Regulation of Age-Associated Heterochromatin Formation

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This application for a Mentored Research Scientist Development Award is to facilitate Dr. Jill Kreiling's transition to an independent investigator in the field of aging biology. Her long-term goals include establishing an independent research program focused on advancing our understanding of heterochromatic changes that occur during the natural aging process. To achieve these goals she will follow a structured career development and training plan that includes training practical laboratory techniques and in bioinformatic analyses; university and extramural courses; attending seminars, meetings and conferences; and training in grant writing and laboratory management skills. This plan will be implemented under the supervision of the primary mentor, Dr. John Sedivy, who is an expert in the field of aging biology and has considerable experience with the analysis of genome-wide studies. Dr. Peter Adams, a leader in the field of chromatin biology, will serve as a co-mentor, along with Dr. Robert Reenan who specializes in RNA biology. In addition, Dr. Charles Lawrence will provide guidance with the bioinformatic approach to genome-wide analyses. There is a growing body of evidence that chromatin undergoes reorganization during normal aging. This is accompanied by a corresponding change in gene expression. It is reasonable to expect that these two processes may be connected. However, the epigenetic mechanisms of this extensive rearrangement of the chromatin are virtually unknown. In this application we propose to study the mechanisms that regulate age- associated heterochromatin formation in post-mitotic tissues. Our preliminary data suggest an increase in facultative heterochromatin incorporating the highly repressive histone variant macroH2A in mouse and baboon skeletal muscle. Recent evidence points towards a role of small non-coding RNAs (ncRNAs) in the deposition of repressive heterochromatin marks. The synthesis of many miRNAs (a subclass of ncRNAs) is regulated by Polycomb group (PcG) repression, and our preliminary data suggest a decrease in PcG repression in old animals. We will study the role of ncRNAs in the regulation of age-associated heterochromatin formation in skeletal and cardiac muscle from mice and rhesus monkeys. Specifically we will correlate specific ncRNAs to sites of age associated heterochromatin formation and we will investigate the regulatory elements present in the ncRNA genes. Our hypothesis is: An age-associated reduction in PcG repression leads to activation of certain miRNA genes, whose products trigger age-associated heterochromatin formation at specific sites on the genome. Specific Aim 1: We will begin these studies by identifying the specific chromatin states that correlate with age in post-mitotic tissues. In addition, we will determine if certain dietary interventions (calorie restriction and resveratrol) can delay the onset of this age- associated phenotype. Specific Aim 2: This information will be used to design ChIP-seq assays to determine the regions of the genome susceptible to age-associated heterochromatin formation. The ncRNA populations will be characterized by RNA-seq of the transcriptome to identify ncRNAs subject to age associated increases in expression. The sequences of the ncRNAs showing an age-dependent up-regulation will be correlated with sequences of the genome showing age-associated heterochromatin to determine potential sites of RNA- dependent deposition of silencing marks. Specific Aim 3: Markers of PcG repression will be investigated to determine if there is a decrease in PcG repression with age. The promoter regions of the associated miRNA genes will be analyzed to determine if PcG repressive elements are present that could regulate expression. Finally, we will verify our results with mechanistic studies in cultured human fibroblasts to show that the expression of specific ncRNAs are repressed by PcG regulation in young cells and this repression is lifted in older cells leading to expression of the ncRNA and age-associated heterochromatin formation. Taken together this project will broaden our understanding of the mechanisms and processes involved in the regulation of age- associated heterochromatin formation.