Abstract
It is not well understood how the human Mediator complex, transcription factor IIH and RNA polymerase II (Pol II) work together with activators to initiate transcription. Activator binding alters Mediator structure, yet the functional consequences of such structural shifts remain unknown. The p53 C terminus and its activation domain interact with different Mediator subunits, and we find that each interaction differentially affects Mediator structure; strikingly, distinct p53–Mediator structures differentially affect Pol II activity. Only the p53 activation domain induces the formation of a large pocket domain at the Mediator–Pol II interaction site, and this correlates with activation of stalled Pol II to a productively elongating state. Moreover, we define a Mediator requirement for TFIIH-dependent Pol II C-terminal domain phosphorylation and identify substantial differences in Pol II C-terminal domain processing that correspond to distinct p53–Mediator structural states. Our results define a fundamental mechanism by which p53 activates transcription and suggest that Mediator structural shifts trigger activation of stalled Pol II complexes.
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References
Holstege, F.C. et al. Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95, 717–728 (1998).
Fondell, J.D., Ge, H. & Roeder, R.G. Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex. Proc. Natl. Acad. Sci. USA 93, 8329–8333 (1996).
Esnault, C. et al. Mediator-dependent recruitment of TFIIH modules in preinitiation complex. Mol. Cell 31, 337–346 (2008).
Davis, J.A., Takagi, Y., Kornberg, R.D. & Asturias, F.A. Structure of the yeast RNA polymerase II holoenzyme: mediator conformation and polymerase interaction. Mol. Cell 10, 409–415 (2002).
Johnson, K.M., Wang, J., Smallwood, A., Arayata, C. & Carey, M. TFIID and human mediator coactivator complexes assemble cooperatively on promoter DNA. Genes Dev. 16, 1852–1863 (2002).
Naar, A.M., Taatjes, D.J., Zhai, W., Nogales, E. & Tjian, R. Human CRSP interacts with RNA polymerase II CTD and adopts a specific CTD-bound conformation. Genes Dev. 16, 1339–1344 (2002).
Tyner, S.D. et al. p53 mutant mice that display early ageing-associated phenotypes. Nature 415, 45–53 (2002).
Maier, B. et al. Modulation of mammalian life span by the short isoform of p53. Genes Dev. 18, 306–319 (2004).
Vousden, K.H. & Prives, C. Blinded by the light: the growing complexity of p53. Cell 137, 413–431 (2009).
Espinosa, J.M. Mechanisms of regulatory diversity within the p53 transcriptional network. Oncogene 27, 4013–4023 (2008).
Wang, G. et al. Mediator requirement for both recruitment and postrecruitment steps in transcription initiation. Mol. Cell 17, 683–694 (2005).
Drane, P., Barel, M., Balbo, M. & Frade, R. Identification of RB18A, a 205 kDa new p53 regulatory protein which shares antigenic and functional properties with p53. Oncogene 15, 3013–3024 (1997).
Ito, M. et al. Identity between TRAP and SMCC complexes indicates novel pathways for the function of nuclear receptors and diverse mammalian activators. Mol. Cell 3, 361–370 (1999).
Jimenez, G.S. et al. A transactivation-deficient mouse model provides insights into Trp53 regulation and function. Nat. Genet. 26, 37–43 (2000).
Johnson, T.M., Hammond, E.M., Giaccia, A. & Attardi, L.D. The p53QS transactivation-deficient mutant shows stress-specific apoptotic activity and induces embryonic lethality. Nat. Genet. 37, 145–152 (2005).
Lin, J., Chen, J., Elenbaas, B. & Levine, A.J. Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein. Genes Dev. 8, 1235–1246 (1994).
Nister, M. et al. p53 must be competent for transcriptional regulation to suppress tumor formation. Oncogene 24, 3563–3573 (2005).
Taatjes, D.J., Naar, A.M., Andel, F., Nogales, E. & Tjian, R. Structure, function, and activator-induced conformations of the CRSP coactivator. Science 295, 1058–1062 (2002).
Johnson, K.M., Wang, J., Smallwood, A. & Carey, M. The immobilized template assay for measuring cooperativity in eukaryotic transcription complex assembly. Methods Enzymol. 380, 207–219 (2004).
Knuesel, M.T., Meyer, K.D., Bernecky, C. & Taatjes, D.J. The human CDK8 subcomplex is a molecular switch that controls Mediator co-activator function. Genes Dev. 23, 439–451 (2009).
Espinosa, J.M., Verdun, R.E. & Emerson, B. p53 functions through stress- and promoter-specific recruitment of transcription initiation components before and after DNA damage. Mol. Cell 12, 1015–1027 (2003).
Gaudreau, L., Adam, M. & Ptashne, M. Activation of transcription in vitro by recruitment of the yeast RNA polymerase II holoenzyme. Mol. Cell 1, 913–916 (1998).
Keaveney, M. & Struhl, K. Activator-mediated recruitment of the RNA polymerase II machinery is the predominant mechanism for transcriptional activation in yeast. Mol. Cell 1, 917–924 (1998).
Yang, F., DeBeaumont, R., Zhou, S. & Naar, A.M. The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator. Proc. Natl. Acad. Sci. USA 101, 2339–2344 (2004).
Mittler, G. et al. A novel docking site on Mediator is critical for activation by VP16 in mammalian cells. EMBO J. 22, 6494–6504 (2003).
Rachez, C. et al. Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 398, 824–828 (1999).
Li, P. et al. Regulation of p53 target gene expression by peptidylarginine deiminase 4. Mol. Cell. Biol. 28, 4745–4758 (2008).
Glover-Cutter, K., Kim, S., Espinosa, J.M. & Bentley, D.L. RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes. Nat. Struct. Mol. Biol. 15, 71–78 (2008).
Donner, A.J., Szostek, S., Hoover, J.M. & Espinosa, J.M. CDK8 is a stimulus-specific positive coregulator of p53 target genes. Mol. Cell 27, 121–133 (2007).
Saunders, A., Core, L.J. & Lis, J.T. Breaking barriers to transcription elongation. Nat. Rev. Mol. Cell Biol. 7, 557–567 (2006).
Taatjes, D.J., Schneider-Poetsch, T. & Tjian, R. Distinct conformational states of nuclear receptor-bound CRSP–Med complexes. Nat. Struct. Mol. Biol. 11, 664–671 (2004).
Chi, T. & Carey, M. Assembly of the isomerized TFIIA–TFIID–TATA ternary complex is necessary and sufficient for gene activation. Genes Dev. 10, 2540–2550 (1996).
Horikoshi, M., Hai, T., Lin, Y.S., Green, M.R. & Roeder, R.G. Transcription factor ATF interacts with the TATA factor to facilitate establishment of a preinitiation complex. Cell 54, 1033–1042 (1988).
Guermah, M., Malik, S. & Roeder, R.G. Involvement of TFIID and USA components in transcriptional activation of the human immunodeficiency virus promoter by NF-κB and Sp1. Mol. Cell. Biol. 18, 3234–3244 (1998).
Roberts, S.G.E. & Green, M.R. Activator-induced conformational change in general transcription factor TFIIB. Nature 371, 717–720 (1994).
Xiao, H. et al. Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol. Cell. Biol. 14, 7013–7024 (1994).
Thut, C.J., Chen, J.L., Klemm, R. & Tjian, R. p53 transcriptional activation mediated by coactivators TAFII40 and TAFII60. Science 267, 100–104 (1995).
Lu, H. & Levine, A.J. Human TAFII31 protein is a transcriptional coactivator of the p53 protein. Proc. Natl. Acad. Sci. USA 92, 5154–5158 (1995).
Kornberg, R.D. Mediator and the mechanism of transcriptional activation. Trends Biochem. Sci. 30, 235–239 (2005).
Malik, S. & Roeder, R.G. Dynamic regulation of pol II transcription by the mammalian Mediator complex. Trends Biochem. Sci. 30, 256–263 (2005).
Di Lello, P. et al. Structure of the Tfb1/p53 complex: insights into the interaction between the p62/Tfb1 subunit of TFIIH and the activation domain of p53. Mol. Cell 22, 731–740 (2006).
Li, A.G. et al. An acetylation switch in p53 mediates holo-TFIID recruitment. Mol. Cell 28, 408–421 (2007).
Momand, J., Zambetti, G.P., Olson, D.C., George, D. & Levine, A.J. The MDM-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 69, 1237–1245 (1992).
Van Orden, K., Giebler, H.A., Lemasson, I., Gonzales, M. & Nyborg, J.K. Binding of p53 to the KIX domain of CREB binding protein. A potential link to human T-cell leukemia virus, type I-associated leukemogenesis. J. Biol. Chem. 274, 26321–26328 (1999).
Ventura, A. et al. Restoration of p53 function leads to tumour regression in vivo. Nature 445, 661–665 (2007).
Xue, W. et al. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature 445, 656–660 (2007).
McKinney, K., Mattia, M., Gottifredi, V. & Prives, C. p53 linear diffusion along DNA requires its C terminus. Mol. Cell 16, 413–424 (2004).
Okorokov, A.L. et al. The structure of p53 tumor suppressor protein reveals the basis for its functional plasticity. EMBO J. 25, 5191–5200 (2006).
Kettenberger, H., Armache, K. & Cramer, P. Complete RNA polymerase II elongation complex structure and its interaction with NTP and TFIIS. Mol. Cell 16, 955–965 (2004).
Batta, K. & Kundu, T.K. Activation of p53 function by human transcriptional coactivator PC4: role of protein-protein interaction, DNA bending, and posttranslational modifications. Mol. Cell. Biol. 27, 7603–7614 (2007).
McKinney, K. & Prives, C. Efficient specific DNA binding by p53 requires both its central and C-terminal domains as revealed by studies with high-mobility group 1 protein. Mol. Cell. Biol. 22, 6797–6808 (2002).
Gomes, N.P. et al. Gene-specific requirement for P-TEFb activity and RNA polymerase II phosphorylation within the p53 transcriptional program. Genes Dev. 20, 601–612 (2006).
Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996).
Bottcher, B., Wynne, S.A. & Crowther, R.A. Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature 386, 88–91 (1997).
De Carlo, S., El-Bez, C., Alvarez-Rua, C., Borge, J. & Dubochet, J. Cryo-negative staining reduces electron-beam sensitivity of vitrified biological particles. J. Struct. Biol. 138, 216–226 (2002).
Ohi, M., Li, Y., Cheng, Y. & Walz, T. Negative staining and image classification—powerful tools in modern electron microscopy. Biol. Proced. Online 6, 23–34 (2004).
Acknowledgements
We thank A. Donner and J. Espinosa for help with the ChIP assays, RT-qPCR experiments and other useful advice, C. Schwartz and M. Morphew for assistance with electron microscopy data collection, J. Blaydes (University of Southampton) for the gift of the HDM2 promoter fragment and J. Goodrich and J. Espinosa for helpful comments on the manuscript. Protein expression in insect cells was completed at the Tissue Culture Core Facility at the University of Colorado Cancer Center (Support Grant #P30 CA046934). This work was supported by the US National Cancer Institute (R01 CA127364) and the Ellison Medical Foundation. K.D.M. and C.B. were supported in part by US National Institutes of Health grant T32 GM065103.
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K.D.M. designed, analyzed and performed most experiments and helped write the paper; S.-c.L. completed immobilized template assays; C.B. assisted with cryo-EM, purified Pol II and provided Mediator–Pol II electron microscopy data; Y.G. mapped Med1 binding domain to p53CTD; D.J.T. designed and performed experiments and helped write the paper.
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Supplementary Figures 1–15, Supplementary Note, Supplementary Results and Supplementary Methods (PDF 2035 kb)
Supplementary Video 1
P53AD-Mediator (MP4 959 kb)
Supplementary Video 2
P53CTD-Mediator (MP4 1921 kb)
Supplementary Video 3
WT p53-Mediator (MP4 1384 kb)
Supplementary Video 4
P53ΔCTD-Mediator (MP4 1016 kb)
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Meyer, K., Lin, Sc., Bernecky, C. et al. p53 activates transcription by directing structural shifts in Mediator. Nat Struct Mol Biol 17, 753–760 (2010). https://doi.org/10.1038/nsmb.1816
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DOI: https://doi.org/10.1038/nsmb.1816
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