Regulation of Transcription at the Human Interleukin-2 Gene:
The mammalian immune system represents a unique model for studying the importance of transcriptional regulation in governing cell growth and differentiation. Studies performed by immunologists have revealed that the development of T-lymphocytes is controlled by the interplay of signal transduction and transcription. Interleukin-2 is a cytokine that acts as an autocrine growth factor promoting the proliferation and development of T cells during the immune response to bacterial and viral infection, as well as tumorigenesis. The IL-2 promoter is relatively compact for mammalian genes, since proper regulation of IL-2 transcription requires only 340 bp of DNA surrounding the transcription start site. Although small, the IL-2 promoter has a complex regulatory region that contains binding sites for at least four families of transcriptional regulatory proteins: NFAT, NF-kB, AP1 (cJun and cFos), and OCT.
We are investigating the roles of both cis-regulatory elements and trans-regulatory factors (e.g., NFAT, AP1, and OCT) in transcription at the IL-2 promoter using in vitro transcription experiments and assays in T cells. Protein-protein contacts are important for transcriptional regulation. For example, contacts between a transcriptional activator and components of the transcription machinery often represent an early step in the process of transcriptional activation. We are performing a series of protein-protein interaction assays to identify and characterize the molecular targets of NFAT, cJun, and cFos in the basal transcription machinery. Our studies revealed that the activators NFATp and cJun target coactivator subunits of the TFIID complex. Human TAFII130 (hsTAF4) is a coactivator for NFATp while cJun derepresses transcription by binding the N-terminal region of TAFII250 (hsTAF1). The functions of these interactions are being investigated through a combination of mutagenesis and transcription assays. The in vitro experiments are being complemented with cell-based experiments to test the effects of specific protein-protein interactions on the development of the mammalian immune response.
Publications
Weaver, J.R., Good, K., Walters, R.D., Kugel, J.F., and Goodrich, J.A. (2007). Characterization of the sequence and architectural constraints of the regulatory and core regions of the human interleukin-2 promoter. Mol. Immunol. 44: 2813-2819.
Hieb, A.R., Baran, S., Goodrich, J.A., and Kugel, J.F. (2006). An 8 nt RNA triggers a rate-limiting shift of RNA polymerase II complexes into elongation. EMBO J. 25: 3100-3109.
Nguyen, T.N. and Goodrich, J.A. (2006). Immobilized protein-protein interaction assays: Eliminating false positive interactions caused by contaminating nucleic acid. Nature Methods. 3: 135-139.
Weaver, J.R., Kugel, J.F., and Goodrich, J.A. (2005). The sequence at specific positions in the early transcribed region sets the rate of transcript synthesis by RNA polymerase II in vitro. J. Biol. Chem. 280: 39860-39869.
Lively, T.N., Nguyen, T.N., Galasinski, S.K., and Goodrich, J.A. (2004). The basic leucine zipper domain of cJun functions in transcriptional activation through interaction with the N terminus of hsTAF1 (human TAFII250). J. Biol. Chem. 279: 26257-26265.
Ferguson, H.A., Kugel, J.F., and Goodrich, J.A. (2001). Kinetic and mechanistic analysis of the RNA polymerase II transcription reaction at the human interleukin-2 promoter. J. Mol. Biol. 314: 993-1006.
Ferguson, H.A. and Goodrich, J.A. (2001). Expression and purification of recombinant human c Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro. Nucleic Acids Res. 29: E98 (6 pages).
Lively, T.N., Ferguson, H.A., Galasinski, S.K., Seto, A.G., and Goodrich, J.A. (2001). c Jun binds the N terminus of human TAFII250 to derepress RNA polymerase II transcription in vitro. J. Biol. Chem. 276: 25582-25588.
Kim, L.J., Seto, A.G., Nguyen, T.N., and Goodrich, J.A. (2001). Human TAFII130 is a coactivator for NFATp. Mol. Cell. Biol. 21: 3503-3513.
Galasinski, S.K., Lively, T.N., Grebe de Barron, A., and Goodrich, J.A. (2000). Acetyl coenzyme A stimulates RNA polymerase II transcription and promoter binding by transcription factor IID in the absence of histones. Mol. Cell. Biol. 20: 1923-1930.