CpG methylation is a major hallmark in mammalian heterochromatin (5
). It plays an important role in gene suppression and is thought to control chromatin structure, for example, by modulating histone tail modifications. We have previously reported that Lsh is a crucial regulator of CpG methylation in mice (12
). Furthermore, we have recently observed abnormal H3-K4 methylation patterns at heterochromatin in the absence of Lsh and reactivation of retroviral gene expression (45
). Thus, Lsh modifies some (although not all, see below) characteristics of heterochromatin in mice. In the present study, we investigated the possibility that Lsh participates directly in the configuration of heterochromatin as opposed to indirectly inducing other protein factors. We demonstrated here a nuclear localization of Lsh, a strong association with chromatin, and a specific accumulation of Lsh at pericentromeric heterochromatin. These results provide an important link with previous results that demonstrated an effect of Lsh on heterochromatin structure and support a model in which Lsh is a direct participant with pericentromeric heterochromatin and plays an important role in normal heterochromatin formation.
Lsh deletion strongly affects DNA methylation at many other repetitive sequences that are not pericentromeric. The effect of DNA hypomethylation on single-copy genes is less pronounced (only five of nine genes show hypomethylation by Southern analysis [see also reference 12
]), a phenomen also reported in DDM1 mutants. These pronounced Lsh effects on repetitive sequences are consistent with a model in which Lsh primarily guards heterochromatin at repetitive sites, and alterations of chromatin structure after Lsh deletion may then spread into single-copy genes.
Since Lsh-deficient cells have residual CpG methylation, functional homologues for Lsh may exist that can also localize to heterochromatin. ATRX, another member of the SNF2 chromatin remodeling family, has been reported to localize to pericentromeric heterochromatin (4
), and ATRX mutations cause DNA methylation defects in patients; however, these defects do not occur at pericentromeric DNA sequences but at other repetitive elements (20
). Possibly, ATRX and Lsh may have redundant roles with respect to DNA methylation at heterochromatic sites such that Lsh substitutes for ATRX function. Another SNF2 family member, SNF2H, is associated with heterochromatin during late S phase of the cell cycle (11
). SNF2H is thought to play a role at heterochromatin by enabling DNA replication through highly condensed regions during S phase. An effect on CpG methylation has not yet been demonstrated. DNA methyltransferases also localize to heterochromatic regions (1
). Dnmt1, thought to be the major “maintenance” methyltransferase, colocalizes to heterochromatin during the late S phase of replication (29
). Since the global DNA methylation deficiency in Lsh−/−
mutants is similar to the one reported for Dnmt1−/−
), we hypothesized cooperative effects of Lsh with Dnmt1. Thus, we tested for the role of Lsh in targeting of Dnmt1 to replication foci. Although we did not see any difference in the composition of replication foci with or without Lsh, this does not exclude a possible role for Lsh in promoting the efficiency of Dnmt1 methylation. However, these questions have to be resolved in an in vitro system utilizing recombinant Lsh.
Heterochromatin has various properties, and it is a current challenge to understand the relationship and sequential requirements of distinct chromatin modifications and chromatin components (39
). A major concern has been the interplay of histone modifications and CpG methylation. Several lines of evidence suggest that histone modifications provide a key signal for DNA methylation in lower organisms. For example, mutants of H3-K9 methyltransferase (such as dim-5 or KYP) lower cytosine methylation in Neurospora crassa
or Arabidopsis thaliana
). Hyperacetylation after treatment with the histone deacetylase inhibitor TSA affects CpG methylation in Neurospora
, a phenomenon not reported for mammalian species (42
). On the other hand, there have been reports supporting the reverse relationship in which DNA methylation affects histone modifications. For example, mutants of met1 (a Dnmt1-like DNA methyltransferase in Arabidopsis
) can reduce H3-K9 methylation (41
). Although treatment with the demethylation agent 5-azacytidine does not alter HP1 binding or staining with the branched H3-K9me antibody (42
), it does alter H3-K4 methylation levels at heterochromatin (45
). In human cancer cells, 5-azacytidine treatment or deletion of DNMT1
can alter the H3-K9 methylation status at the specific promoter region of tumor suppressor genes, thus linking CpG methylation to chromatin modifications in mammals (2
). In the present study we observed that the reverse relationship also exists: disruption of higher-order heterochromatin structure by histone hyperacteylation correlated with dissociation of Lsh from pericentromeric heterochromatin. This suggests a model in which heterochromatin organization constitutes the signal for Lsh localization (and CpG methylation), and Lsh (and CpG methylation) plays a role in perpetuating heterochromatin formation rather than initiating it (7
Pericentromeric heterochromatin plays an important role in spindle fiber attachment and sister chromatid cohesion during mitosis. The association of Lsh at pericentric regions and the effect on the DNA methylation of pericentric satellite sequences suggest a role in mitosis. We have recently found a severe growth defect in MEF derived from Lsh−/−
embryos with signs of abnormal mitosis, including centrosome hyperamplification, abnormal spindle formation, and micronuclei formation (15
). That treatment with the DNA-demethylating agent azacytidine mimicked the absence of Lsh (15
) suggests that Lsh localization to pericentromeric heterochromatin is crucial to ensuring normal DNA methylation patterns that are required for normal mitosis.
At present, we do not know the precise heterochromatic signal that is required for Lsh localization. A three-dimensional configuration may be recognized by Lsh either directly or indirectly through other heterochromatic components. H3-K9 methylation or HP1 may contribute to Lsh recruitment, a hypothesis awaiting further testing. Recently, short RNA molecules have been proposed to be involved in the formation of heterochromatin structure, since deletion of RNA interference machinery inhibits heterochromatin structure in fission yeast (44
). RNA interference leads to short double-stranded RNA that inhibits the accumulation of homologous transcripts. The process appears to be involved in the regulation of H3-K9 methylation and heterochromatic gene silencing (44
). DNA repeats that allow bidirectional transcription may lead to the formation of double-stranded RNA and have been suggested as a nucleation center for heterochromatin (21
). The pairing of RNA molecules with corresponding sequences may serve as target for Suv39h histone methyltransferases (26
). After successful methylation of histone 3 at lysine 9, HP1 is recruited and contributes to establishment of a higher-order heterochromatin structure. This in turn may signal Lsh recruitment, which then facilitates CpG methylation. DNA methylation, occurring at repetitive sequences and at centromeric sites may then fortify heterochromatin structure, thus granting its stable propagation through multiple rounds of cell division. Thus, our results are consistent with a model in which Lsh, although important for heterochromatin formation, serves as a link in maintaining rather than initiating heterochromatin structure.