Meth-QTL amplicon linkage and methylation.

a<p>Gene ID indicates the closest gene to the amplicon.</p>b<p>(CGI) within or overlaps with a CpG island.</p>c<p>(WGBS) whole genome bisulfite sequencing, methylation percentage in BN and SHR strains, or difference between the strains (BN-SHR).</p>d<p>(NSL) No significant linkage is defined as P>0.05 after a minimum of 1000 permutations of the linkage data.</p><p>Assay IDs are provided where there are two identical gene IDs.</p><p>Meth-QTL amplicon linkage and methylation.</p

Differential CpG methylation between the BN and SHR strains.

<p>A) Dendrogram showing the clustering the CpG methylation profiles obtained in four BN and four SHR rats. B) Projection of methylation profiles onto the first principal component. Numbers after the strain label denote biological replicates.</p

Correlation of CpG cytosine meth-QTL linkage and difference in CpG methylation between parental strains.

<p>Methylation data were obtained from PCR amplification of bisulfite-treated DNA followed by Sanger or Illumina sequencing in parental and RI strains. CpGs were classified according to linkage type (<i>cis</i>, <i>trans</i>, no significant linkage) and LOD scores were plotted against the absolute value of the difference in methylation between parental strains. No linkage refers to CpGs for which permutation-based p≥0.05. Correlation was assessed by Pearson <i>r</i> value. The cluster of CpGs with >80% difference in methylation are all from a single amplicon selected because of high differential methylation in the parental strains.</p

Multi-point linkage plots of meth-QTLs.

<p>(A) Linkage plots of 16 <i>cis</i> meth-QTLs with LOD scores >10 for linkage to mean CpG methylation. LOD scores for linkage of average amplicon methylation levels are shown in red, LOD scores for linkage of individual CpG di-nucleotides are shown in grey. The peak of linkage is indicated by a dashed, blue vertical line. (B) Linkage plot for the <i>Odfp2</i> and <i>Asap2</i> meth-QTLs which showed significant <i>trans</i>-linkages to chromosome 2 and 1 respectively whilst the amplicons reside on chromosomes 3 and 6. (C) Linkage plots of <i>Epha2</i> and <i>Ppp1r13b</i> for which the average methylation did not show significant linkage but individual CpG dinucleotides mapped in <i>cis</i>.</p

Summary of differential methylation by source of variation.

<p>*Strand specific methylation.</p><p>Summary of differential methylation by source of variation.</p

Comparison of differential methylation in parental strains and allele-specific methylation in F1 crosses.

<p>(A) Venn diagram showing the number of CpGs tested for differential methylation in the parental strains ONLY or for allele-specific methylation ONLY (grey areas) and the fraction of CpGs tested for BOTH (intersection, blue). The light blue area shows the number of CpGs significantly differentially methylated between the parentals AND/OR showing allele-specific methylation. Areas are not proportional. CpGs on the X chromosome and the mitochondrial chromosome have been excluded. (B) Overlap of differential methylation between the parental strains and allele specific methylation in the F1s. In total, 52,410 out of the 1,705,718 CpG dinucleotides analysed in both the parentals and the F1s (intersection, blue in (A)) showed significant differential methylation. The Venn diagram shows the fraction of CpGs that were differentially methylated between the parents ONLY (purple), between the alleles in the F1s ONLY (orange) or in BOTH (green). The theoretical distribution of frequencies assuming random overlap between the parental and the F1 data sets are shown in italics (<i>X²</i> = 135276.2, <i>P</i><4.9×10⁻³²⁴). The overlap of the two sets is >15 fold higher than expected by chance. (C) Frequency of CpGs with a SNP within 50 base pairs for CpGs showing differential methylation between the parentals only (purple), the F1 alleles only (orange) and both (green) compared to all CpGs analysed in the parentals and the F1s (blue) and the whole genome (red).</p

Hierarchical clustering and principal component analysis of CpG methylation profiles obtained in the F1 reciprocal crosses.

<p>Dendrograms showing the results of clustering the CpG methylation profiles obtained in each of the F1 animals before (A) and after (C) phasing the read data by parental genotype. The prefix of the phased F1 profiles in (C) denotes the reciprocal cross (bnxshr, shrxbn) and the suffix the genotype of the phased read set (bn = Brown Norway, shr = Spontaneously Hypertensive Rat). Numbers after the reciprocal cross prefix denote biological replicates. Profiles were clustered by the Ward's method using the pairwise euclidean distance between the profiles as the distance metric. Panels B and D show the projection of methylation profiles onto the 1^st principal component (PC). Replicates for each cross-genotype combination are separated along the y-axis. The prefix of the label denotes the reciprocal cross (bnxshr, shrxbn), the suffix denotes the parental genotype (bn, shr). While the 1^st PC does not separate crosses in the unphased data it provides complete separation by parental genotype in the phased data. Only CpGs with at least 5× coverage in each replicate/phased read set were included in the analysis and CpG positions affected by SNPs/indels were removed prior to clustering and principal component analysis.</p

Distribution of methylation differences by source of variation.

<p>The boxplots show the the distribution of methylation differences for significantly differentially methylated CpGs (white) and non-significantly differentially methylated CpGs (grey) between the parental strains, the F1 reciprocal crosses, allele specific methylation and parent-of-origin dependent methylation.</p

Sequence variation around differentially methylated CpGs.

<p>(A) Plot showing the frequency of CpGs with a SNP within 1 kb for all CpG dinucleotides in the genome (red), all CpGs tested for differential methylation (blue), CpGs that showed a significant difference in methylation between BN and SHR rats (green), and differentially methylated CpGs with a CpG-disrupting SNP in the vicinity (purple). In the latter case the distance relates to the nearest CpG-disrupting SNP. (B) same plot as (A) zoomed in to the 10 bp interval around the CpG. (C) Sequence logos showing SNP allele frequencies around differentially methylated CpGs for alleles associated with increased (top) and decreased (bottom) methylation.</p

Regulation of class I expression by classical and inverse cim.

<p>(A) CD68+ cells were stained on the cell surface with OX18 (anti-class Ia and Ib). Histograms show representative samples from DA, DA.1H and DA.1H derived strains with different alleles of <i>RT1-A</i> and <i>Tap2</i> as stated on top. Data from all individuals are shown in scatterplot (far right); * significant compared to DA.1H (1H); ** significant compared to DA. (B) CD68+ cells stained with a class Ia specific antibody (F16-4-4). (C) T cells from animals shown in (A) stained intracellularly with OX18 (scatterplot) and F16-4-4 (histogram). (D–E) Subsets of leukocytes from DA and DA.1IR85 spleen stained extracellularly (D) and intracellularly (E) with OX18. Data are representative of 6 individuals per group. (F) Surface expression of MHC class I (OX18) on CD68+ cells from DA and DA.1U congenic strains. (G) CD68+ cells (same as in F) stained with F16-4-4. Vertical lines in scatterplots show mean values. Representative results of at least two independent experiments are shown.</p