Homologous Ets-domain transcriptional repressors demonstrate unique responses to Ras/MAPK signaling

The molecular mechanisms that control the proliferation and differentiation of specific cell types remain poorly understood. Ets transcription factors play important roles in mediating cellular proliferative and differentiation in response to Ras/MAPK signaling in many cell types following mitogenic stimulation. Previous studies provided evidence that PE-1/METS, a member of the ERF sub-family of ETS-domain transcription factors, is able to block the Ras-dependent mitogenic response mediated by Ets target genes during terminal macrophage differentiation. The repressive activity of METS does not inhibit transcription of macrophage-specific genes that are targets of Ras-dependent differentiation. The anti-proliferative action of METS in Rat-1 cells requires interaction with DP103, a DEAD-box protein and a member of the RNA helicase family of proteins. ERF has been identified as an Ets-2 Repressor Factor and is structurally related to METS. These structural similarities have been used to guide a comparative analysis of METS and ERF with respect to functional domains, corepressor requirements, and ability to repress cell cycle control genes in signal and cell-specific contexts. This thesis addresses the question of whether the region of ERF that corresponds to the N-terminal repression domain of METS (RD-N) is able to function as an independent repression domain and whether ERF interacts with the C-terminal repression domain of DP103 in the same manner as METS. The thesis demonstrates the function of ERF as a repressor is inactivated under conditions when METS remains active on natural promoters of the cell-cycle control genes c-myc and cdc-2. Studies of ERF suggest it represses DNA transcription only in the absence of growth factors, and inactivation of ERF is a consequence of phosphorylation on C-terminal threonine residues not conserved in METS. Constitutive activation of the Ras/MAPK pathway in transformed RAW 264.7 cells is associated with constitutive inactivation of ERF in this cell type. Studies of mutant ERF proteins confirm that the C-terminal phosphorylation sites are essential for ERF inactivation and specific Ras/MAPK inhibitors restore the ability of ERF to repress target genes in the presence of growth factors. These findings indicate METS and ERF share some mechanisms for transcriptional repression but they respond differently to growth signaling, suggesting METS and ERF play different biological roles in regulating cellular proliferation.