Regulation of transcription by RNA polymerase II
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.
Non-coding RNA regulators of RNA polymerase II
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.
Single-molecule approaches to understand transcriptional regulation
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.
Transcriptional regulation by cJun and NFAT proteins
NFAT and AP-1 proteins such as cJun have an established role in mediating transcription activation as T cells respond to bacterial and viral infection. We have investigated the roles of these factors and their cis-regulatory elements at the model 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 such as breast cancer, in which cooperation between NFAT and cJun does not yet have an established role.
Walters, R.D., Drullinger, L.F., Kugel, J.F., Goodrich, J.A. (2013). NFATc2 recruits cJun homodimers to an NFAT site to synergistically activate interleukin-2 transcription. Mol Immunol. 56: 48-56
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.
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.
Yakovchuk, P., Goodrich, J.A., Kugel, J.F. (2011) B2 RNA represses TFIIH phosphorylation of RNA polymerase II.. Transcription. Jan;2(1):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 interleukin-2 transcription. Mol Immunol. 47(14):2314-22
Yakovchuk, P., Gilman B., Goodrich, J.A., Kugel, J.F. (2010) RNA polymerase II and TAFs undergo a slow isomerization after the polymerase is recruited to promoter-bound TFIID.. J Mol Biol. 397(1):57-68
Wagner, S.D., Kugel, J.F., Goodrich, J.A. (2010) TFIIF facilitates dissociation of RNA polymerase II from noncoding RNAs that lack a repression domain.. Mol Cell Biol.30(1):91-7
Ponicsan S.L., Kugel J.F., and Goodrich J.A., (2010) Genomic gems: SINE RNAs regulate mRNA production Curr Opin Genet Dev(2):149-55
Kugel J.F., and Goodrich J.A.(2010) Dampening DNA binding: a common mechanism of transcriptional repression for both ncRNAs and protein domains RNA Bio(3):305-9
Yakovchuk, P., Goodrich, J.A., 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 U S A. 106(14):5569-74
Goodrich J.A., Kugel J.F,. (2009) From bacteria to humans, chromatin to elongation, and activation to repression: The expanding roles of noncoding RNAs in regulating transcription. Crit Rev Biochem Mol Biol. 44(1):3-15.
Walters, R.D., Kugel J.F., and Goodrich J.A., (2009) InvAluable Junk: The Cellular Impact and Function of Alu and B2 RNAs. IUBMB Life(8):831-7
Gilman, B., Drullinger, L.F., Kugel, J.F., 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(14):9093-8.
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 RNA Is a Modular Transacting Repressor of mRNA Transcription during Heat Shock. Mol. Cell. 29, 499-509.
Wagner, S.D., Goodrich, J.A. and Kugel, J.F. (2008). RNA and the Regulation of Gene Expression; Chapter 9: The Role of Non-Coding RNAs in Controlling Mammalian RNA Polymerase II Transcription. Caister Academic Press.
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., Halsey W.A., Betterton M.D., Perkins T.T., Kugel J.F., Goodrich J.A. (2007) TFIIA changes the conformation of the DNA in TBP/TATA complexes and increases their kinetic stability. J Mol Biol. 21;372(3):619-32.
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.
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.
Goodrich, J.A. and Kugel, J.F. (2006). Binding and Kinetics for Molecular Biologists. Cold Spring Harbor Laboratory Press. 182 pages. Computer simulations: http://kinetics.cshl.edu/.
Goodrich, J.A. and Kugel, J.F. (2006). Noncoding RNA regulators of RNA polymerase II transcription. Nature Rev. Mol. Cell Biol. 7: 612-616.
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.
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 Structural and Molecular Biology. 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 Structural and Molecular Biology 11: 816-821.