Centromeric Repeats Are Transcribed and Remain in the Nucleus

Arabidopsis cen180 repeat families are 78% to 96% identical in sequence. Variant repeats are located on different chromosomes [49] and may, like human alpha satellites, form subdomains [50], but each repeat contains common conserved regions [2]. We designed cen180 Fc and Rc primers (Figure 1A) to recognize two conserved regions, and these primers amplified a large number of repeats from genomic DNA. Transcripts from centromeric repeats were detected by RT followed by PCR. “F-strand transcripts” are transcribed from the forward (Watson) DNA strand and reverse transcribed into cDNA using an F primer; “R-strand transcripts” are transcribed from the reverse (Crick) DNA strand and reverse transcribed into cDNA using an R primer. Using the highly conserved Fc and Rc primers, transcripts from both strands could be detected (Figure 1B). However, transcripts from each strand could be derived from different subfamilies of repeats. In order to test this possibility, specific primers were used (F primer and R primer, Figure 1A) to amplify only a subset of cen180 repeats [20]. In this case, we found that F-strand transcripts were much more abundant than R-strand transcripts in vegetative and floral tissues, suggesting strand-specific transcription of individual subfamilies of repeats (Figure 1B). Transcription from only one strand was consistent with transcripts initiating in tandem orientation from the repeats, while the amplification of 180-bp multimers indicated that transcripts did not terminate within each repeat. Transcript levels were highest in young seedlings and developing inflorescences, indicating expression in dividing cells.

The same subfamily of cen180 repeats were amplified from genomic DNA and used as strand-specific probes for in situ hybridization (see Materials and Methods). In longitudinal sections of WT inflorescences, F-strand transcripts were more abundant than R-strand transcripts (Figure 2), consistent with RT-PCR (Figure 1B). Young, proliferating tissues (Figure 2A and 2B) had more signal than mature tissue (Figure 2C and 2D). Strand and tissue specificity indicated that the signal came from cellular RNA and was not due to background hybridization with fortuitously denatured DNA. Under higher magnification, small punctate nuclear signals were seen with the F-strand probe, consistent with nascent transcripts remaining at the centromere (Figure 2E). In contrast, what little signal could be detected with the R-strand probe was nuclear but not punctate (Figure 2B and 2D).

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Figure 2. In Situ Hybridization of Centromeric Probes with Longitudinal Sections of Inflorescences

(A) Inflorescence meristem and developing buds probed with digoxigenin-labeled F strand of cen180 F + R PCR products and (B) probed with R strand. Mature tissue about 5 mm below the meristem (C) probed with F strand of cen180 and (D) probed with R strand. The scale bars are 50 μm.

(E) More highly magnified view of nuclei from developing buds probed with F strand (scale bar is 10 μm).

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Satellite Repeat Transcripts Are Regulated by DNA Methylation, Histone Modification, and RNAi

In S. pombe, H3K9me2 depends on RNA silencing of pericentromeric repeats [23], and vice versa [51]. Similarly, in Arabidopsis, H3K9me2, RNAi, and DNA methylations are mutually interdependent [39,45]. We examined the level of pericentromeric transcripts in Arabidopsis mutants defective in DNA and histone methylation as well as in RNAi (Figure 3A). Elevated transcript levels were detected in dcl1–9 (a weak allele of DICER-LIKE1), ago1–9 (a strong allele of ARGONAUTE1), rdr2–1 (a T-DNA insertion in RNA DEPENDENT RNA POLYMERASE2), dcl3–1 (a T-DNA insertion in DICER-LIKE3), kyp-2 (a splice-site mutation in histone H3 K9 methyltransferase), hda6/sil1 (an allele of HISTONE DEACETYLASE6), cmt3-m5662 (a null allele of the CNG DNA methyltransferase), met1–1 (a strong allele of the CG DNA methyltransferase), and ddm1–2 (a hypomorphic allele of the swi/snf chromatin remodeling ATPase). Strand-specific RT-PCR (Figure 3A) indicated that F-strand transcripts were elevated in abundance in cmt3, kyp, hda6, and the RNAi mutants, whereas both F- and R-strand transcripts accumulated in met1 and ddm1. In backcrosses to WT, elevated transcript levels were inherited epigenetically from cmt3, ddm1, and kyp. Reestablishment of silencing was observed for both strands in met1/+ backcrosses and on one strand in hda6/+ backcrosses. dcl1–9 and ago1–9 were not tested in this way due to sterility [39]. Northern analysis of met1 and dcl1 mutants revealed heterogeneous transcripts ranging from approximately 0.1 kb to more than 5 kb, indicating multiple, irregular transcription initiation and/or termination sites (Figure S1).

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Figure 3. Regulation of Centromeric Transcripts

(A) Strand-specific RT-PCR of cen180 repeats. RT was performed with forward and reverse primers indicated to the left of each panel. Mutants in DNA methylation (cmt3, met1), chromatin remodeling (ddm1), histone modification (kyp, hda6), and RNAi (dcl1, ago1) were in the Landsberg erecta (Ler) ecotype. rdr2 and dcl3 were in the Columbia background. m indicates homozygous mutants and b indicates backcrosses of mutants to WT (wt). Positive controls were primers recognizing the ACTIN2 gene; negative controls lacked reverse transcriptase (no RT).

(B) Restriction site analysis of repeats. Repeats were amplified by cen180 F+R primers from genomic DNA (lanes 1), cDNA from met1 mutants (lanes 2), and cDNA from dcl1 mutants (lanes 3). Repeats in genomic DNA that have TaqI sites are not transcribed in either mutant, while those with Sau3AI sites are not transcribed in dcl1.

(C) Oligonucleotide probes specific for the subfamilies of F + R cen180 repeats expressed in dcl1 and met1 mutants.

(D) Strand-specific RT-PCR of cen180 repeats. RT was performed with F or R primers as in (A). Southern hybridization of these products with specific oligonucleotide probes distinguished subfamilies of repeats that were differentially regulated.

(E) Detection of read-through transcripts between cen180 and 106B repeats by strand-specific RT-PCR. 106B Fout (lane 1) and cen180 F (lane 2) primers were used to prime cDNA, followed by PCR amplification. The configuration of the transcripts is shown below each lane. Other combinations of primers did not give detectable products. RNA was prepared from aerial tissues from 28-day-old plants.

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Satellite Repeat Subfamilies Are Silenced by Different Mechanisms

One explanation for this differential regulation was that further subfamilies of F + R repeats were differentially regulated on each strand. We therefore examined which particular repeats were expressed in met1 compared with dcl1, ago1, and WT using restriction and sequence analysis of cDNA. Digestion of PCR products from cDNA and genomic DNA with TaqI revealed that transcribed repeats, which were sensitive to TaqI digestion, were a minority of those amplified from genomic DNA, most of which were not sensitive (Figure 3B). Further, RT-PCR products from met1 were digested by Sau3AI, indicating they had Sau3AI sites, but cDNAs from dcl1 were not digested and must therefore differ in sequence. Transcribed repeats were sequenced and grouped by cDNA sequence similarity using CLUSTALW. Whereas those from WT, dcl1, and ago1 formed mixed clusters, transcripts from met1 were from a distinct subfamily of repeats (Figure S2).

Diagnostic 20-mers were designed from the most highly diverged satellite cDNAs (Figure 3C) and hybridized to strand-specific RT-PCR products from the various mutants (Figure 3D). Hybridization was exclusive and complementary. F-strand transcripts from the subfamily of repeats expressed in WT were elevated in cmt3, kyp, hda6, dcl1, and ago1, but transcripts from the R strand were not seen. Elevated transcript levels were inherited epigenetically when kyp, hda6, and cmt3 mutants were backcrossed to WT. The subfamily of repeats expressed in met1 was transcribed from both strands in met1 and hda6 but only from the F strand in ddm1. These transcripts could not be detected in WT and were epigenetically inherited in ddm1/+ but were substantially resilenced in hda6/+ or met1/+ backcross progeny. In this respect, they resemble ATGP1 gypsy-class LTR retrotransposons [39,41,52]. Additional R-strand transcripts, detected by RT-PCR in ddm1, did not hybridize with either probe, indicating a third subfamily of differentially regulated repeats.

The differentially regulated subfamilies of cen180 repeats are very similar, so sequence differences are unlikely to account for differences in regulation. A more likely explanation was that some cen180 transcripts might be driven by read-through from adjacent retroelements. The 106B LTR-like repeats and Athila-class LTR retroelements are integrated throughout pericentromeric regions [53] and are interspersed in random orientation with respect to cen180s (data not shown). F-strand 106B transcripts were detected in WT but were only weakly up-regulated in cmt3, kyp, and hda6 (Figure 3A). Transcripts from both strands were up-regulated in ddm1 and met1, and R-strand transcripts were up-regulated in hda6. In this respect, 106B repeats resembled both classes of cen180 repeats, and we examined the possibility they were co-transcribed using cDNA that was reverse transcribed with 106B or cen180 primers (Figure 3E). F-strand co-transcripts originating in cen180 repeat arrays could be detected in WT and dcl1. In contrast, reverse strand co-transcripts appeared to originate within 106B and could only be detected in met1. Subsequent RACE PCR showed that these co-transcripts originated in the LTR of Athila2 itself (not shown). Other co-transcripts were not seen. This indicated that differential genetic regulation of each class of cen180 repeat transcript could be explained by differential origin of the transcripts in 106B/Athila2 and cen180 repeats, respectively.

Polymorphic Regulation of Repeats

Most of the mutants described above were isolated in the Landsberg genetic background, but rdr2 and dcl3 were isolated in Columbia, and so this WT strain was also assayed for transcripts. Surprisingly, Columbia and Landsberg transcripts amplified with conserved primers Fc and Rc differed in their regulation (Figures 3A and 4). We therefore sequenced cen180 RT-PCR products from dcl3 and met1 mutants of Columbia and designed diagnostic primers for amplification of genomic DNA and cDNA (Figure 4). Primers corresponding to dcl3-and met1-specific transcripts amplified cDNA from each corresponding mutant in Columbia, as expected. However, no cDNA could be amplified from met1 or dcl1 mutants in Landsberg despite the presence of such repeats in the Landsberg genome (indicated by amplification of genomic DNA). Conversely, specific transcripts detected in dcl1 mutants in Landsberg were found to accumulate in met1 mutants of Columbia, indicating that the regulation of this class of repeats had changed during the divergence of these two ecotypes. Repeats transcribed specifically in met1 mutants of Landsberg do not appear to be present in the Columbia genome. The origin of this natural variation is discussed below.

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Figure 4. Polymorphic Regulation of cen180 Transcripts

Primers were designed to recognize sample cen180 cDNAs from dcl1 and met1 mutants of Landsberg erecta (see Figure 3C) and dcl3 and met1 mutants of Columbia. PCR amplification using genomic DNA templates (DNA) tested for the presence of the particular repeats in the genome of each ecotype. RT-PCRs using RNA detected transcripts in WT (wt) and mutants of each ecotype as indicated. The cen180 Fc + Rc primers served as positive controls in the presence of reverse transcriptase and negative controls in its absence. RNA was prepared from aerial tissues from 28-day-old plants.

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Histone Modifications Associated with Satellite Repeats

We attempted to examine histone methylation associated with centromeric repeats using semiquantitative chromatin immunoprecipitation, although this proved to be very difficult as previously reported [20,40] because of the very high copy numbers involved. Nonetheless, while quantitative changes were not reliable, qualitative changes could be detected (Figure S3). H3K9me2 was substantially reduced in kyp and ago1 over both classes of repeat, resembling transposons such as ATCOPIA4 [39] as well as pericentromeric repeats and retrotransposons in S. pombe and Drosophila [23,31]. In met1 and ddm1, H3K9me2 was largely lost from 106B but only slightly reduced at cen180. H3K4me2 underwent a modest but heritable increase in ddm1 and was found associated with each class of repeat in WT, perhaps reflecting the transcription of the repeats. The association of H3K4me2 with heterochromatic pericentromeric repeats has also been reported in S. pombe [23], while in Drosophila [33] it is interspersed with Cid in more central domains.

DNA methylation of centromeric repeats has been extensively investigated by gel blot analysis, and we have obtained similar results (Figure S4). CNG methylation is partial in WT and reduced in ddm1, cmt3, and kyp [21,37,42,43], but it is unaffected in hda6 [54]. CG methylation is more extensive than CNG methylation at the centromere in WT and is lost only in met1 and ddm1. However, met1 affects transcripts initiating in 106B/Athila2 LTRs and not those initiating within the class of cen180 repeats regulated by RNAi (Figure 3E). hda6 has a similar effect on 106B transcripts but has no effect on CG methylation, although H3K9me2 is reduced ([54] and data not shown). H3K9me2 was retained in rdr2 and dcl3 (data not shown). Thus, neither DNA methylation nor H3K9me2 correlates perfectly with the specific deregulation of 106B/Athila2- or cen180-driven satellite repeat transcripts, although complexes responsible for each modification play a major role.

siRNA from Centromeric Transcripts Depends on DDM1, DCL3, and RDR2

Centromeric repeat transcripts in S. pombe correspond to siRNAs [55] that depend on Dcr1, Rdr1, and Ago1 for their accumulation [56,57]. In Arabidopsis, 24-nucleotide siRNAs corresponding to 180-bp centromeric repeats were detected in WT and were unchanged in met1, hda6, cmt3, ago1, dcl1, and kyp but were reduced in ddm1 and almost entirely absent from dcl3 and rdr2 (Figure 5A and 5B). These blots were deliberately overexposed to show residual levels of 24 nucleotides and smaller classes of siRNA. It is not possible to tell if these are derived from specific subclasses of repeats. Small RNAs corresponding to both strands of 106B repeats were also unchanged in most mutants but were increased in ddm1, decreased in ago1 (Figure 5A), and could not be detected in dcl3 and rdr2 (Figure 5B), resembling siRNA derived from the gypsy class retrotransposons ATGP1 [39] and Athila2 (data not shown). Both dcl3 and rdr2 have been shown to be required for production of endogenous siRNAs, while dcl1 is required for processing of micro-RNAs [58] and trans-acting siRNAs [59,60].

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Figure 5. siRNAs from Centromeric Repeats

Total RNA was purified from plants of the indicated genotypes, and small RNAs were enriched by solubility in PEG (see Materials and Methods). Mutants and WT (wt) were in the Landsberg erecta background (A) or the Columbia background (B). RNA was prepared from aerial tissues from 28-day-old plants.

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Lagging Chromosomes Were Not Observed in Mutants Defective in Silencing

In the fission yeast S. pombe, mutants defective in centromeric silencing recruit, but fail to retain, cohesin in anaphase pericentromeric heterochromatin, because of the loss of H3K9me2 and associated Swi6 [61]. We therefore examined anaphase in centromeric silencing mutants in Arabidopsis. Developing floral or root tissue was fixed in paraformaldehyde and stained with DAPI to detect the presence of lagging or otherwise abnormal chromosomes during anaphase. In WT anaphase cells (N = 20), rdr2 mutant cells (N = 49), and ddm1 mutant cells (N = 23), no abnormality was seen (not shown). Further, these mutants were indistinguishable from WT in growth and fertility, unlike mutants in rad21/cohesin, which are semisterile due to defects in meiosis [34]. One explanation might be that H3K9me2 was largely retained in rdr2 and ddm1, so that any interaction with Swi6 homologs and cohesin was retained. However, mutants in the H3K9 methyltransferase kyp are also fully fertile, and lagging chromosomes were not observed in kyp anaphase cells either (N = 24). Differences between the yeast and plant systems are discussed below.

Silencing of Centromeric Transcripts in Arabidopsis and Fission Yeast

Arabidopsis centromeric repeats are transcribed and, for at least one subfamily, transcripts from one strand accumulate predominantly and are regulated posttranscriptionally by RNAi, similar to the situation in S. pombe. This is consistent with the tandem orientation of these large blocks of repeats: putative promoter sequences found in each repeat would be expected to align in the same orientation as each other (Figure 6). Recently, each strand of the pericentromeric repeats in Arabidopsis was found to differ in DNA methylation [62]. While certainly an intriguing result, these differentially methylated sequences were located outside the satellite repeats and mostly composed of retrotransposons integrated in both orientations. Therefore, the significance of this methylation for strand-specific transcription of the satellite repeats is not yet clear.

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Figure 6. A Model for the Regulation and Evolution of Centromeric Satellite Repeat Transcripts

(A) New tandemly arranged repeats are transcribed on one strand and processed by RNAi.

(B) Older repeats accumulate retrotransposon insertions that interrupt and silence the transcripts via MET1, HDA6, and DDM1.

(C) In met1, hda6, or ddm1 mutants, the silencing complex is lost and promoters from retrotransposon LTRs drive transcription from both strands. cen180 repeats are blue chevrons, and retrotransposons are green boxes (not to scale).

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Even though they are transcribed from only one strand, tandem repeats can theoretically generate siRNA by reiterative rounds of RdRP replication and Dicer degradation [63]. The requirement for both DCL1/AGO1 and RDR2/DCL3 indicates both 21- and 24-nucleotide siRNAs may be involved. Although we could barely detect 21-nucleotide siRNA on blots, rare centromeric siRNA of this size has been detected by sequencing [64]. For other repeat subfamilies, both strands were silent, but transcription could be detected in mutants defective in the DNA methyltransferase MET1 and the type I histone deacetylase HDA6. These transcripts arose by read-out from LTR retrotransposons inserted among the repeats (see Figure 3E).

In fission yeast, RNAi is required to maintain normal levels of H3K9me2 at the outer pericentromeric repeats [23] and mutants have lagging chromosomes at anaphase [24,29]. A similar repeat is responsible for aspects of silencing at the mating type locus, but additional cis-acting elements are required for silencing in the absence of RNAi, requiring histone deacetylation instead [65]. In Arabidopsis, dcl3 and rdr2 mutants had little or no 24-nucleotide centromeric satellite siRNAs, yet had no detectable defects in H3K9me2 accumulation or in centromere function. This is also evidence of redundant mechanisms for maintaining H3K9me2, one RNAi based and the other depending on CG methylation and histone deacetylation of retrotransposons. However, kyp mutants lost most H3K9me2 without anaphase defects, although other H3K9 methyltransferases may be redundant with KYP [19], just as other Dicers are redundant with DCL3 [48]. Nevertheless, we cannot exclude the possibility that heterochromatic association of cohesin via H3K9me2 is not required for mitotic chromosome disjunction in plants [34], which lack centrosomes and may differ from animals in this respect.

If RNAi at the centromere is dispensable but transcripts are still found, this raises the possibility that the transcripts themselves may have some function. Centromeric transcripts from CRM retrotransposons and CentC satellite repeats are associated with immunoprecipitated kinetochores in maize, although neither full-length transcripts nor siRNAs were detected on Northern blots, so that the origin and fate of these transcripts are unknown [66]. Similarly, major and minor satellite repeats are transcribed from mouse centromeres, but in this case RNA interference is thought to play a role [30,67]. Our results indicate that centromeric transcripts can be long, persist in the nucleus, and show some correlation with mitotic activity and may associate with chromosomes. Whether the transcripts serve to recruit factors in addition to the RNAi apparatus remains to be seen.

The role of DDM1 in siRNA production is unclear, but it may stabilize siRNA in a complex [39], or even promote RNA-dependent RNA polymerase, in the same way as other swi/snf helicases promote DNA-dependent RNA polymerases [68]. This could account for the difference between 106B and cen180 siRNA accumulation in ddm1. If siRNA processing was reduced but 106B was transcriptionally up-regulated, this could lead to an overall increase in siRNA in ddm1. In contrast, if cen180 repeats were not transcriptionally up-regulated, siRNA would be decreased in ddm1. Combined losses of siRNA and H3K9me2 are most severe in ddm1, and chromocenters are severely disrupted [69], indicating they may play a role in heterochromatin association even though lagging chromosomes were not observed.

Maintenance and Reestablishment of Silencing

Silencing of centromeric repeats was restored in met1/+ following backcrosses to WT, as was methylation [70]. However, transgene silencing and methylation were not restored in similar backcrosses, although allelic differences could be responsible [52]. Transposon methylation could also be restored in met1/+ but only for those transposons that retained H3K9me2 and siRNA in met1 [39]. siRNA from cen180 repeats was also retained in met1 and hda6, but it was reduced in ddm1 and F-strand silencing was not restored in ddm1/+. These results are consistent with a silencing complex, composed of DDM1, MET1, and HDA6, while de novo histone and DNA modification is guided by siRNA [39]. HDA6 may act downstream of MET1 as CG methylation is unaffected in hda6 mutants [54] (Figure S4), consistent with the interaction of histone deacetylase with CG methyl-binding proteins in mammalian cells [71]. cen180 repeats transcribed in cmt3 and kyp are inherited epigenetically in an active state in backcrosses, indicating silencing cannot be reestablished in trans even in the presence of cen180 siRNA, which is retained in the mutants. H3K9me2 is lost in kyp and may play a role in reestablishment. In cmt3, H3K9me2 is retained but CNG methylation is lost so both marks may be required. However, silencing of some, but not all, of these transcripts is restored in hda6/+, indicating that epigenetic inheritance is not simply a matter of chromosomal modification.

Evolution of Centromeres

Centromeres are dynamic components of genome evolution. In sequenced arrays of human 171-bp satellites, the central repeats appear to be the youngest, with progressively older repeats on the outside. Formation and homogenization of new repeats are hypothesized to occur via unequal recombination, so that one repetitive sequence comes to dominate the centromere [50]. CENP-A has been proposed to co-evolve with such repeats, although binding to new, homogenous repeats has not been directly tested [72]. Transcribed repeats could also be replicated via RT and retrotransposition. New copies, being identical in sequence, would still be recognized by siRNA. As repeats age, retrotransposon insertions would recruit MET1 and DDM1, silencing the repeats transcriptionally and allowing them to diverge (Figure 6). This model predicts that younger RNAi-regulated repeats should be toward the center of the centromere and older MET1-regulated repeats should be on the flanks. In support, BLAST analysis showed that MET1-regulated repeats do indeed predominate in the sequenced portion of the genome, i.e., the exterior regions of the centromere (Figure S5). The regulation of subfamilies of centromeric repeats has diverged in the Columbia and Landsberg ecotypes, which are closely related [73]. Although the centromeric heterochromatin has not been completely sequenced in either strain, the pattern of retrotransposon insertions elsewhere in the genome is quite different [74], and this could account for the differential silencing of individual subfamilies of centromeric repeats in each strain (Figure 6). The transcription of centromeric repeats provides an attractive model for speciation. In wide crosses, paternal chromosomes might be destabilized if the pericentromeric repeats no longer match the sequence of maternal siRNA. This mechanism could contribute to hybrid incompatibility in polyploids as well as the loss of paternal chromosomes in wide crosses [75,76].

Plant material.

cmt3-m5662, met1–1 (E. Richards), ddm1–2 (E. Richards), kyp-2 (S. Jacobsen), hda6/sil1 (I. Furner), dcl1–9 (S. Jacobsen), ago1–9 mutations were introgressed into the Landsberg erecta ecotype. dcl3–1 (J. Carrington) and rdr2–1 (J. Carrington) are in the Columbia ecotype and were obtained from the Syngenta Arabidopsis Insertion Library (SAIL) collection of T-DNA insertions (Syngenta Biotechnology Inc. [SBI], Research Triangle Park, North Carolina, United States). Plants were grown 28 days (16 h light, 8 h dark) for RNA and chromatin preparation. Sources for seed stocks are indicated in parentheses.

Transcript analysis.

RNA was prepared, reverse transcribed into DNA, and amplified as described [39]. RT-PCRs were performed with 100 ng of RNA per reaction using the OneStep kit from Qiagen (Valencia, California, United States) according to the manufacturer's protocol. Negative controls to detect contaminating DNA were performed on the RNA preparations using cen180 F + R or cen180 Fc + Rc primers but no RT. The following reaction conditions were used in each cycle: 94 °C for 20 s, 60 °C for 30 s, and 72 °C for 1 min. Southern hybridization was performed using standard methods [77]. Primer sequences were selected from conserved and variable regions of cen180 repeats, also known as pAL1 and AtCon (see Supplementary Information). The cen180 F (forward) primer is conserved in Arabidopsis and related species, while the R (reverse) primer is specific to subsets of repeats [2,20]. The cen180 Fc and Rc primers match conserved regions within the repeat. For CLUSTALW analysis, sequences were determined for 25 to 30 cDNA clones from each mutant. A cen180 F primer with a T3 promoter and a cen180 R primer with a T7 promoter were used to amplify cen180 repeats, and these products were transcribed in vitro to prepare strand-specific probes for in situ hybridization, performed according to the protocol of Jeffrey Long (Salk Institute for Biological Studies, San Diego, California, United States) (for protocol, see http://www.its.caltech.edu/~plantlab/protocols/insitu.pdf).

Histone H3 methylation.

Chromatin immunoprecipitation (ChIP) was performed as previously described [39,40] using conserved F primers and R primers specific to each class of repeat. Semiquantification of the cen180 repeat ChIP data was performed by comparing PCR results from three different cycle numbers (13, 15, and 17 cycles), which were then analyzed by Southern blotting. In this way, the PCR of such highly repetitive sequences was maintained in the linear phase to avoid PCR saturation. DNA samples from each genotype were then normalized to each other by amplifying dilutions of total input DNA. Semiquantitative data were then obtained by comparing amplification with each set of primer pairs within the same ChIP extraction, which served as internal controls. In this way, control primers such as actin, whose association with lysine-9 is unclear, could be avoided. In all cases, mock precipitation with no antibody yielded little or no product. In Figure S3, PCR conditions were 17 cycles for F and R primers and 28 cycles for dcl- and met-specific primers. PCR products were blotted and probed with radiolabeled cen180 repeats.

siRNA hybridization.

Total RNA was purified, large RNAs were removed by precipitation in 5% PEG-8000/0.5 M NaCl, and 50 μg of the resulting enriched small RNA was loaded per lane in a 10% polyacrylamide/urea gel and separated by electrophoresis [39]. The gel was electroblotted and probed with in vitro transcribed, radiolabeled RNA. Imaging was performed on a PhosphorImager (Fuji, Tokyo, Japan).

Accession Numbers

The dBEST accession numbers in GenBank (http://www.ncbi.nlm.nih.gov/Genbank) for the centromeric satellite cDNA sequences are DV671393 to DV671628.