Download Project Summary The single nucleotide polymorphisms (SNPs) are

Project Summary The single nucleotide polymorphisms (SNPs) are found to have important roles in the pathogenesis of diseases, gene-­‐ environment interactions, susceptibility to many diseases, and drug-­‐protein interactions. The relation between diseases and identified SNPs are commonly investigated by linkage analysis. In genome screening studies like HGD and 1000Genomes, several SNPs were identified in genes involved in biological clock mechanisms. SNPs in the CLOCK, BMAL1, CRY2, and PER2 genes are associated with many physiological disorders like sleep disorders, metabolic disorders, and infertility. However, the effects of SNPs on biological clock and the related disorders are not well understood. CLOCK/BMAL1 heterodimers bind DNA of clock target genes at E-­‐boxes and initiate the transcription of their RNA and 15% of genes (positive feedback). The resulting PER and CRY proteins dimerize in the cytoplasm and translocate to the nucleus where they inhibit CLOCK/BMAL1 proteins from initiating further transcription (negative feedback). This rhythm repeats every 24 hours. In the proposed project, the effects of SNPs on 4 core clock (CLOCK, BMAL1, CRY2 AND PER2) protein’s function and thus, on the biological clock will be investigated. In the scope of the project; •
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For each gene, 8-­‐10 SNPs that placed in functional domains like HLH and PAS1-­‐2 will be chosen from the published 1000genomes project database. Additionall the SNPs will be chosen based on the SIFT and PolyPhen bioinformatics tools. Gene variants will be created by site-­‐directed mutagenesis, and the interaction between CLOCK variants and other proteins will be investigated. The effects of the SNPs will be examined by looking the interaction between CLOCK and BMAL1 dimer, CRY2 and PER2 dimer, and interaction between CLOCK/BMAL and CRY2/PER2 complex. Luciferase assays will be performed using HEK293T cell-­‐lines. Finally, the effect of variant clock proteins on 24-­‐hours circadian rhythm of U2OS cells will be investigated. We performed preliminary studies with Bmal1 gene. Based on lusiferase analyses, selected variations result in decrease in transactivation capacity of BMAL1-­‐CLOCK dimer. Additionally, CRY2-­‐PER2 dimer has different inhibition effect on variant BMAL1-­‐CLOCK dimer than wild type BMAL-­‐CLOCK dimer. These preliminary results are encouraging and give hope about the success of proposed project. As a result of the proposed basic science project, the effects of identified clock gene SNPs on the biological clock mechanism will be learned. The findings of the proposed project will encourage us and other researcher to propose new project(s) to investigate the role of the SNP in the other biological clock related disorders. If a candidate SNP was found to significantly affect the biological clock mechanism, further molecular analysis and linkage studies can be performed to understand the role of the SNP in the other biological clock related disorders. As far as we know, the suggested project will be the first study investigating the effects of SNPs resulting in amino acid changes in CLOCK protein. Additionally the findings of the study will lead other similar studies and may provide beneficial data for medical researches. This proposal will decipher the relationship between the SNPs and clock at the molecular level and will enable us and other research groups to investigate the relation between clock and clock related diseases such as cancer, metabolic disorders, diabetes, and obesity. This study will encourage us and other research groups to investigate similar studies with other clock genes and SNPs, and enable us to understand relation between clock and SNPs.