ectopic de novo DNA methylation and transcriptional repression. The BNS locus encodes a homolog of a cell cycle regulator, Swm1/APC13 (Ufano et al. 1999; Yoon et al. 2002; Hall et al. 2003). The 3′ downstream from BNS is flanked by a truncated long interspersed nuclear element (LINE) retrotransposon in many Arabidopsis ecotypes. In wild-type plants, the LINE sequence is heavily methylated and transcriptionally silent, whereas BNS is free of cytosine methylation and is expressed. BNS transcripts extend into the LINE over its target site duplication (TSD) sequence. Strikingly, in bns lines, BNS was found heavily methylated in all sequence contexts, including CHH methylation, which is a characteristic of RdDM.

Consistent with the hypothesis that RdDM is responsible for the ddm1-induced BNS methylation, 24-nt small RNAs corresponding to the methylated BNS coding region were detected in bns, but not wild-type, lines. Remarkably, the ddm1 mutation did not induce the BNS methylation in the Cvi ecotype background, which lacks the LINE flanking BNS (Saze and Kakutani 2007). The findings imply that transcriptional reactivation of the LINE flanking BNS in the ddm1 background results in the production of read-through BNS antisense transcripts that form dsRNAs with BNS mRNAs, which then trigger RdDM of BNS.

Identification of another mutation, ibm1, which induces BNS methylation

A genetic screen of a mutagenized plant population using methylation-sensitive restriction enzymes and PCR—seeking novel mutations that, like ddm1, induce de novo DNA methylation in the BNS sequence—led to the identification of IBM1 (Saze et al. 2008). The IBM1 gene turned out to encode a JmjC domain-containing protein that belongs to the JHDM2/KDM3 family conserved from plants to mammals. It is noteworthy that the BNS sequence was found methylated in the first generation in which ibm1 became homozygous. This contrasts the situation in the ddm1 homozygous mutant in which several generations of self-pollination were required to detect BNS methylation. Unlike the ddm1 mutation, the ibm1 mutation did not cause DNA hypomethylation in heterochromatic sequences, including 180CEN repeats and 5S ribosomal DNA. Moreover, whereas the ddm1 mutation induced dense methylation at the BNS locus in all sequence contexts, the ibm1-induced de novo BNS methylation was found primarily at CHG sites, leaving CHH methylation negligible. These findings suggested that the mechanisms by which IBM1 and DDM1 limit DNA methylation in the BNS sequence are distinct, and that the primary targets for IBM1 are euchromatic sequences (Saze et al. 2008).

Taken together with the previous findings that KYP HMT controls CHG methylation by CMT3 DMT in A. thaliana, it was hypothesized that loss of IBM1 allows ectopic H3K9me by KYP at BNS, which subsequently directs CHG methylation by CMT3 (Saze et al. 2008). Supporting this proposal, ChIPs detected enrichment of H3K9me2 mark that colocalized with DNA methylation in the BNS sequence. Convincingly, the kyp and cmt3 mutations suppressed BNS methylation in the ibm1 background. Interestingly, the ibm1 mutant showed more complex developmental defects than that caused by a loss-of-function allele of the BNS gene, and the kyp and cmt3 mutations rescued most, if not all, of the phenotypes, suggesting that the ibm1 mutation induces ectopic de novo methylation of multiple genes, including BNS, thereby resulting in those abnormalities (Saze et al. 2008).

IBM1 guards transcribed genes from CHG methylation

Genome-wide analyses of the distribution of 5mC in wild-type and ibm1 mutant lines revealed ectopic methylation in numerous genes, primarily at CHG sites (Miura et al. 2009). In contrast, the ibm1 mutation did not increase DNA methylation of transposons, demonstrating that heterochromatic sequences are not the targets for IBM1. Importantly, unlike the ddm1 mutation, the ibm1 mutation induces genic methylation in the absence of adjacent transposons, and it typically causes heavier methylation at the central region of transcription units than that at the 5′- and 3′-terminal regions (Miura et al. 2009). The findings reinforce the notion that DDM1 and IBM1 use different mechanisms to control genic DNA methylation.

Interestingly, transcriptome analyses revealed that each region of increased DNA methylation coincides with a transcription unit, moderately transcribed genes and constitutively expressed genes are the most susceptible to the ibm1-induced CHG methylation, and long genes are susceptible to stronger methylation. This stronger methylation of longer genes resulting by mutation of ibm1 was less evident in genes with low levels of expression, suggesting that the effect of the gene length also depends on transcription (Miura et al. 2009). It seems reasonable to speculate that the mechanism by which the ibm1 mutation leads to genic CHG methylation is coupled with transcription, and that IBM1 functions to block KYP HMT or CMT3 DMT activity or remove inappropriately established H3K9me2 by KYP during transcription (Fig. 3). It would be interesting to know whether KYP and/or IBM1 interact with the transcription machinery. Consistent with the observation that the ibm1 mutation does not cause lethality in Arabidopsis, preliminary results revealed that transcriptional repression is not always an outcome of the ibm1-induced global de novo CHG methylation of the gene body (Miura et al. 2009). Why the Arabidopsis genome does not employ mechanisms to keep the bodies of active genes completely free of CG methylation by MET1, as it does for CHG methylation by CMT3, remains a mystery.

Possible functional interaction between IBM1 and DDM1

Curiously, methylation at the BNS locus was found enriched in the 3′ region of transcribed sequence near the flanking LINE in the ibm1 mutant (Saze et al. 2008). One could imagine that IBM1 and DDM1 might cooperate to prevent the spreading of methylation over genic regions in rare cases where genes are flanked by transposons. It would be interesting to know whether the pattern of BNS methylation induced by the ibm1 mutation changes in the absence of the neighboring LINE in the Cvi ecotype background. Remarkably, ddm1 ibm1 double mutants exhibit dramatically enhanced developmental defects, involving smaller leaves, slower growth, and complete sterility, compared with ddm1 or ibm1 single mutants (Saze et al. 2008). Similar genome-wide mapping of 5mC and transcription profiling in the ddm1 ibm1 double mutant in comparison with the ibm1 or ddm1 single mutant may provide insights into functional interactions between DDM1and IBM1.

Are Epe1, IBM1, and DMM-1 histone demethylases?

The JmjC domain has been implicated in active removal of methyl groups from histones (Klose et al. 2006a,b; Tsukada et al. 2006). JmjC histone demethylases can demethylate mono-, di-, and trimethylated lysine by an oxidative mechanism that requires Fe(II) and α-ketoglutarate (αKG) as cofactors. Demethylation is thought to occur by direct hydroxylation of the methyl group, resulting in an unstable hydroxymethyl product that is released spontaneously as formaldehyde (Trewick et al. 2005; Klose et al. 2006a). Three amino acid residues within the JmjC domain bind to the Fe(II) cofactor, and two additional residues bind to αKG. The JmjC domain of the Neurospora DMM-1 and the Arabidopsis IBM1 have the highly conserved residues within the predicted cofactor-binding sites known to be required for demethylase activity, whereas that of the S. pombe Epe1 lacks a conserved residue within the Fe(II)-binding site (Ayoub et al. 2003; Zofall and Grewal 2006; Trewick et al. 2007). Histone demethylase activity has not been detected for either of the proteins. Nonetheless, conserved residues in the JmjC domain of Epe1 and DMM-1 have been shown to be essential for their in vivo functions (Zofall and Grewal 2006; Trewick et al. 2007; Honda et al. 2010).

Based on structural homology between Epe1 and another JmjC domain protein, FIH, which hydroxylates itself at Asn803 to repress its binding to the histone acetylase p300 (Schofield and Ratcliffe 2005), it has been proposed that Epe1 may also hydroxylate itself or another heterochromatin protein, such as Swi6, to modulate the stability of heterochromatin (Trewick et al. 2005, 2007). Moreover, the lysine demethylase LSD1, which was originally identified as a histone H3K9me1/2 demethylase in human cells, has been found to demethylate K370me1/2 of the p53 tumor suppressor in vitro (J Huang et al. 2007). The question of whether JmjC domain proteins are able to demethylate nonhistone proteins is still open. Interestingly, a dmm-1 substitution mutation (H216A) in a conserved residue within the Fe(II)-binding site of the JmjC domain, which presumably abolishes the catalytic activity of DMM-1, results in the spreading of DMM-1 from RIP'd regions to adjacent sequences, phenocopying the effect of the dmm-2 mutation. This raises the possibility that DMM-1 hydroxylates/demethylates itself or DMM-2 to promote its interaction with DMM-2.

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