|Molecular Epi Breakfast meeting at the Montreal EMS Meeting
Agenda and Program for Molecular Epi SIG Breakfast in Montreal
7AM October 19, 2011
Agenda – 7:00-7:30AM
Breakfast and discussion of activities for the year.
Potential Usefulness of ChIP-Chip and miRNA Methodologies in Molecular Epidemiology - Talks as follows:
An Overview of ChIP-chip Technology and its Application in the Discovery of Thyroid Hormone Receptor Binding sites in the Mouse Genome
Martin Paquette1,2, Hongyan Dong1, Rémi Gagné1, Andrew Williams1, Mike Wade1, Carole Yauk1,2,, 1 Environmental Health Sciences and Research Bureau, Healthy Environments and Consumer Safety, Health Canada. 2 Department of Biology, Carleton University, Ottawa, ON.
Large numbers of proteins function through direct or indirect interactions with DNA. Chromatin immunoprecipitation can be used in combination with DNA microarrays (ChIP-chip), or with next generation sequencing (ChIP-seq), to characterize on a genomic scale, the DNA sequences that mediate regulatory control for a specific protein. ChIP-chip involves cross-linking DNA with proteins, sonicating the DNA to produce small DNA fragments, and immunoprecipitation of the protein-DNA complex using an antibody specific to a protein of interest. Enriched DNA is labelled and hybridized to microarrays that contain probes complimentary to the genome of interest. Hybridization signals from enriched samples (immunoprecipitated) are compared to labelled non-enriched DNA samples (total input). Statistical analyses are used to determine sites of protein binding enrichment, and bioinformatics is used to identify the genomic location and predict the precise DNA binding sites of the protein. In ChIP-seq, the enriched and total input samples are analyzed using next generation sequencing technologies.
In our laboratory, ChIP-chip is being used to profile nuclear receptor binding sites. The profiling of transcription factors is of interest because they have the ability to regulate the expression of large numbers of target genes. As such, perturbations in the interaction between transcription factors and their genomic targets can greatly impact overall cellular function. The focus of our research is to characterize the thyroid hormone (TH) induced transcriptome to understand the molecular consequences of toxicant-induced hypothyroidism. THs bind to thyroid receptors (TRs) and recruit co-regulators to control target gene expression. THs exert effects on growth, development and metabolism of practically every cell and organ. Disruption of TH physiology during critically sensitive periods in development can lead to adverse outcomes. We have identified TR binding sites and target genes using a ChIP-chip approach in juvenile euthyroid mice. The analysis has revealed novel TH responsive genes, which have been confirmed to bind to TR in vitro. Electrophoresis mobility shift assays and reporter assays have been used to demonstrate that mutation or deletion of the DNA binding sites for select newly identified binding sites abolishes TR-DNA interaction. In addition to providing insight into the molecular processes regulated by TH, the ChIP-chip data are being used to develop an improved consensus sequence for the binding of TR to DNA.
MicroRNAs: Biology, Technology and Toxicology
Julie Bourdon, Health Canada, Environmental Health Centre, Tunney's Pasture, 0803A, Ottawa, ON, Canada.
MicroRNAs (miRNAs) are short (~ 22 nucleotide long), highly conserved, non-coding RNAs that are important post-transcriptional regulators of mRNA levels. MiRNAs operate through binding to the 3’ untranslated regions of target mRNA, leading to degradation or translational repression. Individual miRNAs have been shown to repress hundreds of target genes. Moreover, miRNAs are known to affect at least one third of all human genes. The increasing amount of information available on miRNA targets and modes of action has enabled researchers to link specific miRNAs to various important biological processes. Altered miRNA profiles have also been associated with a number of diseases, including cancer, diabetes, neurological diseases and viral infections. As such, miRNAs are considered potentially important biomarkers of exposure and effect.
This talk will focus on the biology behind miRNAs, the experimental applications employed to identify differentially expressed miRNAs and their mRNA targets, and the biological implications of changes in miRNA abundance. The techniques reviewed will include genomic approaches (miRNA arrays and sequencing) to identify up- and down-regulation of miRNAs, miRNA target prediction and pathway identification techniques, as well as in vitro approaches used to identify biological miRNA effects (transfection of mimics and inhibitors). Demonstrations on the application of these approaches will be given using work conducted as part of Health Canada’s initiative to apply systems biology approaches to investigate mechanisms of action and risk of adverse health outcomes following exposure to toxic chemicals. This will include work on nanoparticle exposures, including carbon black and titanium dioxide, and the importance of miRNAs 135b, 146a and 146b in resolution of particle induced inflammation. Finally, literature on the use of miRNAs as potential serum biomarkers of effect and exposure will be reviewed.