PROJECT SUMMARY Activation of E2F transcription factors is the most downstream event in the retinoblastoma protein (Rb) tumor suppressor pathway, which controls entry into the cell cycle and proliferation. E2F stimulates expression of genes needed for DNA synthesis during S phase and for further progression into mitosis. Inactivation of Rb and aberrant activation of the E2F-controlled transcription program is a hallmark of cancer. While Rb repression of E2F has been extensively characterized, little is known regarding how E2F activates transcription and how additional regulatory mechanisms control E2F function. Here we explore the structural mechanisms underlying the function and posttranslational regulation of E2F. Our first aim is to determine how E2F associates with chromatin and influences chromatin architecture to induce gene expression. We will use structural, biochemical, and cellular assays to investigate how E2F binds nucleosomes and how those interactions are critical for modulating chromatin architecture as cells activate the transcription program for S phase entry. Our second aim is to identify novel E2F posttranslational modifications and how modifications including phosphorylation and acetylation modulate E2F association with chromatin and co-activator proteins. Our third aim is to reveal how E2F proteins are recognized by the E3 ligase adaptor protein Cyclin F (CycF) for ubiquitylation and degradation. A structural and cell biology approach will be applied to define the CycF-binding motif, and we will test the hypothesis that chemical inhibition of CycF can deregulate the cell cycle in model cancer cells. In collaboration with the other program projects and cores, our study will provide unprecedented molecular analysis of E2F function and regulation. Our experimental results will provide foundational knowledge that can be applied to design new therapeutic strategies that target E2F activity in diverse cancers.