The major chicken vitellogenin gene (VTGII) serves as a model for studying three integrated transcriptional control mechanisms. The transcription of this gene is estrogen-dependent and liver-specific. In addition, the promoter has a negative element which maps to a consensus heat shock element (HSE) and binds a constitutively expressed heat shock factor (HSF). To our knowledge this is the only context where an HSE is implicated in the regulation of a gene that is not a heat shock gene. Since this HSE is in close apposition to essential positive control elements, we hypothesize that negative control may be achieved through a competition between this HSF and positive factors for binding to this promoter. This will be tested using in vitro binding and transient cotransfection assays. The analysis of positive control elements within the VTGII promoter led us to clone three transcription factors and we will test whether they are relevant to the liver-specificity of this promoter. Two of the factors (GATA-4 and HLF) are liver-enriched. The third factor (VBP) is encoded by a complex gene that is differentially spliced to yield factors which have distinct N-terminal and C-terminal domains. One of these isoform- specific N-terminal domains renders the internal generic transactivation domain inactive in chick embryo fibroblasts (where the target VTGII promoter is inactive) but not in chicken hepatoma cells (where the target VTGII promoter is active). The basis for this novel mode of cell- specific transactivation will be determined and we will test whether the other isoform-specific domains also differentially modulate the transactivation or binding of VBP in different cell types. One of the reasons for cloning liver-enriched transcription factors that regulate abundantly expressed liver-specific genes (such as the chicken VTGII gene) is that they provide an entry point for elucidating how the hepatocyte phenotype is determined. this has clear relevance to health as any disruption in such a transcription factor hierarchy would likely result in some type of liver dysfunction. We will analyze the promoters of the GATA-4 and HLF genes to identify liver-enriched transcription factors that may act earlier in this hierarchy. In the event that novel liver-enriched factors are implicated in regulating either of these promoters, we will attempt to clone them.
