CELL-CYCLE ANALYSIS OF E2F IN PRIMARY HUMAN T-CELLS REVEALS NOVEL E2FCOMPLEXES AND BIOCHEMICALLY DISTINCT FORMS OF FREE E2F

The transcription factor E2F activates the expression of multiple gene s involved in cell proliferation, such as c-myc and the dihydrofolate reductase gene. Regulation of E2F involves its interactions with other cellular proteins, including the retinoblastoma protein (Rb), the Rb- related protein p107, cyclin A, and cdk2. We undertook a detailed anal ysis of E2F DNA-binding activities and their cell cycle behavior in pr imary human T cells. Three E2F DNA-binding activities were identified in resting (G0) T cells with mobilities in gel shift assays distinct f rom those of previously defined E2F complexes. One of these activities was found to be a novel, less abundant, Rb-E2F complex. The most prom inent E2F activity in resting T cells (termed complex X) was abundant in both G0 and G1 but disappeared as cells entered S phase, suggesting a possible role in negatively regulating E2F function. Complex X coul d be dissociated by adenovirus EIA with a requirement for an intact El A conserved region 2. However, X failed to react with a variety of ant ibodies against Rb or p107, implicating the involvement of an ElA-bind ing protein other than Rb or p107. In addition to these novel E2F comp lexes, three distinct forms of unbound (free) E2F were resolved in gel shift experiments. These species showed different cell cycle kinetics . UV cross-linking experiments suggested that a distinct E2F DNA-bindi ng protein is uniquely associated with the S-phase p107 complex and is not associated with Rb. Together, these results suggest that E2F cons ists of multiple, biochemically distinct DNA-binding proteins which fu nction at different points in the cell cycle.