Functional characterization of transcriptional inhibitory domains in the C/EBP-epsilon basic region/leucine zipper transcription factor



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Texas Tech University


C/EBPE is a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors and is primarily expressed in neutrophils. Multiple functional domains were previously identified in the C/EBPe polypeptide including a regulatory domain (RD or RDI) that negatively regulates the activity of C/EBPe. Within the RD a five amino acid motif (the regulatory domain motif (RDM)) is conserved in three other C/EBP family members and is critical for the inhibitory function of the RD. Interestingly the RDM is similar to the recognition sequence for the small ubiquitin-like modifier protein (SUMO) and attachment of SUMO to the RDM can activate C/EBPe. The goal of this study was to explore the molecular mechanisms by which the RD controls C/EBPe activity. These experiments may provide important information about general transcriptional regulatory mechanisms as RD-like elements, and SUMO attachment sites, exist in inhibitory domains of a large number of transcriptional regulators.

Two models were designed to explain the mechanism of the function of the RD. One model proposes that intra-molecular interactions exist between the RD and its linked activation domain (AD), thereby blocking access of the AD to components of the transcriptional machinery. In this model, the attachment of SUMO to the RDM releases the AD and activates C/EBPe. Physical interactions between the RD and AD were not detected in either a mammalian two-hybrid assay or a direct interaction assay. The validity of this first model was also assessed by examining the activation domain specificity of the RD. The C/EBPe RD was capable of inhibiting linked ADs from three different classes, suggesting that the RD is unlikely to function according to model one. The second model assumes the RD functions by recruiting as yet unknown inhibitory RDM binding factor(s) (RDM-BF). In this model, SUMO attachment would displace the RDM-BFs thereby releasing the inhibitory effect of the RD. The first candidate RDM-BF tested was the family of proteins with histone deacetylase (HDAC) activity as these proteins are classically associated with co-repressor activity. Although the activity of C/EBPe was increased in the presence of the general HDAC inhibitor Trichostatin A, this effect was not dependent on the integrity of the RDM. These results indicate that C/EBPe may recruit HDACs through a domain separate from the RDM. In conclusion, the AD and RD of C/EBPe appear not to directly interact, however, additional candidate RDM-BFs must be identified and examined to determine whether the RDM functions by recruiting accessory, inhibitory proteins.