The research and career development activities outlined in this application have been designed to equip the candidate, Dr. Archana Unnikrishnan, with the scientific and technical expertise necessary to become an independent investigator in the field of aging. The proposed research aims to study the role of DNA methylation in dietary restriction (DR) mediated insulin sensitivity. The goals of this training program are: 1. gain hands on experience in the cutting-edge technology available to study DNA methylation; Oxidative Bisulfite Oligonucleotide Sequencing; 2. determine the role of DNA methylation in the effect of DR on glucoregulation; 3. determine the effect of DR on the enzymes involved in DNA methylation; and 4. gain experience in writing grants and have a R01 funded grant by the end of the K01 award. The candidate will receive training in novel bisulfite based next generation sequencing technology and biology of aging through intensive coursework and hands on laboratory experience under the supervision of Drs. Arlan Richardson and Willard Freeman. The candidate will also gain training in proteomic analysis by using mass spectrometry by collaborating with Dr. Mike Kinter. The other major goal of this application is to enable the candidate, as a junior faculty, to secure protected time for research activities, establish new collaborations, and develop, and develop a novel line of research that produces competitive grant proposals for future funding. The research objective is to characterize and discover novel changes in DNA methylation that occur with DR that have an effect on the transcriptome. DR is believed to retard aging and has become the ?gold standard? by which other manipulations that increase lifespan are compared. Many mechanisms have been proposed for the life-extending action of DR; however, it is still unclear as to the molecular basis of DR?s action. An important facet of DR that has been largely overlooked by the research community is that DR can have early effects that create a metabolic memory, which persists even when the restriction is discontinued. The purpose of this grant is to gather the first data on the effect of DR on DNA methylation and metabolic memory that will allow us to test the following hypothesis: DR induces metabolic memory by inducing changes in DNA methylation (5mC) at specific genomic regions that regulate the expression of genes that are involved in insulin sensitivity. This hypothesis will be tested in the following aims: 1) assess the effect of DR on DNA methylation and hydroxymethylation across gene regulatory regions of tissues involved in glucoregulation; 2) determine the transcriptional phenotype related to the changes in DNA methylation in tissues involved in glucoregulation; 3) Determine the effect of DR on the pathway involved in DNA methylation and demethylation.
One of the hallmark features of DR is the improvement of glucose and insulin tolerance and the purpose of this study is to use cutting edge novel next generation sequencing approach to quantitate DNA methylation to investigate the role of DNA methylation in DR. The findings from this study will advance our understanding of how DR can have early effects that can induce alterations in DNA methylation which potentially leads to improvement in insulin sensitivity & can persist even when DR is discontinued. This would be a major discovery in aging, demonstrating a novel mechanism through which DR can potentially alter aging and age-related diseases.