A comprehensive understanding of growth control in normal mammalian cells requires that we know how gene regulatory circuits operate as a function of the cell cycle. The transcription factor, E2F, and pRB, a tumor suppressor known to restrain E2F activity, are among the best-studied regulators of cell cycle dependent gene expression. Pathways that involve pRB are known to be de-regulated in a majority of human cancers. Despite the fact that E2F and pRB have been linked genetically to growth control, the mechanisms by which these factors modulate endogenous gene expression and globally regulate cell cycle progression are not well understood. A complete description of their function requires that we understand not only physiological changes that occur at a given cell cycle regulated promoter but also the intricate biochemical mechanisms that switch a gene from the 'OFF' to 'ON' state in a reversible manner during the cell cycle. A detailed description can be achieved only by studying the regulation of E2F targets in a natural (chromatin) setting under conditions where cells are either stimulated to grow or are continuously cycling. Our hypothesis suggests that discrete mechanisms must exist that distinguish gene expression profiles during each stage of the cell cycle. Given the pivotal role of pRB in tumor suppression, an understanding of these mechanisms will provide crucial clues necessary for understanding the regulation of normal cell growth and the ways in which controls may go awry in human cancer. This Research Proposal takes advantage of multiple, complementary approaches to test this idea.
In Aim 1, a combination of genomics and biochemistry will be used to identify targets of E2F and pRB families and chromatin modifying enzymes in proliferating cells at each stage of the cell cycle to determine those factors that regulate genes in both active and inactive states. Using rapidly emerging bioinformatics and computational tools, approaches in Aim 2 will attempt to identify those patterns that link genes co-regulated by E2F, pRB, and chromatin modifying enzymes. Biochemical experiments in Aim 3 will test predictions about these patterns and will reveal those mechanisms that distinguish expression profiles of native E2F target genes at different stages of the cell cycle. ? ?
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