Unknown,Transcriptomics,Genomics,Proteomics

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Different DNA methylation between hypothalamus paraventricular (PVH) and ventromedial (VMH) nuclei

ABSTRACT: The goal is to compare the DNA methylation patterns between ARH and PVH to examine to what extent DNA methylation is region specific in genome scale. ARH and PVH were micro-dissected from mice brains. Each 7 pieces of ARH or PVH were pooled and used for DNA methylation comparison by methylation specific amplification microarray (MSAM). As a brief description of the MSAM: 500ng of genomic DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification and then cohybridization. Two-color cohybridizations were performed as paired comparison of PVH vs. ARH with two biological replicates including dye swap.

ORGANISM(S): Mus musculus

SUBMITTER: Ge Li

PROVIDER: E-GEOD-48197 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:BACKGROUND & AIMS: Toll-like receptor 2 (Tlr2) is important in bacterial pattern recognition and has been recognized as a modifier of intestinal inflammation. In this study we sought to determine the epigenomic, transcriptomic and microbiomic consequences of Tlr2 deficiency in the colonic mucosa of mice to gain insights into biological pathways that shape the interface between the gut microflora and the mammalian host. METHODS: Colonic mucosa from C57BL/6 and Tlr2-/- mice was interrogated by methylation specific amplification microarray (MSAM) to screen for changes in DNA methylation, with bisulfite pyrosequencing validation. Transcriptomic changes in the same tissue were analyzed by microarray expression profiling and real time RT-PCR. The mucosal microbiome was studied by high throughput, detailed pyrosequencing of 16S RNA. RESULTS: Tlr2 deficiency influenced the methylation of about 1% of the interrogated genome and resulted in a significant expression change in a similar percentage of all transcripts studied. Importantly, gene ontology analysis revealed that the expression of genes involved in immune processes is significantly modified by the absence of Tlr2, some of which have been already linked to inflammatory bowel diseases (Stat1, Anpep), for example. Overlaps between DNA methylation and gene expression changes were confirmed at Anpep and Ifit2. The epigenomic and transcriptomic modifications associated with alteration in mucosal microbial composition, affecting 11% of the detected bacterial species in the Tlr2-/- animals. CONCLUSIONS: Tlr2 deficiency induces colonic mucosal epigenomic, transcriptomic and microbiomic changes underscoring the intricate network of biological processes that provide the link between genotype and phenotype in mammals. Our findings bare implications for common gastrointestinal disorders such as IBD and colon cancer. [MSAM]: Methylation profiling. Two independent Tlr2-/- to WT comparisons were performed as follows. Samples were labelled with Cy3 (WT) and Cy5 (Tlr2-/-) and two independent Tlr2-/- to WT comparisons were done on a 2x105k microarray containing probes, covering 33,404 (81% of all) SmaI intervals between 100 bp and 2.2 kb in the mouse genome. [mRNA]: mRNA profiling. 4 independent Tlr2-/- to WT comparisons were performed as follows. RNA (0.4ug) samples were processed and labelled with Cy3 and Cy5 (Quick Amp labelling kit, two color, Agilent technologies) and 4 independent Tlr2-/- to WT comparisons were done on Agilent technologies 4x44k whole genomic expression microarray G2519F, Amadid:014868). The Supplementary files below contain processed data from the MSAM and expression microarrays. For the MSAM: an interval was considered a 'hit' if the averaged probe ratios within an interval showed a >1.6 fold change in both Tlr2-/- vs. WT comparisons. For the expression microarrays: transcripts were considered a 'hit' if probe average intensities showed a >1.6 fold change in at least two microarray comparisons out of four, with the remaining arrays not contradicting the increase or decrease of expression (i.e.: 1 or above 1 in case at least two other >1.6; 1 or less than 1 in case at least two other <0.625).

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Different DNA methylation between hypothalamus paraventricular (PVH) and ventromedial (VMH) nuclei

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