Project description:Purpose: The goal of this study was to compare the genome-wide promoter methylation alterations in macrophages and endothelial cells during hindlimb ischemia among normal, hyperlipidemic and type-2 diabetic mice. Methods: Unilateral hindlimb ischemia was induced by ligating femoral artery proximal to the bifurcation of superficial and deep femoral artery in mice deficient of LDL receptor and expressing only apolipoprotein B100 (LDLR-/-ApoB100/100, C57BL/6J background) (The Jackson Laboratory, Bar Harbor,USA) and mice with β-cell specific over-expression of insulin-like growth factor-2 in atherosclerotic background (IGF-II/LDLR-/-ApoB100/100, C57BL/6J background) with type 2 diabetic features on high-fat diet (TD 88173, Harlan Teklad: 42% of calories from fat and 0.15% from cholesterol, no sodium cholate) 8 weeks prior to surgery and continued throughout the study 1. C57BL/6J (WT) mice fed with regular chow-diet (R36, Lactamin) served as controls. All animals were aged between 20 to 24 weeks at the time of hindlimb operations. For sorting macrophages from ischemic muscles, ischemic gastrocnemius muscles were minced and enzymatically dissociated using a cocktail containing 450 U/mL Collagenase I, 125 U/mL Collagenase XI, 60 U/mL DNAseI, and 60 U/mL hyaluronidase (Sigma Aldrich) for 1 h at 37°C. The cells were then counted and divided into CD31+ve and CD31-vefractions using CD31 magnetic bead enrichment (Miltenyi Biotec). For macrophage sorting CD31-ve fraction was incubated for 15 minutes with rat anti-mouse CD16/32 mAb (Fc Block, BD-pharmingen) and stained with FITC conjugated rat anti-mouse F4/80 antibody (Serotec) for 20 minutes at 4˚C. For endothelial sorting CD31+ fraction was incubated for 15 minutes with rat anti-mouse CD16/32 mAb (Fc Block, BD-pharmingen) and stained with APC conjugated rat anti-mouse CD31 antibody (BD-pharmingen) and FITC conjugated rat anti-mouse CD45 ((BD-pharmingen) for 20 minutes at 4˚C. FACS sorting was performed on FACS AriaIII (BD Biosciences). Genomic DNA was isolated from FACS sorted macrophages and endothelial cells using AllPrep DNA/RNA/Protein Mini Kit (Qiagen Finland, Helsinki, Finland) according to manufacturer's instructions. Results: The sample similarity as assessed by Pearson’s correlation matrix and Hierarchial clustering showed high correalation among macrophages, as well as endothelial cells. There was a clear clustering of macrophages and endothelial cells as evidence by their CpG methylation clustering, furthermore macrophages from HL and T2DM mice showed clear clustering compared to control macrophages. Differential methylation analysis of RRBS methylation data from macrophages and endothelial cells was performed using Methylkit. Using a threshold of adjusted p value (Q) <0.05 and percentage methylation difference of >5%, we identified 198 and 272 genes whose promoters were hypomethylated in HL and T2DM macrophages. Similarly, there were 102 and 136 gene promoters were hypermethylated in HL and T2DM macrophages, respectively compare to control macrophages. Thus, proximal promoter methylation suggested that HL and T2DM have convergent influences on the proximal promoter methylation of numerous macrophage specific genes. In order to find out whether these genes with differential methylated promoters were differentially expressed at mRNA expression level in purified macrophages, we further compared our data with the GEO datasets as above. Of the 198 genes with promoter hypomethylation in HL macrophages 72 genes were suggested to be upregulated in M1- Mϕs; whereas, of the 102 genes with promoter hypermethylation, 51 genes were suggested to be upregulated in M2- Mϕs. Similarly, out of 272 genes with differentially methylated promoters in T2DM macrophages 88 genes were suggested to be upregulated in M1-Mϕs; whereas, out of 136 genes with promoter hypermethylation 60 genes were suggested to be upregulated in M2- Mϕs. Thus a significant promoter hypomethylation of M1-Mϕ and hypermethylation of M2-Mϕ genes suggested the predominance of proinflammatory M1-Mϕs in ischemic muscles of HL and T2DM compared to M2-Mϕs in control mice. Conclusions: We found significant promoter hypomethylation of genes typical for proinflammatory M1-Mϕs and hypermethylation of anti-inflammatory, proangiogenic M2-Mϕ associated genes in HL and T2DM ischemic muscles. Epigenetic alterations skewing Mϕ phenotype towards proinflammatory as opposed to anti-inflammatory, proangiogenic and tissue repair phenotype may contribute to impaired adaptive vascular growth in these pathological conditions. Macrophages and endothelial whole genome DNA methylation was performed in triplicates (Each sample was pooled from 3-4 mice) by RRBS Sequencing approach using Illumina HiSeq 2500. qRT–PCR validation was performed using TaqMan assays.
Project description:The reprogramming of parental methylomes is essential for embryonic development. In mammals, paternal 5-methylcytosines (5mCs) have been proposed to be actively converted to oxidized bases. These paternal oxidized bases and maternal 5mCs are believed to be passively diluted by cell divisions. By generating single-base resolution, allele-specific DNA methylomes from mouse gametes, early embryos, and primordial germ cell (PGC), as well as single-base-resolution maps of oxidized cytosine bases for early embryos, we report the existence of 5hmC and 5fC in both maternal and paternal genomes and find that 5mC or its oxidized derivatives, at the majority of demethylated CpGs, are converted to unmodified cytosines independent of passive dilution from gametes to four-cell embryos. Therefore, we conclude that paternal methylome and at least a significant proportion of maternal methylome go through active demethylation during embryonic development. Additionally, all the known imprinting control regions (ICRs) were classified into germ-line or somatic ICRs. The cross of two mouse strains was performed using DBA/2J as the paternal strain and C57BL/6J as the maternal strain. The hybrid embryos were collected at 2-cell, 4-cell, ICM, E6.5, E7.5 stages. Female and male E13.5 PGC samples (B6; 129S4-Pou5f1tm2Jae/J) were collected from timed mating of C57BL/6J female mice. MethylC-Seq: oocytes (C57BL/6J), sperm (DBA/2J), 2-cell embryos, 4-cell embryos, ICM, E6.5 embryos, E7.5 embryos, E13.5 female PGCs and E13.5 male PGCs. TAB-Seq: 2-cell embryos. fCAB-Seq: 2-cell embryos. RNA-Seq: oocytes (C57BL/6J).
Project description:At sites of inflammation, certain Foxp3+ Tregs have the ability to alter their phenotype and become pro-inflammatory helper/effector cells, without losing Foxp3 expression. We show that this functional reprogramming is controlled by the transcription factor Eos (Ikzf4), an obligate co-repressor for Foxp3. The ability to reprogram was restricted to a specific subset of Foxp3+ Tregs, arising as early as the thymus and identifiable by short half-life of Eos at rest, characteristic cell-surface markers (CD38+CD69+CD103NEG) and a distinct pattern of DNA methylation. Mice made selectively deficient in this subset of Eos-labile Tregs became markedly impaired in their ability to cross-present new antigens and prime CD8+ T cells. Downregulation of Eos and consequent Treg reprogramming was prevented by the immunoregulatory enzyme IDO, via activation of the aryl hydrocarbon receptor (AhR). Thus, the Foxp3+ lineage contains a committed subset of Tregs that are constitutively primed for conversion into biologically important helper cells. Cells from thymus or spleen were incubated for 1 hr with cycloheximide (CHX), then CD4+GFP+ Tregs were FACS-sorted into Eos-labile (CD38+CD103NEG) and Eos-stable (CD103+CD38NEG) subsets. Control CD4+GFPNEG (non Treg) cells were sorted from spleen. Genome-wide differential methylation analysis was performed using Reduced Representation Bisulfite Sequencing (RRBS). The genomic DNA from each sample was digested with the methylation-insensitive restriction enzyme MspI (restriction site, CCGG) and ligated to Illumina sequencing adaptors containing methylated cytosine residues. The ligated MspI fragments were size-selected, treated with sodium bisulfite, and amplified by PCR. The PCR products were purified and sequenced using Illumina HiSeq 2000 sequencer with a read length of 100bp.
Project description:These mouse strains differ in absolute numbers of hematopoietic stem cells but differ genetically only at the Chr 5 congenic locus. We used microarray analysis to identify candidate regulators of hematopoietic stem cells based on differential gene expression patterns. Triplicate RNA samples, except for duplicate DBA/2J samples, were isolated from 80,00-137,000 LSK cells from each strain. Each of the 11 samples were analyzed on individual microarrays on two Illumina Sentrix-Mouse 6 Whole Genome Expression Beadhips.
Project description:The host response to influenza A infections is strongly influenced by host genetic factors. Animal models of genetically diverse mouse strains are well suitable to identify host genes involved in severe pathology, viral replication and immune responses. Here, we have utilizing a dual RNAseq approach that allowed us to investigate both viral and host gene expression in the same individual from a single expression assay after H1N1 infection. We performed a comparative expression analysis to identify (i) correlations between host genes and the viral gene expression, (ii) host genes involved in viral replication, and (iii) genes showing differential expression between the two mouse strains after infection. These genes may be key players involved in regulating the differences in pathogenesis and host defense mechanisms after influenza A infections. Expression levels of influenza segments correlated well with the viral load and may thus be used as surrogates for conventional viral load measurements. Furthermore, we investigated the functional role of two genes, Reg3g and Irf7, in knock-out mice and found that deletion of the Irf7 gene renders the host highly susceptible to H1N1 infection. Female, 10-12 weeks old mice were anesthetized by intra-peritoneal injection with Ketamine/Xylazine (85% NaCl (0.9%), 10% Ketamine, 5% Xylazine) with doses adjusted to the individual body weight. Mice were then intra-nasally infected with 20µl virus solution (2x10³ FFU PR8M) or mock-treated with PBS.
Project description:DNA methylation is involved in many biological processes during plant growth and development. Here, we report a novel annual growth rhythm that is found in cotton plants grown in different time-of-year. To further study this rhythm in other plants, we use Arabidopsis thaliana for genome-wide bisulfite sequencing. Two A. thaliana DNA samples were extracted from 20 days old whole plant in Feburary and August for bisulphite treatment and further Illumina sequencing.
Project description:Obesity is a highly heritable complex disease that results from the interaction of multiple genetic and environmental factors. Formerly obese individuals are susceptible to metabolic disorders later in life, even after lifestyle changes are made to mitigate the obese state. This is reminiscent of the metabolic memory phenomenon originally observed for persistent complications in diabetic patients, despite subsequent glycemic control. Epigenetic modifications represent a potential mediator of this observed memory. We previously demonstrated that a high fat (HF) diet leads to changes in chromatin accessibility in the mouse liver. The regions of greatest chromatin changes in accessibility are largely strain dependent, indicating a genetic component in diet-induced chromatin alterations. We have now examined the persistence of diet-induced chromatin accessibility changes upon diet reversal in two strains of mice. We find that a substantial fraction of loci that undergo chromatin accessibility changes with HF diet remain in the remodeled state after diet reversal in C57BL/6J mice. In contrast, the vast majority of diet-induced chromatin accessibility changes in A/J mice are transient. Our data also indicate that the persistent chromatin accessibility changes observed in C57BL/6J are associated with specific transcription factors and histone posttranslational modifications. The persistent loci identified here are likely to be contributing to the overall phenotype and are attractive targets for therapeutic intervention. Examination of chromatin remodeling with FAIRE-seq in livers of C57BL/6J and A/J mice on three diet regimen: 1) control diet for 16 weeks, 2) high fat diet for 16 weeks, or 3) high fat diet for 8 weeks with control diet for 8 weeks. These chromatin profiles were complemented with gene expression data (RNA-seq)
Project description:We report a single-cell DNA methylation analysis using BS-Seq, which reveals enrichment of demethylating epimutations in gene bodies and repeat regions. Comparison of single cells vs. a reference cell pool from Mouse liver tissue using an Illumina HiSeq machine.
Project description:Aims: Epidemiological and animal studies have shown that maternal diet can influence metabolism in adult offspring. However, the molecular mechanisms underlying these changes remain poorly understood. Here, we aim to explore phenotypes induced by maternal obesity in a mouse model and examine gene expression and epigenetic alterations in adulthood induced by maternal diet. Methods: We analyzed genetically identical male mice born from dams fed a high- or low-fat diet throughout pregnancy and until day 21 postpartum. After weaning, half of the males of each group were fed a high-fat diet, the other half a low-fat diet. We first characterized the genome-wide gene expression patterns of six tissues of adult offspring - liver, pancreas, white adipose, brain, muscle and heart [GSE40903] . We then measured DNA methylation patterns in liver at selected loci and throughout the genome. Results: Maternal diet had a significant effect on the body weight of the offspring when they are fed an obesogenic diet after weaning. Our analyses showed that maternal diet had a pervasive effect on gene expression, with a pronounced effect in liver where it affected many genes involved in inflammation, cholesterol synthesis and RXR activation. Maternal diet had no detectable effect on DNA methylation in the liver. Conclusions: Overall, our findings highlighted the persistent influence of maternal diet on adult tissue regulation and suggested that the transcriptional changes were unlikely to be caused by DNA methylation differences in adult liver. Methylation is compared between nine week old animals fed a common diet as adults, but derived from mothers fed different diets.
Project description:In this study we use RNAseq to explore allele specific expression (ASE) in adipose tissue of male and female F1 mice, produced from reciprocal crosses of C57BL/6J and DBA/2J strains. Comparison of the identified cis-eQTLs, to local-eQTLs, that were obtained from adipose tissue expression in two previous population based studies in our laboratory, yields poor overlap between the two mapping approaches, while both local-eQTL studies show highly concordant results. Specifically, local-eQTL studies show ~60% overlap between themselves, while only 15-20% of local-eQTLs are identified as cis by ASE, and less than 50% of ASE genes are recovered in local-eQTL studies. Utilizing recently published ENCODE data, we also find that ASE genes show significant bias for SNPs prevalence in DNase I hypersensitive sites that is ASE direction specific. We suggest a new approach to analysis of allele specific expression that is more sensitive and accurate than the commonly used fisher or chi-square statistics. Our analysis indicates that technical differences between the cis and local-eQTL approaches, such as differences in genomic background or sex specificity, account for relatively small fraction of the discrepancy. Therefore, we suggest that the differences between two eQTL mapping approaches may facilitate sorting of SNP-eQTL interactions into true cis and trans, and that a considerable portion of local-eQTL may actually represent trans interactions. 4 samples - male and female, BxD and DxB adipose of pooled RNA (3 animals per pool) were analyzed with high coverage RNAseq data.