| The c-myc gene is upregulated in many different types of cancer, and it has been shown that high levels of c-Myc protein is required for tumor maintenance. Thus, knowledge of its mechanism of action could provide information which could lead to novel forms of cancer therapy. Myc is a transcription factor that activates the transcription of a number of genes important for tumorigenesis. In this thesis, I have used the Myc target gene cad as a model to determine how Myc activates transcription. I began my studies by using the chromatin immunoprecipitation (ChIP) assay to examine which proteins and protein modifications associated with the cad promoter correlate with Myc binding and activation of transcription. I found that high levels of histone acetylation are present in the absence of activated transcription, and RNA polymerase II (RNAP II) is bound constitutively to the cad promoter. These results indicate that Myc does not activate cad transcription by stimulating histone acetylation or formation of the preinitiation complex. In contrast, when I performed the ChIP assay using an antibody that recognizes the phosphorylated form of the RNAP II carboxyl-terminal domain (CTD), I found that phosphorylation of the CTD correlated with Myc binding on the cad promoter, which indicates that Myc activates transcription at a post-RNAP II recruitment step, possibly through binding of a CTD kinase. Using a GST pull-down assay, I found that the Myc transactivation domain binds to a number of cyclins and cyclin dependent kinases involved in CTD phosphorylation, most notably cdk8 and cyclin T1. Deletion analysis showed that cyclin T1 binds to the Myc box I domain of the Myc transactivation domain, and transfections with various regions of the Myc transactivation domain fused to the Ga14 DNA binding domain showed that the cyclin T1 interaction is important for Myc-mediated activation of the cad promoter. Furthermore, direct recruitment of cyclin T1 and its cdk partner, cdk9, to the cad promoter resulted in activation of cad to levels similar to those seen with Myc. Finally, the HIV protein Tat was able to activate cad when recruited to the promoter, and this activation was dependent upon interaction with cyclin T1 and cdk9. Therefore, I propose a model in which Myc activates the cad promoter by stimulating promoter clearance of the RNAP II complex through recruitment of the CTD kinase complex cyclin T1/cdk9. |
