Cells must avoid senescence, an irreversible process in which a cell exits the cell cycle, to allow for tissue repair and renewal. On the other hand, senescence must be induced in cells that have acquired genetic damage to provide a barrier to propagation of deleterious mutations. Sin3 proteins have recently been shown to play an important role in inducing senescence in mammalian cells. Sin3 proteins assemble into complexes that globally regulate gene expression by recruiting chromatin-modifying and remodeling enzymes. This project seeks to understand how the mammalian Sin3 isoforms, Sin3A and Sin3B, regulate oncogene-activated and replicative senescence in IMR90 human fibroblasts and how Sin3 interacts with two well-studied senescence-inducing pathways involving the pRb and ASF1a proteins.
In Specific Aim 1, Sin3A- and Sin3B-specific complexes will be biochemically isolated from cycling and senescent IMR90 cells, and components that uniquely characterize senescent cells will be identified to potentially explain changes in the chromatin landscape that underlie senescence. Using quantitative assays, the spatial and temporal binding patterns of any novel or unique Sin3A- or Sin3B-complex components will be correlated with the expression and histone modifications of select genes before and after induction of senescence. The functional importance of these components in chromatin remodeling and senescence will be assessed after gene ablation. Markers of senescence, including assembly of senescence-associated heterochromatin foci (SAHF), will be examined to determine how the biochemical role of Sin3 complexes relates to changes in chromatin and gene expression and how Sin3 function is mechanistically related to senescence. Since Sin3 proteins are known to interact with pRb and ASF1 complexes, each of which plays a known role in senescence, Specific Aim 2 will determine how Sin3 function is linked to pRb and ASF1 activities during induction of senescence. We will study the recruitment of Sin3 and Sin3-associated proteins to genes regulated during senescence, focusing on the unique components identified in Aim 1. The presence of pRb, ASF1a, HIRA, and heterochromatin histone markers at these genes will be determined before and during senescence. The role Sin3 in the heterochromatinization of pRb-bound E2F target genes and HIRA/ASF1a-mediated SAHF formation will also be determined using gene ablation coupled with ChIP and gene expression studies. In vitro studies of senescence are useful as they represent an established model for biological aging. To strongly correlate the impact of our findings to human aging and age-related disease, studies from both Aims will be extended to aging, normal human fibroblasts at low, intermediate, and high population doublings and to fibroblasts from patients with Hutchinson-Gilford Progeria Syndrome (HGPS), a genetic disorder that results in premature aging. Overall, the goal of the proposed research is to determine the role of each Sin3 isoform in senescence, the mechanisms by which senescence-specific Sin3 complexes regulate senescence via chromatin remodeling, and how Sin3 interacts with other senescence-inducing pathways.
Recently, Sin3 proteins have been implicated in the induction of senescence, an important cellular model for biological aging. Understanding the mechanisms by which Sin3 normally alters the genetic environment to initiate the aging program will have immense impact on the understanding of disorders like Hutchinson-Gilford Progeria Syndrome (HGPS), a rare genetic disorder that results in premature aging in children, and age-related pathologies in which cells bypass the normal aging process, such as in coronary artery disease and cancer. Knowledge about the molecular processes that allow a cell to either enter or bypass the aging process is valuable in the design of potential drugs to treat diseases such as HGPS, coronary artery disease, and cancer.
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