Dataset 2.tar

Zipped; raw ABI sequence data files

Intronic Parent-of-Origin Dependent Differential Methylation at the <em>Actn1</em> Gene Is Conserved in Rodents but Is Not Associated with Imprinted Expression

<div><p>Parent-of-origin differential DNA methylation has been associated with regulation of the preferential expression of paternal or maternal alleles of imprinted genes. Based on this association, recent studies have searched for parent-of-origin dependent differentially methylated regions in order to identify new imprinted genes in their vicinity. In a previous genome-wide analysis of mouse brain DNA methylation, we found a novel differentially methylated region in a CpG island located in the last intron of the <em>alpha 1 Actinin (Actn1)</em> gene. In this region, preferential methylation of the maternal allele was observed; however, there were no reports of imprinted expression of <em>Actn1</em>. Therefore, we have tested if differential methylation of this region is common to other tissues and species and affects the expression of <em>Actn1</em>. We have found that <em>Actn1</em> differential methylation occurs in diverse mouse tissues. Moreover, it is also present in other murine rodents (rat), but not in the orthologous human region. In contrast, we have found no indication of an imprinted effect on gene expression of <em>Actn1</em> in mice: expression is always biallelic regardless of sex, tissue type, developmental stage or isoform. Therefore, we have identified a novel parent-of-origin dependent differentially methylated region that has no apparent association with imprinted expression of the closest genes. Our findings sound a cautionary note to genome-wide searches on the use of differentially methylated regions for the identification of imprinted genes and suggest that parent-of-origin dependent differential methylation might be conserved for functions other that the control of imprinted expression.</p> </div

Bisulfite sequencing analysis of the <i>Actn1</i> DMR in mouse, rat and human tissues.

<p>Panel A shows bisulfite sequencing results from clones isolated from rat liver, mouse liver, and human hepatocytes. Each horizontal line represents a unique clone. Red and blue lines represent maternal and paternal parent-of-origin, respectively, based on five strain-specific variants. Open circles are unmethylated CpGs, while closed circles are methylated CpGs. Green and yellow circles shown in human hepatocyte clones represent variant rs11557769 and distinguish parental alleles, although parent-of-origin is unknown. Orthologous CpGs are connected by dotted lines (in relation to mouse). Panel B shows bisulfite sequencing results from clones isolated from rat right brain hemisphere (top) and mouse right brain hemispheres (bottom).</p

Maternal methylation of a novel DMR at the <i>Actn1</i> gene in diverse mouse tissues.

<p>A) A detailed map of the novel maternal <i>Actn</i>1 DMR is shown in the lower part. The diagram directly above shows the design for the MS-RFLP and bisulfite sequencing validation assays. Also included in this diagram are the locations of the methylation-sensitive enzyme restriction sites tested with MS-RFLP (<i>BsaAI, EagI</i> and <i>HpaII)</i>, the strain-specific cut sites (<i>AhdI</i> (present in 129S1 but not in PWK, due to SNP rs32640406) and <i>StyI</i> (present in PWK but not in 129S1, due to SNP rs32640412)), and the strain-specific resulting restriction fragments (see Methods). B) MS-RFLP results of four mouse liver samples. The matrix above the gel shows the different conditions for each individual lane. The plus sign (+) indicates addition, while the minus sign (−) indicated no addition of each corresponding endonuclease. C) Percent maternal methylation of an individual CpG (targeted by the <i>BsaAI</i> endonuclease) within different tissues. Circles represent individual (PWK×129S1)F₁ mice, while triangles represent individual (129S1×PWK)F₁ mice. Horizontal bars represent percent maternal methylation averages.</p

LOD curves and allelic distribution of QTLs for plaque lesion at arch.

<p>(A-C) LOD curves of QTL for plaque lesion size at arch in sex-combined scan with sex as additive covariate of a cross between B6-apoE and DBA-apoE (red lines), a cross between B6-apoE and 129-apoE mice (black lines) and a cross between DBA-apoE and 129-apoE mice (blue lines) on chromosome Chr2 (A), Chr10 (B) and Chr1 (C). The horizontal dashed and dotted lines represent thresholds for suggestive QTL (p = 0.63) and significant QTL (p = 0.05) determined in single locus scan of the cross between B6-apoE x DBA-apoE mice. The significance thresholds for LOD scores were determined by 1000 permutations using R/qtl software. (D) Allelic distribution of the QTLs for plaque lesion sizes at arch at the nearest marker to the peaks in Chr1 and Chr14 in both sexes in the cross between B6-apoE and 129-apoE mice. Data represent mean ± SE. Comparison of lesion sizes were done by one-way analysis of variance (ANOVA), followed by Tukey-Kramer’s HSD-test. Only mice homozygous for 129 allele at both Chr1 and Chr14 have significantly larger plaques compared to those with other combinations of the loci by one-way ANOVA (p = 0.001), followed by Tukey-Kramer’s HSD-test: *** p<0.001–129/129 genotype vs B6/B6 genotype, ^## p<0.01–129/129 vs 129/B6 genotype genotype. **** p<0.0001–129/129 genotype vs B6/B6 genotype, ^#### p<0.0001–129/129 genotype vs B6/129 genotype; (E) Allelic distribution of the QTLs for plaque lesion sizes at arch at the nearest marker to the peaks in Chr1 and Chr8 in both sexes in the cross between B6-apoE and 129-apoE mice. If Chr8:13 cM is homozygous for the 129 allele, genotype effects were not present, but that if at least one allele of Chr8;13cM derived from B6, a strong effect of genotypes is revealed. Data represent mean ± SE. Comparison of lesion sizes were done by one-way analysis of variance (ANOVA), followed by Tukey-Kramer’s HSD-test. * p<0.05–129/129 genotype vs B6/B6 genotype; ^# p<0.05–129/129 genotype vs B6/129 genotype, **** p<0.0001–129/129 genotype vs B6/B6 genotype; ^#### p<0.0001–129/129 genotype vs B6/129 genotype; § p<0.05- B6/B6 genotype vs B6/129 genotype. (F) Allelic distribution of the QTLs for plaque lesion sizes at arch at the nearest marker to the peaks Chr10 and Chr12 in both sexes in the cross between B6-apoE and 129-apoE mice. Data represent mean ± SE. 129 allele of the Chr12 has significant protective effect only when Chr10 is homozygous for B6 allele Comparison of lesion sizes were done by one-way analysis of variance (ANOVA), followed by Tukey-Kramer’s HSD-test. * p<0.05- B6/B6 genotype vs 129/129 genotype; ^# p<0.05- B6/B6 genotype vs B6/129 genotype; **p<0.01- B6/B6 genotype vs 129/129 genotype; ^## p<0.01- B6/B6 genotype vs B6/129 genotype.</p

QTLs for atherosclerosis at the aortic root in F2 mice from intercross between DBA2-apoE and B6-apoE.

<p>QTLs for atherosclerosis at the aortic root in F2 mice from intercross between DBA2-apoE and B6-apoE.</p

QTLs for atherosclerotic lesions at root in three F2 intercrosses of Apoe-null mice on B6, DBA and 129 backgrounds.

<p>QTLs for atherosclerotic lesions at root in three F2 intercrosses of Apoe-null mice on B6, DBA and 129 backgrounds.</p

Genetic architecture of atherosclerosis dissected by QTL analyses in three F2 intercrosses of apolipoprotein E-null mice on C57BL6/J, DBA/2J and 129S6/SvEvTac backgrounds

<div><p>Quantitative trait locus (QTL) analyses of intercross populations between widely used mouse inbred strains provide a powerful approach for uncovering genetic factors that influence susceptibility to atherosclerosis. Epistatic interactions are common in complex phenotypes and depend on genetic backgrounds. To dissect genetic architecture of atherosclerosis, we analyzed F2 progeny from a cross between apolipoprotein E-null mice on DBA/2J (DBA-apoE) and C57BL/6J (B6-apoE) genetic backgrounds and compared the results with those from two previous F2 crosses of apolipoprotein E-null mice on 129S6/SvEvTac (129-apoE) and DBA-apoE backgrounds, and B6-apoE and 129-apoE backgrounds. In these round-robin crosses, in which each parental strain was crossed with two others, large-effect QTLs are expected to be detectable at least in two crosses. On the other hand, observation of QTLs in one cross only may indicate epistasis and/or absence of statistical power. For atherosclerosis at the aortic arch, <i>Aath4</i> on chromosome (Chr)2:66 cM follows the first pattern, with significant QTL peaks in (DBAx129)F2 and (B6xDBA)F2 mice but not in (B6x129)F2 mice. We conclude that genetic variants unique to DBA/2J at <i>Aath4</i> confer susceptibility to atherosclerosis at the aortic arch. A similar pattern was observed for <i>Aath5</i> on chr10:35 cM, verifying that the variants unique to DBA/2J at this locus protect against arch plaque development. However, multiple loci, including <i>Aath1</i> (Chr1:49 cM), and <i>Aath2</i> (Chr1:70 cM) follow the second type of pattern, showing significant peaks in only one of the three crosses (B6-apoE x 129-apoE). As for atherosclerosis at aortic root, the majority of QTLs, including <i>Ath29</i> (Chr9:33 cM), <i>Ath44</i> (Chr1:68 cM) and <i>Ath45</i> (Chr2:83 cM), was also inconsistent, being significant in only one of the three crosses. Only the QTL on Chr7:37 cM was consistently suggestive in two of the three crosses. Thus QTL analysis of round-robin crosses revealed the genetic architecture of atherosclerosis.</p></div

LOD curves and allelic effect of QTL for root plaque size.

<p>(A) LOD curve for root lesion size with sex as additive covariate in F2 mice from a cross B6-apoE X DBA-apoE. The horizontal dashed and dotted lines represent thresholds for suggestive (p = 0.63) and significant (p = 0.05) QTLs. The significance thresholds for LOD scores were determined by 1000 permutations using R/qtl software. (B) A LOD curve for root lesion size in female-only (green) and male-only (purple) scans in F2 mice from cross B6-apoE X DBA-apoE. The horizontal dashed and dotted lines represent thresholds for suggestive (p = 0.63) and significant (p = 0.05) QTLs. The significance thresholds for LOD scores were determined by 1000 permutations using R/qtl software. (C) Allelic distribution of the main QTLs for plaque lesion size at root at the nearest marker to the peak at Chr 7 in female F2 mice from a cross B6-apoE X DBA-apoE. Data represent mean ±SE. Comparison of lesion sizes were done by one-way analysis of variance (ANOVA) (p<0.01) followed by Tukey-Kramer’s HSD-test, **p<0.01 (D) Allelic distribution of the main effect QTLs for plaque size (μm²) subjected to square root transformation (sqrt) at the root in Chr 4 and Chr 6 in both sexes in F2 mice from cross B6-apoE X DBA-apoE. Data represent mean ±SE. Comparison of lesion sizes were done by one-way analysis of variance (ANOVA), followed by Tukey-Kramer’s HSD-test. *** p = 0.001- B6/B6 genotype vs DBA/DBA genotype; # p<0.05- B6/B6 genotype vs B6/DBA genotype, ## p<0.01- B6/B6 genotype vs B6/DBA genotype.</p

LOD curves and allelic distribution of QTLs for arch plaque lesion.

<p>(A) LOD curve for arch lesion size with sex as additive covariate in F2 mice from a cross B6-apoE x DBA-apoE. The horizontal dashed line represents a threshold for a suggestive (p = 0.63) QTL and a dotted line represents a threshold for a significant QTL (p = 0.05). The significance thresholds for LOD scores were determined by 1000 permutations using R/qtl software. (B) Allelic distribution of the QTL on Chr2 for plaque lesion size at the arch at the nearest marker to the peak in F2 mice from the cross B6-apoE x DBA-apoE. Data represent mean ± SE. Lesion size comparison was performed by one-way analysis of variance (ANOVA) (p<0.0001) followed by Tukey-Kramer’s HSD-test. **** p<0.0001 (C) Allelic distribution of QTL on Chr10 for plaque lesion size at the arch at the nearest marker to the peak in F2 mice from the cross B6-apoE x DBA-apoE. Data represent mean ± SE. Lesion size comparison was performed by one-way analysis of variance (ANOVA) (p<0.001) followed by Tukey-Kramer’s HSD-test. p = 0.06 between B6/B6 and DBA/DBA genotypes, **** p<0.0001 between B6/B6 and B6/DBA genotypes (D) Allelic distribution of the main effect QTL for plaque lesion size at the arch in Chr 4 and Chr 6 in both sexes in F2 mice from the cross B6-apoE x DBA-apoE. Data represent mean ±SE. Only mice homozygous for B6 allele at both Chr4 and Chr6 have increased plaque size by one-way ANOVA (p = 0.001), followed by Tukey-Kramer’s HSD-test: * p<0.05- B6/B6 genotype vs DBA/DBA genotype, ### p<0.001- B6/B6 genotype vs B6/DBA genotype.</p