Enrichment for histone H3 lysine 9 methylation at Alu repeats in human cells.

The aim of this study was to identify in human cells common targets of histone H3 lysine 9 (H3-Lys9) methylation, a modification that is generally associated with gene silencing. After chromatin immunoprecipitation using an H3-Lys9 methylated antibody, we cloned the recovered DNA and sequenced 47 independent clones. Of these, 38 clones (81%) contained repetitive elements, either short interspersed transposable element (SINE or Alu elements), long terminal repeat (LTR), long interspersed transposable element (LINE), or satellite region (ALR/Alpha) DNA, and three additional clones were near Alu elements. Further characterization of these repetitive elements revealed that 32 clones (68%) were Alu repeats, corresponding to both old Alu (23 clones) and young Alu (9 clones) subfamilies. Association of H3-Lys9 methylation was confirmed by chromatin immunoprecipitation-PCR using conserved Alu primers. In addition, we randomly selected 5 Alu repeats from the recovered clones and confirmed association with H3-Lys9 by PCR using primer sets flanking the Alu elements. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine rapidly decreased the level of H3-Lys9 methylation in the Alu elements, suggesting that H3-Lys9 methylation may be related to the suppression of Alu elements through DNA methylation. Thus H3-Lys9 methylation is enriched at human repetitive elements, particularly Alu elements, and may play a role in the suppression of recombination by these elements.

Post-translational modifications of histone tails play a critical role in the assembly of heterochromatin, and accumulating evidence suggests that the pattern of histone modifications specify regulation of gene expression (1,2). Acetylation of lysine residues on histone H3 and H4 usually leads to the formation of an open chromatin structure, with transcription factors accessible to promoters. Phosphorylation of serine 10 and acetylation of lysine 14 on histone H3 result in gene activation (3). Lys 4 methylation on histone H3 localizes to sites of active transcription, and this modification may be stimulatory for transcription (4). The different combinations of histone tail modifications influence transcription by affecting chromatin structure. Recent studies have shown that methylation at Lys 9 on histone H3 is a marker of heterochromatin and is specifically associated with inactivation of gene expression (5). H3-Lys 9 methylation is enriched on the inactive X chromosome in women (6) and at loci silenced in cancer (7)(8)(9). Beyond this, the distribution of H3-Lys 9 in human DNA has not been well characterized.
Alu repeats represent the most frequent repetitive element in the human genome (10). Alu elements are ϳ280 bp in length and consist of two similar, but distinct, monomers linked by an oligo(dA) tract. Presumably, the 1 million Alu elements that are fixed within the human genome represent the fraction of insertions that were neutral or at least tolerable functionally. Alu elements possess an RNA polymerase (pol III) promoter (11). Despite this, the steady state abundance of pol III-directed Alu transcripts is usually very low in tissues and cultured cells indicating transcriptional silencing (12). However, cellular stress can dramatically increase the abundance of human Alu RNA (12), although it is unknown how and how many Alu elements are induced by stress. Accumulating evidence indicates that regulation of Alu expression is determined on many levels, some of which act locally upon individual Alu elements (13,14). DNA methylation and chromatin configuration might each globally repress transcription of this repetitive sequence family and therefore potentially direct regulation of the Alu stress response.
Here we performed an unbiased approach to clone targets of H3-Lys 9 methylation using chromatin immunoprecipitation (ChIP) 1 assay and found that most such clones contain Alu elements. Thus, Alu expression might be crucially regulated by H3-Lys 9 methylation and associated chromatin remodeling.

EXPERIMENTAL PROCEDURES
Cell Lines and Culture Conditions-SW48 cells were grown in L-15 medium (Invitrogen) plus 10% fetal bovine serum in plastic tissue culture plates in a humidified atmosphere containing 5% CO 2 at 37°C. RKO cells were grown in high glucose Dulbecco's modified Eagle's medium (Invitrogen) plus 10% fetal bovine serum (Intergen). The cells were grown to a density of 5.0 ϫ 10 5 to 3.0 ϫ 10 6 cells per dish before being harvested for cross-linking experiments. Cell lines were obtained from the American Type Culture Collection.
5-Aza-dC Treatment of Cells-Cells were split 12-24 h before treatment. Cells were then treated with either 5-aza-dC, 5 M (Sigma), or phosphate-buffered saline for 72 h. Media containing 5-aza-dC or phosphate-buffered saline was changed every 24 h.
Chromatin Immunoprecipitation-The protocols for ChIP have been described previously (15). Briefly, SW48 and RKO cells are treated with 1% formaldehyde for 8 min to cross-link histones to DNA. After washing by cold phosphate-buffered saline, the cell pellets are resuspended in * This work was supported in part by National Institutes of Health Grant R33CA89837 and by the George and Barbara Bush fund for innovative cancer research. DNA sequencing in the M. D. Anderson Cancer Center core sequencing facility is supported by Core Grant CA16672 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 550 l of lysis buffer (150 mM NaCl, 25 mM Tris-Cl, pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate) and sonicated for 7 ϫ 8 s. The lysate (500 l) is then divided into three fractions; the first and second ones (230 l each) are diluted in 260 l of lysis buffer, and the third one (40 l) is used for input control. The first lysate is incubated with 10 l of anti H3-Lys 9 methylated histone H3 antibody (Upstate Biotechnology) at 4°C overnight. The second lysate is incubated with Tris/EDTA buffer (10 l) at 4°C overnight as a negative control. To collect the immunoprecipitated complexes, protein A-Sepharose beads (Amersham Biosciences) are added and incubated for 1 h at 4°C. After washing, the beads are treated with RNase (50 g/ml) for 30 min at 37°C and then proteinase K overnight. The cross-links are then reversed by heating the sample at 65°C for 6 h. DNA is extracted by the phenol/chloroform method, ethanol-precipitated, and resuspended in 100 l of water. We performed a total of six individual chromatin immunoprecipitations in the SW48 cell line to obtain DNA enriched with methylated H3-Lys 9 for cloning purposes.
Cloning of H3-Lys 9 -enriched DNA Fragments-SW48 ChIP products were ethanol-precipitated and treated with mung bean nuclease to make blunt ends, then cloned into the zero-blunt vector (Invitrogen). Colonies having inserts were identified by EcoRI restriction enzyme digestion and were sequenced at the MDACC core sequencing facility.
ChIP-PCR-ChIP products from SW48 and RKO were used for confirmation PCR using the oligonucleotide primers as follows. Alu11-F, CAGCCTGGGTGATAGAGCAAG; Alu11-R, AGAGAAAGAGGAAACA-CAAGGAGC; Alu21-F, GCACCCAGCTGAATTTTCCA; Alu21-R, CTG-  The PCR products are visualized by agarose or 6% polyacrylamide gel electrophoresis and quantitated by capillary electrophoresis using the Agilent 2100 bioanalyzer (Agilent Technologies). To ensure that PCR amplification was in the linear range, each reaction was initially set up at different dilutions of DNA for varying amplification cycle numbers, and we selected the final PCR conditions accordingly. The assays were done in duplicate.

RESULTS
To identify genes linked to methylated histone H3-Lys 9 , we performed ChIP using an anti-Lys 9 methylated histone H3 antibody and cloned the immunoprecipitated DNA (strategy described in Fig. 1). Initially, we verified that the library contained methylated H3-Lys 9 enriched DNA fragments by PCR using primers encompassing the promoter regions of P16, hMLH1, P21, and GAPDH. P16 and hMLH1 are silenced, and P21 and GAPDH are expressed in the SW48 cell line (7). In our library, compared with genomic DNA, the former two promoters showed a high degree of enrichment and the latter two promoters showed specific depletion, in accordance with known levels of H3-Lys 9 methylation (data not shown). Having verified the quality of the library, we then randomly selected 47 clones, sequenced them, and characterized the sequences using BLAST (www.ncbi.nlm.nih.gov/) and BLAT (genome.ucsc.edu/) searches.
The results of sequencing are summarized in Table I, and a representative example is shown in Fig. 2. Twenty-four (51%) of forty-seven clones contained Alu elements only. Five clones had both Alu and long terminal repeats (LTR) or Alu and long interspersed transposable element (LINE). Three additional clones had Alu elements within 200 -300 bp of the recovered fragment (clones 43, 44, and 45 had AluSg, -Ye, and -Jb, respectively). Some of the sequences began or ended in the middle of Alu elements, and the range of Alu element length in the recovered clones was 95-282 bp in each clone. Thus, overall, 32 of 47 (68%) clones were in or within 200 bp of an Alu element. Considering that Alu elements form about 10% of the human genome (16), this result indicates marked enrichment for Alu elements in this library, suggesting a high degree of H3-Lys 9 methylation at this repetitive sequence. Twenty-three (72%) of thirty-two Alu elements belonged to the old Alu subfamily, and In the assays, DNA combined with methylated histone H3-Lys 9 antibody is immunoprecipitated and detected by PCR amplification. The promoters of P21 and MLH1 show a low and high level of H3-Lys 9 methylation in SW48 and RKO cell lines, respectively. The intensity of the PCR bands was quantitated by densitometry or using the Agilent 2100 bioanalyzer. C, ratios of precipitated DNA over input DNA were calculated as a relative precipitated fold shown on the y axis. nine (28%) Alu elements were classified into young Alu subfamilies. We also obtained 1 clone related to a short interspersed transposable element (SINE) other than Alu. Three clones contained LTRs only, and four clones contained LINE elements only, respectively. Satellite region (ALR/Alpha) DNA was present in only 1 clone. Including Alu elements, a total of 41 clones (87%) were related to repetitive elements, which then form the bulk of DNA modified at H3-Lys 9 . We found no CpG island within 3 kb of any clone.
To verify these results, we randomly selected 5 Alu clones and prepared primer sets encompassing the Alu elements (ChIP-PCR). In each case, at least one primer was located in the flanking region to ensure that we were studying that specific Alu element ( Fig. 2A). ChIP-PCR experiments using these primers in SW48 and RKO colorectal cancer cells are shown in Fig. 2B. MLH1 and P21 were used as controls. As we reported previously (7), both SW48 and RKO show high levels of H3-Lys 9 methylation in the hMLH1 promoter and low levels of H3-Lys 9 methylation in the P21 promoter, corresponding to gene suppression and activation, respectively. All Alu elements showed a pattern similar to the hMLH1 promoter in both cell lines. We summarized these quantitative analyses in Fig. 2C. Ratios of precipitated DNA over input DNA were high in all the Alu elements and hMLH1 and low in P21, suggesting that Alu elements are suppressed by H3-Lys 9 methylation in a manner similar to hMLH1 gene silencing in cancer. Primers that amplify many Alu elements simultaneously (i.e. in the conserved Alu sequence) showed similar results (data not shown).
Alu elements show high degrees of DNA methylation in human cells (14,17). Therefore, it is possible that the H3-Lys 9 methylation we observed was related to DNA methylation and recruitment of a silencing complex. To test this hypothesis, we examined the effects of inhibition of DNA methylation on Alu H3-Lys 9 methylation. To this end, we performed ChIP on SW48 cells treated with the DNA methyltransferase inhibitor 5-aza-dC daily for 3 consecutive days. As shown in Fig. 3, all Alu elements and MLH1 showed a decreasing level of H3-Lys 9 methylation shortly after 5-aza-dC. These results are consistent with our previous reports on non-repetitive, silenced genes and suggest that H3-Lys 9 methylation of Alu elements is partially targeted by DNA methylation. DISCUSSION In this study we identified Alu elements as major targets for histone H3-Lys 9 methylation using chromatin immunoprecipitation. Methylation at Lys 9 on histone H3 has been shown to be a marker of heterochromatin and to be associated with inactivation of gene expression (5). In the promoter regions of P16, hMLH1, and MGMT, we recently found increased H3-Lys 9 methylation in cells with promoter DNA methylation-associated silencing (7), and others have reported similar findings (8,9). Thus, H3-Lys 9 methylation appears to be a ubiquitous marker of silencing in human cells and likely leads to transcriptional repression by recruitment of a protein complex such as the HP1-associated complex (18).
The repetitive DNA at yeast centromeres is maintained in a transcriptionally silent state by methylation of H3-Lys 9 and binding of Swi6 or heterochromatin protein 1 (HP1) to the modified chromatin (19). A recent study of centromere chromatin silencing in the fission yeast, Schizosaccharomyces pombe, demonstrated that silencing of homologous repeats by H3-Lys 9 methylation is initially mediated by components of the RNA interference machinery (20). Intriguingly, gene silencing in inactivation of the X chromosome in mammalian female cells involves the expression of Xist RNA, which coats the X chromosome, followed soon after by H3-Lys 9 methylation, gene inactivation, and ultimately methylation of DNA (21). The finding of Alu elements as a target of H3-Lys 9 methylation raises the possibility that by analogy to yeast silencing of homologous repeats, mechanisms involving RNA interference also operate in the establishment of silenced Alu elements.
Among 32 Alu clones recovered here, the frequency of old Alu elements (23 clones, 72%) and young Alu elements (9 clones, 28%) are consistent with the prevalence of old and young Alu elements in the human genome. In older Alu subfamilies, many of the CpG sites have mutated to TpG or CpA because of deamination of 5-methylcytosine. In contrast, newly retrotransposed Alu elements are CpG-rich and extensively methylated. Our results show that histone H3-Lys 9 methylation is characteristic of both old and young Alu subfamilies and suggests that, once established, H3-Lys 9 Alu methylation persists over long periods of time, despite eventual inactivation of the Alu elements by promoter mutations.
We also found another repetitive element, LINE, in 7 clones (15%). LINE-1 elements comprise roughly 15% of the human genome and are concentrated in AT-rich regions (16). LINE-1 promoters are also usually methylated at DNA (22), thus providing an explanation for H3-Lys 9 methylation. Nevertheless, the frequency of LINE elements in our clones was almost similar to that in the human genome, suggesting that LINE elements are less specifically targeted than Alu elements, al- FIG. 3. A, results of histone H3-Lys9 methylation ChIP assays in SW48 cells treated with 5-aza-dC (DAC). Assays were conducted as in Fig. 2. B, ratios of precipitated DNA over input DNA is shown on the y axis. Clone identity is shown on the x axis. All Alu elements, MLH1, and P21 show decreasing levels of H3-Lys 9 methylation. though the number of clones analyzed overall in this study is small.
It remains unclear which protein complexes are involved in Alu silencing when these sequences are highly methylated. The SNF2h-NuRD-cohesin complex, which is a chromatin remodeling complex, was recently reported to specifically associate with some Alu elements, accompanied by histone H3-Lys 4 methylation and either histone H3 or H4 acetylation (23). In that study, the binding of the cohesin complex to chromatin around Alu elements could be increased by inhibition of DNA methylation. In our study, treating cells with 5-aza-dC decreased the degree of H3-Lys 9 methylation in all 5 Alu elements tested. It is possible then that the SNF2h-NuRD-cohesin complex counteracts the activity of a silencing complex recruited by histone H3-Lys 9 methylation and that normally reside on Alu elements, possibly as a result of DNA methylation in these regions.
An obvious functional significance of these findings is to provide a molecular mechanism for the known silencing of Alu elements. This silencing is thought to be essential in reducing the mutational load associated with active Alu elements in dividing cells, as has been proposed in the genome defense hypothesis (24). It is of interest, however, that H3-Lys 9 methylation can spread in cis, as seen in fission yeast (25). If similar spreading occurs in human cells, one might observe time-dependent spreading of silencing, a hypothesis consistent with the observations of age-related DNA methylation in normal tissues (26,27). Furthermore, such Alu element H3-Lys 9 methylation may serve as an initial seed for gene silencing in cancer, as suggested by previous studies (28 -30).