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| Cristol 256 303/492-3273 303/492-5894 (fax) | Cristol 220/218 303/492-2508 |
Assistant Professor (Biochemistry). Ph.D., Carnegie Mellon University, 1992; Fellow of the Damon Runyon-Walter Winchell Cancer Fund, 1992-94; University of California Berkeley, 1992-1996; Leukemia Society Special Fellow, 1995-1998; Pew Scholar in the Biomedical Sciences, 1999-Present.
The growth, differentiation, and development of all living things are controlled by elaborate cascades of differential gene expression. A major switch point for regulating the expression of genes is at the level of transcription. Transcription of protein encoding genes in eukaryotes is orchestrated by a host of protein factors, including RNA polymerase II, general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH), coactivators, chromatin remodeling factors, and gene-specific transcriptional regulators (activators and repressors). The underlying goal of our work is to uncover molecular mechanisms governing human RNA polymerase II transcription. To this end, we use a combination of biochemistry, molecular biology, and molecular genetics to investigate mechanisms of transcriptional regulation at mammalian promoters.
Regulation of the Eukaryotic Transcription Cycle
The goal of this line of research is to understand what limits the rate and extent of transcription at human promoters and to understand how transcriptional activators stimulate distinct steps in the transcription reaction. We have reconstituted human RNA polymerase II transcription in vitro using highly purified transcription factors and used this transcription system to measure the kinetics of discrete steps of transcription: including, preinitiation complex formation, initiation, escape commitment, promoter escape, and transcript elongation. Our studies revealed that promoter escape can limit the rate of transcription (1,4). The finding that promoter escape is rate limiting in vitro suggests that this may be an important step for regulation at natural promoters in the human genome. In performing kinetic studies we identified a transition, termed escape commitment, which occurs after initiation and prior to promoter escape. Upon completion of escape commitment, ternary complexes are stable and slowly proceed forward through promoter escape. Escape commitment involves translocation between synthesis of the third and fourth phosphodiester bonds. We propose that a conformational change in ternary transcription complexes occurs during translocation after synthesis of a 4 nucleotide RNA. With a kinetic model for the transcription reaction in place, we now have the tools and level of understanding to examine the mechanisms by which transcriptional activators regulate the rates and extents of completion of distinct steps in human mRNA synthesis.
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. Work performed by immunologists has 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 350 bp of DNA surrounding the transcription start site. The IL-2 promoter, however, has a complex regulatory region that contains binding sites for at least four families of transcriptional regulatory proteins: NFAT, NF-kB, AP1, and OCT. The compact but complex character of this promoter makes it amenable to a variety of both in vitro and in vivo studies.
We are currently investigating the roles of both cis-regulatory elements and trans-regulatory factors (e.g., NFAT, AP1, and OCT) on transcription at the IL-2 promoter using in vitro transcription experiments and assays in T cells. One paradigm that has been revealed by studies performed in many laboratories is the importance of protein-protein contacts in mediating regulated transcription. It is thought that contact between a transcriptional activator and components of the transcription machinery represents 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 have revealed that the activators NFATp and cJun target coactivator subunits of the TFIID complex (3,5,6). Human TAFII130 is a coactivator for NFATp while cJun derepresses transcription by binding the N-terminal region of TAFII250. The functions of the interactions found are being investigated through the combination of mutagenesis and transcription assays. Interesting observations made in these in vitro experiments are being complemented with in vivo experiments in order to test the effects of specific protein-protein interactions on the development of the mammalian immune system.
Selected Publications
1. Kugel, J.F. and J.A. Goodrich. (1998). Promoter escape limits the rate of transcription from the adenovirus major late promoter on negatively supercoiled templates. Proc. Natl. Acad. Sci. 95:9232-9237.
2. Galasinski, S.K., T.N. Lively, A. Grebe de Barron, and J.A. Goodrich. (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.
3. Kim, L.J., H.A. Ferguson, A.G. Seto, and J.A. Goodrich. (2000). Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp. BMC Immunology. 1: 1.
4. Kugel, J.F. and J.A. Goodrich. (2000). A kinetic model for the early steps of RNA synthesis by human RNA polymerase II. J. Biol. Chem. 275:40483-40491.
5. Kim, L.J., A.G. Seto, T.N. Nguyen, and J.A. Goodrich. (2001). Human TAFII130 is a coactivator for NFATp. Mol. Cell. Biol. 21:3503-3513.
6. Lively, T.N., H.A. Ferguson, S.K. Galasinski, A.G. Seto, and J.A. Goodrich. (2001). cJun binds the N’Äëterminus of human TAFII250 to derepress RNA polymerase II transcription in vitro. J. Biol. Chem. In press.