Levels of proteins required for DNA replication must be increased when a non-replicating cell begins neoplastic growth. Our lab has shown that this regulation is controlled by increased transcription mediated by members of the myc and E2F gene families. Each of these gene families consists of multiple proteins and, to date, it has been very difficult to clearly link one member of a family of transcription factors to a specific target promoter.
In Aim 1, we proposes to use immunoprecipitation of transcription factors crosslinked to chromatin to determine which E2F and myc family members regulate G1/S-phase transcription. Although it is clear that deregulation of the activity of transcription factors such as E2F and myc is associated with cancer, it is not yet clear how this deregulation contributes to a neoplastic phenotype. We believe that development of a technique that can determine which transcription factor binds to a specific promoter in the natural chromatin context of a normal versus a tumor cell will aid in our understanding of how nuclear oncogenes transform cells. A model has been developed to explain how E2F family members specify differential transcription of Go versus S phase. However, the current model does not accurately account for results obtained from analysis of mutated dhfr promoter constructs. The experiments proposed in Aim II will attempt to determine which domain of E2F plays a unique, yet critical, role in dhfr transcription. Novel aspects of our assay system include the exact replacement of the dhfr E2F site with a GAL4 binding site and the expression of GAL4/E2F fusion proteins from a growth-regulated promoter.
In Aim III, the investigator proposes experiments that will determine how the E2F family of transcription factors, interacts with other transcriptional regulators and with the basal transcriptional machinery. The preliminary data suggests that only a subset of transcription factors can synergize with E2F to mediate S phase transcriptional activation; the investigator will continue the experiments by analyzing the ability of well-characterized transcriptional regulators to cooperate with E2F factors.
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