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Laboratory of Molecular Immunoregulation, Basic Research Program,1 Image Analysis Laboratory, SAIC-Frederick, National Cancer Institute, Frederick, Maryland 217022
Received 19 June 2003/ Returned for modification 30 June 2003/ Accepted 15 August 2003
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ABSTRACT |
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INTRODUCTION |
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Another hallmark of mammalian heterochromatin is DNA methylation (5-7, 9, 34, 39). The addition of methyl groups to cytosines within the CpG dinucleotide by DNA methyltransferases is involved in regulating transcription, maintaining genome stability, imprinting, and X chromosome inactivation. In mammalian cells, DNA methylation is catalyzed by two important classes of DNA methyltransferases (5). DNA methyltransferase 1 (Dnmt1) resides at the replication fork and methylates CpG dinucleotides in the newly synthesized daughter strand. The function of Dnmt1 is thought to be essential for maintaining DNA methylation patterns in proliferating cells (30). There are two members of the second class of methyltransferases, Dnmt3a and Dnmt3b, which are required for the initiation of de novo methylation during embryonic development (37).
Although DNA methylation and heterochromatin are closely associated, their precise relationship has not been elucidated. Recently, several members of the SNF2 family of chromatin remodeling enzymes have been found to regulate DNA methylation, suggesting that chromatin structure influences DNA methylation pattern (34).
SNF2 family members are marked by the presence of seven conserved "helicase" motifs involved in ATP binding and hydrolysis (13, 14). Many ATP-dependent chromatin remodeling complexes have been identified, such as SWI/SNF/BRM, ISWI, or Mi-2/NuRD, and each chromatin remodeling complex contains at least one member of the SNF2 superfamily. Chromatin remodeling complexes disrupt histone-DNA interactions and allows for the physical movement, or sliding, of nucleosomes on the DNA utilizing the energy derived from ATP hydrolysis (3). Thus, they alter the accessibility of chromatin to various proteins that regulate DNA-based processes such as DNA replication, transcription, and repair. DDM1, ATRX, and Lsh all belong to the SNF2 superfamily and have been demonstrated to regulate DNA methylation (12, 20, 25). Recombinant DDM1 has been recently demonstrated to induce nucleosome repositioning on a short DNA fragment, thus confirming its membership in the SNF2 family of chromatin remodeling proteins (8). However, neither DDM1, ATR-X, nor Lsh have been reported as yet to interact with large protein complexes, a characteristic of other SNF2 family members.
The DDM1 gene (named for decrease in DNA methylation) deficiency causes a 70% reduction of genomic DNA methylation in the plant Arabidopsis thaliana (27). The loss of DNA methylation is most pronounced at repetitive sequences, followed by hypomethylation at single-copy genes. ATRX deficiency gives rise to changes in the pattern of methylation of several highly repeated sequences, including ribosomal DNA arrays, a Y-specific satellite sequence, and subtelomeric repeats. Mutations in the human ATRX gene result in the inherited disease 
-thalassemia (19, 20).
Lymphoid-specific helicase (Lsh) is another member of the SNF2 family of chromatin remodeling proteins (17, 24, 28). Lsh is a nonredundant member of the SNF2 family and is essential for normal murine development and survival. Targeted deletion of Lsh results in perinatal lethality, with abnormal lymphoid development and renal defects (16, 18). DNA derived from Lsh-/- tissue shows a substantial loss of CpG methylaton (12). This suggests that Lsh is an important factor in determining DNA methylation and thus crucial for heterochromatin organization in mammals. However, it is not known whether Lsh controls methylation directly or indirectly, for example, via induction of other enzymes involved in DNA methylation. We have previously reported that Lsh does not influence methyltransferase activity or Dnmt1, Dnmt3a, or Dnmt3b protein levels, suggesting a more direct role of Lsh in establishing or maintaining methylation (12). Here we sought to examine the cellular localization of Lsh and its association with chromatin. We report a close association of Lsh with pericentromeric heterochromatin, suggesting that Lsh via CpG methylation directly participates in the organization of heterochromatin. Disruption of higher-order heterochromatin structure by prolonged exposure to the histone acetylase inhibitor trichostatin A (TSA) is followed by delocalization of Lsh. These results are consistent with a model in which the histone tail modifications that shape higher-order heterochromatin structure provide a signal for Lsh recruitment that in turn is crucial for establishing methylation patterns and the formation of heterochromatin.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vector construction.
Based on the mouse Lsh cDNA (U25691) sequence, three primers were designed. The first, a 5' primer designated LSHN01 (5'-AGAGATGGCCGAACAAACGGAG-3'), encoded the N-terminal of LSH, including the initiation codon. The second, a 3' primer designated LSHFC1 (5'-CCGGCTAGCTCACTTGTCATCGTCGTCCTTGTAGTCAAATAAACATTCAGCACTGG-3'), encoded the C-terminal sequence of the Lsh peptide, followed by a DNA sequence encoding the eight-amino-acid FLAG tag (DYKDDDDK) and a stop codon. The third was another 3' primer, LSHFC2 (5'-CCTTGTCATCGTCGTCCTTGAGTCAAATAAACATTCAGCACTGG-3'), that contained a C-terminal sequence for the Lsh protein followed by a DNA sequence encoding the Flag tag without a stop codon. The complete open reading frame of the full-length mouse Lsh cDNA was amplified by reverse transcription-PCR with total RNA purified from PD31 cells as a template and the primer pair LSHN01 and LSHFC1. A 2.5-kb portion of PCR-amplified product was subcloned directly into pCRBLUNT (Invitrogen), forming the vector pCRLSHF1. By using the pCRLSHF1 as a template and two primers, LSHN01 and LSHFC2, the PCR-amplified product was subcloned into pCRBLUNT (Invitrogen), and the new vector was termed pCRLSHF2. The plasmid pCRLSHF1 was cut with EcoRI, and the LSH DNA fragment was subcloned into the EcoRI site of pGene/V5-his (Invitrogen), designating the vector pGeneLSHF. The same EcoRI DNA fragment was subcloned into the EcoRI site of pEGFP-C1 (Clontech), generating the vector pEGFPLSH that encoded the EGFP-tagged Lsh at the C terminus. The pEGFPLSH was partially digested with EcoRI, MunI, and BamHI, respectively. The appropriate DNA fragments were recollected and self-ligated. The serial deletion mutants of Lsh were generated as follows. After EcoRI digestion, the deletion mutant, 
1-176, did not contain the DNA sequence encoding the 1 to 176 of amino acids of Lsh. The vector generated after MunI digestion had the DNA fragment deleted that encoded amino acids 244 to 469 of Lsh and was thus designated 244-469. The vector generated after BamHI digestion did not encode amino acids 475 to 768 of Lsh and was therefore designated 475-768. The plasmid pCRLSHF2 was digested with EcoRI and cloned into the EcoRI site of pEGFP-N1 (Clontech), producing the vector pLSHEGFP that encoded the enhanced green fluorescent protein (EGFP)-tagged Lsh at the N terminus. The pLSHEGFP vector was digested with XhoI, and the appropriate DNA fragments were collected and self-ligated. The deletion vector, termed 1-31, generated by XhoI digestion did not encode amino acids 1 to 31 of Lsh.
Stable and transient transfections. The GeneSwitch system (Invitrogen), a mifepristone (Invitrogen)-regulated expression system for mammalian cells, was chosen to produce a cell line with inducible expression of the Lsh gene. The GeneSwitch 3T3 cells express the GeneSwitch regulatory protein from the pSwitch vector. GeneSwitch 3T3 cells were seeded at a concentration of 106 in 10-cm dishes overnight before transfection. Cells were transfected with 2.5 µg of Lsh-inducible vector pGeneLSHF or empty vector pGene/V5-his as a control, by using FuGENE 6 transfection reagent (Roche) according to the manufacturer's recommendations. Stably transfected cells were selected with 500 to 1,000 µg of Zeocin (Invitrogen)/ml. Zeocin-resistant cells were induced with 100 pM mifepristone for induction of the Lsh protein. For EGFP expression NIH 3T3 cells were seeded at a concentration of 1 x 105 to 2 x 105 cells per well in a six-well plate (Costar cluster multiple-well plates) for 15 h before transfection. Cells were transfected with 2 to 4 µg of EGFP-tagged Lsh vector or control vector by using SuperFect transfection reagent (Qiagen) or FuGENE 6 transfection reagent (Roche) according to manufacturers' recommendations. Transiently transfected cells were cultured overnight, and Lsh-GFP expression was observed by fluorescence microcopy or Lsh expression was examined by Western analysis.
Cell cycle analysis. For cell cycle synchronization, EL4 cells were seeded at ca. 105/ml in a total volume of 10 ml per 25-cm2 flask, with L-mimosine (400 µM; Sigma), aphidicolin (250 ng/ml; Sigma), and nocodazole (400 ng/ml; Sigma). The treated cells were incubated for 24 h in the presence of the blocking reagents, and the drugs were removed for flow cytometric analysis or Western analysis. For cell cycle synchronization, NIH 3T3 cells were incubated at 3 µg of aphidicolin/ml for 18 h and then released into aphidicolin-free medium (10). For cell cycle analysis, cell suspensions were washed twice in phosphate-buffered saline (PBS) and fixed with 70% of ethanol at -20°C for at least 2 h. Cells were washed once with PBS and then incubated in 25 µg of propidium iodide (Sigma)/ml with 100 µg of RNase A/ml in PBS at 37°C for 30 min. Flow cytometric analysis was performed on a Coulter EPICS XL-MCL cytometer. The data were collected and analyzed by using the Coulter System II software provided by the manufacturer.
Indirect immunofluorescence studies.
MEFs were seeded on glass coverslips to 50 to 60% confluency. Cells were fixed in 2% paraformaldehyde solution and permeabilized with 0.5% Triton X-100 (Triton X) solution. Coverslips were blocked with 5% goat serum for 30 min at room temperature and then incubated with primary antibodies in a humidifying chamber at room temperature for 1 h or at 4°C overnight. After the coverslips were washed five times, they were incubated with Cy5 and fluorescein isothiocyanate-conjugated secondary antibodies (Jackson Immunoresearch) in PBS with 1.0 mM MgCl2 at room temperature for 1 h. After an extensive washing, coverslips were mounted onto glass microscope slides by using mounting reagent (Molecular Probes). To immunostain newly synthesized DNA and related proteins, cell staining was performed as previously described (29, 40). The following primary antibodies were used. Dnmt1 was kindly provided by T. Bestor (Department of Genetics and Development, Columbia University, New York, N.Y.) as published before (29), PCNA (PC10; 05-347) was purchased from Upstate Biotechnology, and M2 (anti-Flag) was purchased from Sigma, monoclonal bromodeoxyuridine (BrdU) antibody was from Pharmingen (catalog no. 347580) or Roche (catalog no. 1296736); HP1 (1H5; MAb3584) was purchased from Chemicon, and branched anti-histone 3 lysine 9 methylation antiserum was kindly provided by T. Jenuwein (Research Institute of Molecular Pathology, Vienna, Austria). The cells were observed under a fluorescence microscope (Zeiss) or by three-dimensional confocal microscopy (LSM 510 confocal microscope; Zeiss).
Western analysis and protein fractionation.
Protein extracts were separated on NuPAGE Bis-Tris Pre-Cast gels (for low- to proteins), or NuPAGE Tris-Acetate gels (for high-molecular-weight proteins) by electrophoresis and transferred onto nitrocellulose. Each lane contained 10 µg of protein extracts, with the exception of the cell cycle experiments or fractionation experiments, in which proteins were extracted from equal amounts of cells (i.e., 105). After a blocking step, the membrane was incubated with primary antibodies to distinct proteins, followed by incubation with secondary horseradish peroxidase-coupled antibodies (Amersham Pharmacia). Detection of horseradish peroxidase was performed by using the enhanced chemiluminescence Western blotting detection reagents according to the manufacturer's instructions, followed by visualization on Hyperfilm ECL chemiluminescent film. The following antibodies were used for detection of proteins as the primary antibodies. Lsh antiserum was used as described previously (18). Brg-1 (sc-8749), HDAC1 (sc-7872), MBD1 (sc-10751), CBP (sc-369), and p300 (sc-584) were purchased from Santa Cruz Biotechnology. M2 (anti-Flag; F3165) and Vimentin (LN-6; V2258) were purchased from Sigma. MeCP2 (07-013), PCNA (PC10; 05-347), histone H3 (05-499), acetylated histone 3 (06-599), histone4 (07-108), and acetylated histone 4 (06-598), and dimethyl-histone H3 lysine 9 (MeK9; 07-212) were purchased from Upstate Biotechnology. HP1 (2G9; MAB3446) was purchased from Chemicon, EFGP (JL-8; 8873) was from Clontech, and DNMT3a (64B1446; IMG-268) was from Imgenex. Cell extraction with Triton X, chromatin fractionation, and nuclear matrix isolation were modified as described previously (22, 38). After two washes in PBS, cells were incubated and extracted for 3 min at 4°C in cytoskeleton buffer (CSK; 10 mM PIPES [pH 6.8], 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA) supplemented with leupeptin, aprotinin, and pepstatin (1 µg/ml each), 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 0.5% (vol/vol) Triton X. The nuclear skeleton that was resistant to Triton X was separated from soluble proteins by centrifugation at 5,000 x g for 3 min. Chromatin proteins were solubilized by DNA digestion with 1 mg of RNase-free DNase I/ml in CSK buffer (with adjusted 50 mM NaCl) in the presence of proteinase inhibitors for 15 min at room temperature, and the supernatant, containing chromatin proteins, was collected by centrifugation. The pellet was washed with 0.25 M ammonium sulfate for 5 min at 4°C and pelleted again. The second pellet was washed with 2 M NaCl in CSK buffer for 5 min at 4°C and then centrifuged. The nuclear matrix was then solubilized in 8 M urea buffer.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lsh localizes to pericentromeric heterochromatin.
In order to examine to which nuclear structure Lsh is recruited, the Triton X-resistant fractions were further eluted by digestion with DNase I and high-salt washes. Under these conditions chromatin-bound fractions are separated from the nuclear matrix (22). Vimentin, for example, a component of the nuclear matrix scaffold, resists elution of high-salt concentrations (Fig. 4A). DNA-binding proteins (MecP2 and MBD1) or transcriptional regulators (p300) or the replication factor PCNA are only loosely associated with the nuclear components and are readily eluted by detergent. In contrast, about half of Lsh resists Triton X washes, and most of this fraction is detectable in the "chromatin" fraction which also contains histones, HP1 and Dnmt3a (Fig. 4A) (10). This suggests that the bound Lsh protein primarily associates with chromatin, as opposed to binding to the nuclear scaffold (for comparison, see lanes 2 and 5 in Fig. 4A). However, a small portion of induced Lsh appears in the scaffolding fraction and, at present, it cannot be determined whether this is of physiologic relevance or an artifact due to overexpression of Lsh.
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, a major component of pericentromeric heterochromatin (Fig. 4C). In addition, we have demonstrated that chromatin immunoprecipitated with anti-Lsh/Flag antibodies in 3T3 cells showed a close association of Lsh with minor and major satellite sequences that are the main components of pericentromeric DNA (Q. Yan and K. Muegge, unpublished data). Thus, our data show that Lsh is localized at pericentromeric heterochromatin, and previous work indicated that Lsh is crucial for CpG methylation at pericentromeric DNA (12). Taken together, these results support the idea that Lsh plays a direct role in the formation of pericentromeric heterochromatin. Lsh is not required for targeting of Dnmt1 to replication foci. Lsh-deficient cells show a global methylation defect that is similar to the one reported in Dnmt1-/- embryos. Thus, we hypothysed that Lsh and its presumed chromatin remodeling activity maybe important for targeting Dnmt1 to sites of methylation. Dnmt1 has a significant preference for hemimethylated DNA relative to unmethylated DNA in vitro. Dnmt1 associates with sites of DNA synthesis, the replication foci, throughout the S phase but shows a diffuse nucleoplasmic distribution pattern during other phases of the cell cycle. The direct interaction with PCNA allows Dnmt1 to track along the newly synthesized strands and to ensure proper copying of the methylation "code." Thus, Dnmt1 is thought to play a role in the maintenance of CpG methylation (5). Based on Lsh's nuclear expression pattern, we tested whether Lsh could directly participate in the maintenance of genomic methylation. Therefore, we first determined whether Lsh could associate with replication foci, the site of newly synthesized DNA. Lsh-induced 3T3 fibroblasts were synchronized with a high concentration of the cell cycle inhibitor aphidicolin. Cells were then released into normal cell cycle and at different time points pulsed with BrdU in order to detect newly synthesized DNA. Costaining for detection of incorporated BrdU, as well as Lsh, allowed the identification of different stages of the cell cycle. As shown in Fig. 5A, Lsh associated with replication foci only during late S phase, when few, large foci are visible by BrdU incorporation. Lsh did not colocalize with replication foci at early S phase, characterized by a fine punctate staining pattern, or mid-phase with a staining pattern of BrdU at the periphery of the nucleus and perinucleolar sites (29). Lsh also colocalized with Dnmt1 in late S phase (Fig. 5B). Thus, Lsh potentially may facilitate maintenance of methylation during the late phase of replication.
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after cross-linking (Yan and Muegge, unpublished), suggesting a close association of Lsh with HP1 on heterochromatic nucleosomes. However, Lsh is not directly interacting with HP1, since Flag-tagged Lsh cannot directly precipitate HP1 without cross-linking. In order to understand better the signal that recruits Lsh to pericentromeric heterochromatin, we examined whether Lsh requires higher-order heterochromatin organization for its localization. The pericentromeric heterochromatin structure depends on the presence of underacetylated histones. Prolonged treatment with the histone deacetylase inhibitor TSA results in disruption of pericentromric heterochromatin structure and centromere function (42). HP1, which is recognized by methylated lysine 9 residue of histone 3, is no longer retained at centromeric heterochromatin. Also, the binding of the H3-K9me branched peptide antibody, recognizing a close configuration of methylated lysine residues at histone 3 tails, is disturbed (31, 42). To test whether Lsh's pericentric localization was dependent on higher-order heterochromatin organization, we treated 3T3 cells with TSA to increase global levels for histone 3 and 4 acetylation (Fig. 7B). After induction of hyperacetylation, 3T3 cells displayed an altered DAPI staining pattern with relocation of centromeric regions toward the nuclear periphery (Fig. 8), as has been previously reported (42). Similarly, staining with the branched anti-H3-K9 methylation antibody was disturbed, indicating a successful disruption of the heterochromatin structure (31) (Fig. 8B). Using anti-Flag antibody as a probe, indirect immunofluorescence staining revealed a diffuse staining pattern for Lsh and a failure of Lsh to associate with DAPI. This indicated that Lsh localization requires a higher-order heterochromatin structure (Fig. 8A). In order to confirm that Lsh binding to pericentromeric heterochromatin depends on a higher-order structure, we examined the Triton X resistance of the Lsh protein. As shown in Fig. 7C, almost all Lsh was extractable with Triton X, indicating a loss of the ability to bind to chromatin. Under the same conditions HP1 was flushed by Triton X and no longer retained by heterochromatin. To demonstrate that cells did not simply succumb to histone hyperacteylation after TSA treatment, cells were washed with PBS and cultured for 24 to 48 h to allow for recovery. The association of either Lsh (Fig. 7C) or HP1 (data not shown) with heterochromatin was reversible and returned to levels of untreated cells within a day, as judged by Western analysis. The colocalization of Lsh with DAPI could also be restored within a day of recovery (Fig. 8A). Similarly, the higher-order structure as indicated by staining with the branched H3-K9me antibody reversed after 1 day of recovery (Fig. 8B). Therefore, the signal that is required for recruitment of Lsh was not erased even after TSA treatment, and removal of the drug resulted in the relocalization of Lsh. These results suggest that binding of Lsh to pericentromeric heterochromatin is dependent on histone modifications and closely associated with normal higher-order structure of heterochromatin.
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association with pericentromeric heterochromatin is not altered in the absence of Lsh, despite changes in histone methylation levels (45). In order to determine whether HP1 reassembly is delayed in the absence of Lsh, we disrupted HP1 association with pericentromeric chromatin by TSA treatment in wild-type and Lsh-deficient cells. Table 1 demonstrates that the dissociation of HP1 with DAPI occurs in ca. 77 to 85% of cells in both cultures. The recovery rate of HP1 localization in Lsh-deficient cells after 3, 6, or 12 h resembles that of wild-type cells, further supporting the idea that Lsh plays a role in maintaining rather than initiating heterochromatin formation, as further discussed below.
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DISCUSSION |
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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, 33), 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, 10, 32). Dnmt1, thought to be the major "maintenance" methyltransferase, colocalizes to heterochromatin during the late S phase of replication (29, 40). Since the global DNA methylation deficiency in Lsh-/- mutants is similar to the one reported for Dnmt1-/- mutants (12, 30), 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 (23, 43). 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, 45), it does alter H3-K4 methylation levels at heterochromatin (45). In human cancer cells, 5-azacytidine treatment or deletion of DNMT1 and DNMT3b 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, 35). 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.
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ACKNOWLEDGMENTS |
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The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.
This project was funded in whole or in part with federal funds from the National Cancer Institute and the Alternative Medicine Program, National Institutes of Health, under contract no. NO1-CO-12400.
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FOOTNOTES |
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