麻豆淫院

Mammalian mRNA Transcription and its Regulation

Controlling gene expression is essential to growth, development, and sustained life. A critical control point for regulating gene expression is at the level of transcription. The proper regulation of transcription is essential for maintaining normal pathways of cell growth and differentiation, thereby avoiding the rampant cell proliferation observed in tumors. 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, gene-specific transcriptional regulatory proteins (activators and repressors), as well as non-coding RNAs. The underlying goal of our work is to uncover molecular mechanisms governing mammalian RNA polymerase II transcription and its regulation. To this end, we use a combination of biochemistry, molecular biology, molecular genetics, cell-based assays, genomics, and single molecule techniques to investigate mechanisms of mammalian transcriptional regulation.

Regulation of RNA Polymerase II by Non-coding RNAs

All cells respond to stress, and do so in part by altering gene expression. When eukaryotic cells are subjected to heat shock, general RNA polymerase II transcription decreases at the same time as transcription of a set of heat shock specific genes increases. Two non-coding RNAs (mouse B2 RNA and human Alu RNA) are transcriptionally upregulated upon heat shock. We have found that these ncRNAs bind RNA polymerase II with high affinity (low nM) and block the formation of functional initiation complexes in vitro. Our studies show that each of the ncRNAs binds the catalytic cleft of RNA polymerase II and that it is recruited with the polymerase into complexes assembling at promoters where it keeps the polymerase from properly engaging the DNA. Surprisingly, B2 RNA serves as a substrate and a template for the RNA-dependent RNA polymerase (RdRP) activity of RNA polymerase II. Specifically, RNA polymerase II adds 18 nucleotides of defined sequence to the 3' end of B2 RNA, which results in a dramatic decrease in the stability of the ncRNA in cells. We are now studying the function of B2 RNA and Alu RNA as transcriptional repressors genome-wide during heat shock, investigating the roles of these ncRNAs in controlling host cell transcription during herpes virus infection, and determining the general role of the RNA polymerase II RdRP activity in mammalian cells.

Regulation of Human mRNA Transcription by NFAT and AP-1 Proteins

The mammalian immune system represents a unique model for studying the importance of transcriptional regulation in governing cell growth and differentiation. 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. We have investigated the roles of both cis-regulatory elements and trans-regulatory factors (e.g., NFAT, AP1) in transcription at the IL-2 promoter using in vitro transcription experiments and assays in T cells. We have found that NFAT1 and cJun homodimers have a unique ability to interact with one another and that this interaction is critical for the synergistic activation of IL-2 transcription. We are now studying the role of these transcription factors in mediating synergy at other promoters and in other biological systems.

Single Molecule Approaches to Understanding Protein-Nucleic Acid Binding and the RNA polymerase II Transcription Reaction

The goal of this line of research is to investigate the assembly mechanism, dynamics, and heterogeneity of human transcription factor/DNA complexes, and how these parameters contribute to transcriptional control. Single-molecule studies have emerged as essential contributors to understanding the dynamic behavior, conformational states, and heterogeneity of biological complexes, thus allowing unprecedented insight into their function. Single-molecule experiments complement the knowledge gained from ensemble biochemical experiments by allowing the observation of sub-populations of molecules that exist in distinct states and also the measurement of dynamic behavior in individual molecules, which are obscured by averaging across all the molecules present in an ensemble. We are leveraging the unique abilities of the techniques of single-molecule fluorescence co-localization and single-molecule fluorescence resonance energy transfer (smFRET), which together can provide unprecedented insight into the biological function of nucleoprotein complexes. We are using these techniques to investigate the assembly and activity of complexes containing promoter DNA, transcriptional regulatory proteins, general transcription factors, and RNA polymerase II.

Research

Ponicsan, S.L., Houel, S., Old, W.M., Ahn, N.G., Goodrich, J.A., Kugel, J.F. (2013). The non-coding B2 RNA binds to the DNA cleft and active site region of RNA polymerase II. J. Mol. Biol. Epub ahead of print.

Wagner, S.D., Yakovchuk, P., Gilman, B., Poniscan, S.L., Drullinger, L.F., Kugel, J.F., Goodrich, J.A. (2013). Mammalian RNA polymerase II acts as an RNA-dependent RNA polymerase to extend and destabilize a non-coding RNA. EMBO J. Epub ahead of print.

Kassube, S.A., Fang, J., Grob, P., Yakovchuk, P., Goodrich, J.A., Nogales, E. (2012). Structural insights into transcriptional repression by ncRNAs that bind to human Pol II. J. Mol. Biol. Epub ahead of print.听

Blair, R.H. Goodrich, J.A., and Kugel, J.F. (2012). Single molecule FRET shows uniformity in TBP-induced DNA bending and heterogeneity in bending kinetics. Biochemistry. 51: 7444-7455.

Dridi, S., Hirano, Y., Tarallo, V., Kim, Y., Fowler, B.J., Ambati, B.K., Bogdanovich, S., Chiodo, V.A., Hauswirth, W.W., Kugel, J.F., Goodrich, J.A., Ponicsan, S.L., Hinton, D.R., Kleinman, M.E., Baffi, J., Gelfand, B.D., and Ambati, J. (2012). ERK1/2 activation is a therapeutic target in age-related macular degeneration. Proc. Natl. Acad. Sci. USA. 109:13781-13786.听

Tarallo, V., Hirano, Y., Gelfand, B.D., Dridi, S., Kim, Y., Kerur, N., Cho, W.G., Kaneko, H., Fowler, B.J., Bogdanovich, S., Albuquerque, R.J.C., Hauswirth, W.W., Chiodo, V.A., Kugel, J.F., Goodrich, J.A., Ponicsan, S.L., Chaudhuri, G., Dunaief, J., Ambati, B.K., Ogura, Y., Yoo, J.W., Lee, D.-K., Provost, P., Hinton, D.R., N煤帽ez, G., Baffi, J., Kleinman, M.E., and Ambati, J. (2012). DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88. Cell. 149:847-859.

Kaneko, H., Dridi, S., Tarallo, V., Fowler, B.J., Gelfand, B.D., Cho, W.G., Kleinman, M.E., Ponicsan, S.L., Hauswirth, W.H., Chiodo, V.A., Karik贸, K., Yoo, J.W., Lee, D.-K., Hadziahmetovic, M., Song, Y., Chaudhuri, G., Buaas, F.W., Braun, R.E., Hinton, D.R., Zhang, Q., Grossniklaus, H.E., Provis, J.M., Madigan, M.C., Milam, A.H., Justice, N.L., Albuquerque, R.J.C., Blandford, A.D., Bogdanovich, S., Hirano, Y., Witta, J., Fuchs, E., Littman, D.R., Ambati, B.K., Rudin, C.M., Chong, M.M.W., Provost, P., Kugel, J.F., Goodrich, J.A., Dunaief, J.L., Baffi, J.Z., Ambati, J. (2011). DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration. Nature. 471:325-330.

Titov, D.V., Gilman, B., He, Q.-L., Bhat, S., Low, W.-K., Dang, Y., Smeaton, M., Demain, A.J., Miller, P.S., Kugel, J.F., Goodrich, J.A., Liu, J.O. (2011). XPB, a subunit of TFIIH, is a target of the natural product triptolide. Nature Chemical Biology. 7:182-188. PMID: 21278739

Yakovchuk, P., Goodrich J.A., and Kugel, J.F. (2011). B2 RNA represses TFIIH phosphorylation of RNA polymerase II. Transcription. 2:45-49.

Nguyen, T.N., Kim, L.J., Walters, R.D., Drullinger, L.F., Lively, T.N., Kugel, J.F., and Goodrich, J.A. (2010). The C-terminal region of human NFATc2 binds cJun to synergistically activate IL-2 transcription. Mol. Immunol. 47: 2314-2322.

Yakovchuk, P., Gilman, B, Goodrich, J.A., and Kugel, J.F. (2010). RNA polymerase II and TFIID undergo a rate limiting isomerization after recruitment to promoter DNA. J. Mol. Biol. 397: 57-68.

Wagner, S.D., Kugel, J.F., Goodrich, J.A. (2010). TFIIF facilitates dissociation of RNA polymerase II from ncRNAs that lack a repression domain. Mol. Cell. Biol. 30: 91-97.

Yakovchuk, P., Goodrich, J.A., and Kugel, J.F. (2009). B2 RNA and Alu RNA repress transcription by disrupting contacts between RNA polymerase II and promoter DNA within assembled complexes. Proc. Natl. Acad. Sci. USA. 106: 5569-5574.听

Gilman, B., Drullinger, L.F., Kugel, J.F., and Goodrich, J.A. (2009). TATA binding protein and transcription factor IIB induce transcript slipping during early transcription by RNA polymerase II. J Biol Chem. 284: 9093-9098.听

Mariner, P.D., Walters, R.D., Espinoza, C.A., Drullinger, L.F., Wagner, S.D., Kugel, J.F., and Goodrich, J.A. (2008). Human Alu ncRNA is a modular transacting repressor of mRNA transcription during heat shock. Mol. Cell. 29: 499-509.

Hieb, A.R., Halsey, W.A., Betterton, M., Perkins, T., Kugel, J.F., and Goodrich, J.A. (2007). TFIIA changes the conformation of the DNA in TBP/TATA complexes and increases their kinetic stability. J. Mol. Biol. 372: 619-632.

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.

Espinoza, C.A., Goodrich, J.A., and Kugel, J.F. (2007). Characterization of the structure, function and mechanism of B2 RNA, an ncRNA repressor of RNA polymerase II transcription. RNA. 13: 583-596.

Hofmann, W.A., Vargas, G.M., Ramchandran, R., Ljuba, S., Goodrich, J.A., and de Lanerolle, P. (2006). Nuclear myosin I is necessary for the formation of the first phosphodiester bond by RNA polymerase II. J. Cell. Biochem. 99: 1001-1009.

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.

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.

Hofmann, W.A., Stojiljkovic, L., Fuchsova, B., Vargas, G.M., Mavrommatis, E., Philimonenko, V., Kysela, K., Goodrich, J.A., Lessard, J.L., Hope, T.J., Hozak, P. and de Lanerolle, P. (2004). Actin is part of pre-initiation complexes and is necessary for transcription by RNA polymerase II. Nature Cell Biol. 6: 1094-1101.

Espinoza, C.A., Allen, T.A., Hieb, A.R., Kugel, J.F., and Goodrich, J.A. (2004). B2 RNA binds directly to RNA polymerase II to repress transcript synthesis. Nature Struct. Mol. Biol. 11: 822-829.

Allen, T.A., Von Kaenel, S., Goodrich, J.A., and Kugel, J.F. (2004). The SINE encoded mouse B2 RNA represses mRNA transcription in response to heat shock. Nature Struct. Mol. Biol. 11: 816-821.

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.

Galasinski, S.C., Resing, K.A., Goodrich, J.A., and Ahn, N.G. (2002). Phosphatase inhibition leads to histone deacteylase 1/2 phosphorylation and disruption of co-repressor interactions. J. Biol. Chem. 277: 19618-19626.

Ehley, J.A., Melander, C., Herman, D., Baird, E.E., Ferguson, H.A., Goodrich, J.A., Dervan, P.B., and Gottesfeld, J.M. (2002). Promoter scanning for transcription inhibition with DNA-binding polyamides. Mol. Cell. Biol. 22: 1723-1733.

Kugel, J.F. and Goodrich, J.A. (2002). Translocation after synthesis of a four nucleotide RNA commits RNA polymerase II to promoter escape. Mol. Cell. Biol. 22: 762-773.

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 cFos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro. Nucleic Acids Res. 29: E98 (6 pages).听

Lively, T.N., Ferguson, H.A., Galasinski, S.K., Seto, A.G., and Goodrich, J.A. (2001). cJun binds the N-terminus of human TAF250 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.

Kugel, J.F. and Goodrich, J.A. (2000). A kinetic model for the early steps of RNA synthesis by human RNA polymerase II. J. Biol. Chem. 275: 40483-40491.

Kim, L.J., Ferguson, H.A., Seto, A.G., and Goodrich, J.A. (2000). Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp. BMC Immunology 1: 1 (10 pages).

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.

Kugel, J.F. and Goodrich, J.A. (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.

Reviews, Methods, Chapters, News and Views

Blair, R.H. Goodrich, J.A., and Kugel, J.F. 听(2013). 听Using FRET to monitor protein-induced DNA bending: the TBP-TATA complex as a model system. 听Methods Mol. Biol. 听997:203-215.

Kugel, J.K. and Goodrich J.A. 听(2013). 听The regulation of mammalian mRNA transcription by long non-coding RNAs: Recent discoveries and current concepts. 听Epigenomics. 听5:95-102.

Kugel, J.F. and Goodrich, J.A. 听(2012). 听Non-coding RNAs: key regulators of mammalian transcription. 听Trends Biochem. Sci. 听37:144-151.

Goodrich, J.A. and Tjian, R. 听(2010). 听Unexpected roles for core promoter recognition factors in cell-type specific transcription and gene regulation. 听Nature Reviews Genetics. 听11: 549-558.

Goodrich, J.A. and Kugel, J.F. 听(2010). 听Dampening DNA binding: 听an effective means to control transcription for both ncRNAs and protein domains. 听RNA Biology. 听7: 305-309.

Goodrich, J.A. and Kugel, J.F. 听(2010). 听Genome-wide insights into eukaryotic transcriptional control. 听Genome Biology. 听11: 305.

Ponicsan, S.L., Kugel, J.F., and Goodrich, J.A. 听(2010). 听Genomic gems: 听SINE RNAs regulate mRNA production. 听Curr. Opin. Genetics & Dev. 听20: 149-155.

Walters, R.D., Kugel, J.F., and Goodrich, J.A. 听(2009). 听InvAluable Junk: 听The cellular impact and function of Alu and B2 RNAs. 听IUBMB Life. 听61: 831-837.

Kugel, J.F. and Goodrich, J.A. 听(2009). 听In new company: 听U1 snRNA associates with TAF15. 听EMBO Reports. 听10: 454-456.

Goodrich, J.A. and Kugel, J.F. 听(2009). 听From bacteria to humans, chromatin to elongation, and activation to repression: The expanding roles of noncoding RNAs in regulating transcription. 听Crit. Rev. Biochem. Mol. Biol. 听44: 3-15. 听

Wagner, S.D., Kugel, J.F., and Goodrich, J.A. 听(2008). 听The role of non-coding RNAs in controlling mammalian RNA polymerase II transcription. 听In RNA and the regulation of gene expression: A hidden layer of complexity. 听Morris, K., ed. 听(Norwich: Horizon Scientific Press). 133-147.

Kugel, J.F. and Goodrich, J.A. 听(2007). 听An RNA transcriptional regulator templates its own regulatory RNA. 听Nature Chem. Biol. 听3: 89-90.

Goodrich, J.A. and Kugel, J.F. 听(2006). 听Noncoding RNA regulators of RNA polymerase II transcription. 听Nature Rev. Mol. Cell Biol. 听7: 612-616.

Kugel, J.F. and Goodrich, J.A. 听(2006). 听Beating the heat: a translation factor and an RNA mobilize the heat shock transcription factor HSF1. 听Mol. Cell. 听22: 153-154.

Nguyen, T.N. and Goodrich, J.A. 听(2006). 听Immobilized protein-protein interaction assays: Eliminating false positive interactions caused by contaminating nucleic acid. 听Nature Methods. 听3: 135-139.

Goodrich, J.A. and Tjian, R. 听(2006). 听Transcription: The never ending story. 听In Gene Expression and Regulation. Ma, J., ed. (Beijing: Higher Education Press and Springer).听

Kugel, J.F. and Goodrich, J.A. 听(2003). 听In vitro studies of the early steps of RNA synthesis by human RNA polymerase II. 听Methods Enzymol. 听370: 687-701.

Book

Goodrich, J.A. and Kugel, J.F.听 (2006).听听Binding and Kinetics for Molecular Biologists.听Cold Spring Harbor Laboratory Press.听 182 pages.听 Computer simulations:听.