The PAX gene family encodes a group of transcription factors that play important roles during embryogenesis with mutations resulting in developmental abnormalities. Normally expression of these genes is downregulated prior to terminal differentiation with a select few tissues maintaining expression in the adult. Expression of PAX genes has also been observed in a variety of different cancers, although the functions of the aberrantly expressed PAX proteins have yet to be determined. Previous research has shown that knockdown of PAX8 in cancer cell lines results in the downregulation of E2F1 mRNA and protein levels. E2F1 is the major regulator of the cell cycle and its expression is often deregulated in cancer.
The overall aim of this study was to investigate the relationship between PAX8 and E2F1 in cancer cells. The first step was to determine the effects of PAX8 knockdown and overexpression on the activity of the E2F1 promoter, using two E2F1 promoter constructs, pE2F1(-242) and pE2F1(-728). The results suggested that PAX8 was involved in the regulation of the E2F1 promoter as the activity levels of both E2F1 constructs decreased following PAX8 knockdown and increased following PAX8 overexpression.
The next step was to determine if PAX8 was regulating the E2F1 promoter directly using chromatin immunoprecipitation (ChIP). Six potential binding sites were predicted in the region of the E2F1 promoter contained in the longer of the two constructs, pE2F1(-728). Of these six predicted sites, PAX8 was found to bind to site 3, thereby demonstrating that PAX8 activates E2F1 transcription by binding directly to the promoter.
Finally, deletion constructs and site-directed mutagenesis were employed in an attempt to validate the ChIP results as well as to determine if PAX8 was able to bind to site 2, which was not included in the ChIP experiment as successful primers were not able to be designed. Unfortunately these experiments were unsuccessful and thus no definitive conclusions could be made. Despite this, it was determined that PAX8 binds to and activates the E2F1 promoter in cancer cells. The involvement of PAX8 in the regulation of proliferation (by activating E2F1 transcription) reveals a potential function for PAX8 in cancer.
