| The study of mammalian gene expression is complicated by the fact that DNA-binding transcription factors often belong to large families of highly-related proteins. In this thesis, I have used the cad gene ( carbamoyl-phosphate synthase/aspartate carbamoyl transferase /dihydroorotase) as a model system to investigate transcriptional regulation by members of the basic helix-loop-helix leucine zipper (bHLHzip) family of transcription factors. bHLHzip proteins, including the proto-oncogene c-Myc and the USF factors, influence gene expression by binding E box sequences (the consensus E box is CACGTG) within promoters of target genes. Previously, our lab has shown that an E box in the hamster cad promoter is required to activate cad gene expression in response to growth signals. To further evaluate which bHLHzip factor regulates cad transcription, I examined which factor(s) binds the endogenous cad promoter in living cells using a UV crosslinking and immunoprecipitation procedure. Crosslinking performed on asynchronous cells revealed that both c-Myc and USF1 bind the hamster cad promoter. To evaluate factor binding during the growth cycle of mouse NIH 3T3 cells, I cloned the mouse cad promoter and showed that a conserved E box is required for growth regulated transcription. Using a PCR-based formaldehyde crosslinking procedure on serum-synchronized NIH 3T3 cells, I showed that both c-Myc and USF1 are bound specifically at the E box of the cad promoter during late G1 phase in living cells. However, the pattern of Myc binding, but not USF1 binding, correlates with cad expression suggesting that Myc is the dominant regulatory factor. In support of this model, I have shown that (1) mutant cad promoter constructs which are bound by USF1, but not Myc, are no longer growth responsive and (2) the transactivation. domain of Myc, but not USF1, activates transcription when brought to the cad promoter as a Gal4 fusion protein. From my experiments, I conclude that cad is indeed a c-Myc target gene. I suggest that cad represents one class of target genes where discrimination between bHLHzip family members is not achieved at the level of DNA binding but through highly specialized protein-protein interactions at the core promoter. |
