Controlling levels of transcription is of central importance to all aspects of life. Indeed, the regulatory regions of higher eukaryotic promoters are frequently larger than the genes that they control with multiple binding sites for many distinct regulatory proteins that act in concert to orchestrate levels of transcription and, therefore, protein production in cells. To fully understand mammalian growth and development it is essential that we obtain a detailed understanding of the molecular mechanisms governing transcriptional regulation. To study the regulation of human mRNA synthesis we will apply kinetic experiments in a reconstituted transcription system for an entirely natural promoter. This unique approach is critical because of the importance of promoter sequence and architecture in the proper regulation of transcription. We reconstituted an in vitro system for studying the activation of transcription at the promoter of the human interleukin-2 gene. IL-2 is a key signaling molecule in the mammalian immune response. The IL-2 promoter is complex but compact and is perfectly suited to mechanistic studies in vitro and to controlled examination in cultured T lymphocytes. We have expressed and purified transcriptional activators, including NFATp, cJun, cFos; we have identified activator- coactivator interactions; and we have established transcriptional activation at the IL-2 promoter in vitro. We are now poised to perform kinetic studies on the regulation of transcription at the natural human IL-2 promoter. Another aspect of the proposed research is that we will evaluate the function of activator-coactivator interactions in IL-2 transcriptional activation in cultured human T lymphocytes. It is critical to test in vitro observations in the context of cells, since a reconstituted in vitro transcription system lacks some factors that affect transcriptional regulation, such as nucleosomes, post- translational modifications, and perhaps yet unidentified proteins or cofactors. Our studies will provide detailed insight into the mechanism of transcriptional regulation at a natural promoter that is of great importance to the development of the human immune system.
