The identification and characterization of the transcriptional repressor Related to GEF/PBF

Within the 258-bp MyoD Core Enhancer, we identified a 40-bp sequence, referred to as Required Element 2 (RE2), that is essential for enhancer activity in all skeletal muscle lineages. Based on DNase I protection and electrophoretic mobility shift assays, RE2 is a target for DNA binding proteins. In a screen for factors that interact with RE2, we isolated a previously undescribed murine gene called Related to GEF/PBF (Rgp) that is expressed in the developing dermomyotome and myotome. The Rgp gene encodes for a single C2H2 zinc finger protein that shares a high degree of sequence similarity with two known DNA binding proteins, Glut4 Enhancer Factor (GEF) and Papillomavirus Binding Factor (PBF). Rgp is actively shuttled between the nucleus and the cytoplasm by competing nuclear import and export signals. Our data indicates that nuclear localized Rgp can repress transcription in a sequence specific manner while cytoplasmic Rgp is transcriptional inert; these results suggest that the transcriptional activity of Rgp is regulated by its subcellular localization. The Rgp repression domain has been mapped to a conserved central region that contains the C2H2 zinc finger. This regulatory region has no similarity to any known repression motifs. Further analysis has shown that an intact zinc finger is required for transcriptional repression but is dispensable for sequence specific DNA interactions. Finally, we have determined that the C-terminus of Rgp contains an RE2 recognition motif. A CR1/CR2 motif located in this C-terminal domain is necessary but not sufficient to mediate RE2 specific interactions. The CR1/CR2 motif has been shown to mediate the promoter specificity of TCF E-tail isoforms. A detailed understanding of how CR1/CR2 modulates transcription factor activity is lacking, but previous work suggests it may be modifying the DNA binding site preference and/or mediating protein:protein interactions. We also demonstrate that GEF and PBF are transcriptional repressors that are actively imported and exported from the nucleus. Based on structural and functional conservation, we have defined a new family of transcriptional repressors referred to as the Rgp family