The profile of repeat-associated histone lysine methylation states in the mouse epigenome

Abstract

Histone lysine methylation has been shown to index silenced chromatin regions at, for example, pericentric heterochromatin or of the inactive X chromosome. Here, we examined the distribution of repressive histone lysine methylation states over the entire family of DNA repeats in the mouse genome. Using chromatin immunoprecipitation in a cluster analysis representing repetitive elements, our data demonstrate the selective enrichment of distinct H3-K9, H3-K27 and H4-K20 methylation marks across tandem repeats (e.g. major and minor satellites), DNA transposons, retrotransposons, long interspersed nucleotide elements and short interspersed nucleotide elements. Tandem repeats, but not the other repetitive elements, give rise to double-stranded (ds) RNAs that are further elevated in embryonic stem (ES) cells lacking the H3-K9-specific Suv39h histone methyltransferases. Importantly, although H3-K9 tri- and H4-K20 trimethylation appear stable at the satellite repeats, many of the other repeat-associated repressive marks vary in chromatin of differentiated ES cells or of embryonic trophoblasts and fibroblasts. Our data define a profile of repressive histone lysine methylation states for the repetitive complement of four distinct mouse epigenomes and suggest tandem repeats and dsRNA as primary triggers for more stable chromatin imprints.

Figure 1

Overview of repetitive elements in the mouse genome. (A) Schematic diagram of a mitotic mouse chromosome illustrating the distribution of major (pericentric) and minor (centromeric) satellite repeats and of the various interspersed repetitive elements. (B) Summary description of repeat classes, highlighting repeat organization, length, copy number and overall abundance in the mouse genome. Specific primers (black arrows) were designed to generate PCR fragments from within the repetitive elements, thereby allowing a cluster analysis that detects the sum of chromatin modifications for a given repeat class. The sequences of these primers are indicated in Supplementary data. Although the entire repeat units are shown, most repetitive elements in the mouse genome are not full length (Waterston et al, 2002).

Figure 2

Cluster analysis for repeat-associated histone lysine methylation states. ChIP of wt and Suv39h dn ES cell chromatin with antibodies detecting H3-K9, H3-K27 and H4-K20 mono-, di- and trimethylation. As controls, ChIP was also performed with H3-K4 trimethyl (an active mark) antibodies and for the tubulin gene. Purified DNA from enriched chromatin fragments was amplified by real-time PCR with the repeat-specific primer sets (see Figure 1B), and values are indicated as percentage precipitation relative to the input. The dashed line represents the threshold signal of 0.5%, above which significant enrichment for distinct histone lysine methylation states was scored.

Figure 3

Repeat-associated H3-K9, H3-K27 and H4-K20 methylation states in chromatin of Dnmt-deficient ES cells. ChIP profile of wt (J1), Dnmt1-/- and Dnmt3ab-/- ES cell chromatin as described in Figure 2. Also indicated is the degree of DNA methylation (5mC) that is present at the distinct repeat classes in wt and mutant Dnmt chromatin. For this analysis, genomic DNA was digested with the methylation-specific restriction enzyme McrBC, and relative DNA methylation was measured by the inverse ability of the remaining DNA fragments to generate PCR products (Rabinowicz et al, 2003) with the repeat-specific primer sets (see Figure 1B).

Figure 4

Elevated transcript levels for repetitive DNA elements in Suv39h dn ES cells. RT–PCR analysis for repeat-derived transcript levels in total RNA from wt and Suv39h dn ES cells (A) or from wt (J1) and Dnmt1-/- or Dnmt3ab-/- ES cells (B). cDNA was generated by random priming and then amplified with the repeat-specific primer sets (see Figure 1B). Linear range amplification of cDNA was calibrated by real-time PCR (between 22 and 27 cycles; see Supplementary Figure S2), and PCR products are visualized by inverse images of EtBr-stained 2% agarose gels. As controls, reactions were performed without reverse transcriptase (−RT). The relative average increase in transcription of two distinct elements within a repeat class is indicated in the middle of the two columns shown in panel A.

Figure 5

Tandem satellite repeats generate dsRNA. Total RNA from wt and Suv39h dn ES cells was treated for the indicated time points with RNAseONE™ to digest ssRNAs (A) or with RNAseV1 to cleave dsRNAs (B). The remaining pool of RNA molecules was then converted to cDNA and amplified by real-time PCR with the repeat-specific primer sets (see Figure 1B). The histograms indicate the percentages, relative to undigested RNA, of repeat-derived RT–PCR products that can be generated after RNAseONE™ (% dsRNA) or RNAseV1 (% ssRNA) treatment.

Figure 6

Repeat-associated transcript levels in four distinct mouse epigenomes. (A) Images of cultured mouse ES cells, RA-ES cells, day 13.5 MEFs and TS cells. (B) RT–PCR analyses for repeat-derived transcript levels in the four distinct cell populations. Total RNA was converted into cDNA, which was then amplified by real-time PCR with the repeat-specific primer sets (see Figure 1B). The histogram indicates the relative difference (after normalization for tubulin expression) in the abundance of repeat-derived transcripts with respect to levels observed in wt ES cells (see Figure 4 and Supplementary Figure S2), which were set at 1.

Figure 7

Stability of repressive histone lysine methylation marks at tandem but not at interspersed repeats. ChIP profile for repeat-associated histone lysine methylation states in wt chromatin of ES cells, RA-ES cells, MEFs and TS cells. The dashed line represents the threshold signal of 0.5%, above which significant enrichment for distinct histone lysine methylation states was scored. To adjust for differences in the ChIP efficiency with chromatin of distinct cell populations, samples were spiked with Drosophila chromatin as an internal standard. For clarity, enriched marks are colour-coded.

Figure 8

Summary of repeat-associated histone lysine methylation marks in mouse chromatin. Repressive histone lysine methylation marks with prominent enrichment over the 0.5% threshold in wt ES cell chromatin (see Figures 2 and 7) were added to give the sum of these ‘signature' modifications for a distinct repeat class. For example, the major satellite repeats accumulate H3-K9 tri-, H3-K27 mono- and H4-K20 trimethylation at ∼6% of precipitated material, whereas SINEs and LINEs do not display a specific signal. For the satellite repeats and for some DNA transposons, H3-K9 tri-, H3-K27 mono- and H4-K20 trimethylation are significantly reduced in Suv39h dn ES cell chromatin (see Figure 2); these Suv39h-dependent marks are highlighted by yellow-coded Me in the respective methyl hexagons. In chromatin of differentiated cell types, most of the histone lysine methylation profiles at interspersed repeats differ from those observed in ES cell chromatin (see Figure 7), while they appear more stable at tandem satellite repeats. An example for this variability and the accumulation of additional marks is schematically indicated for RA-induced alterations in chromatin of wt ES cells (bottom panel). While these combinations of marks are, on average, enriched as detected by our cluster analysis, these groupings do not necessarily reflect their combined presence over individual repeats in distinct chromosomal locations.