The JNKs differentially regulate RNA polymerase III transcription by coordinately modulating the expression of all TFIIIB subunits Shuping Zhong and Deborah L. Johnson1 Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, and the Norris Comprehensive Cancer Center, 2011 Zonal Avenue, Los Angeles, CA 90033 Edited by Robert G. Roeder, The Rockefeller University, New York, NY, and approved June 12, 2009 (received for review May 4, 2009) RNA polymerase (pol) III-dependent transcription is subject to strin- III transcription has been extensively observed in transformed and gent regulation by tumor suppressors and oncogenic proteins and tumor cells supporting the idea that it plays a crucial role in enhanced RNA pol III transcription is essential for cellular transfor- tumorigenesis (11). Recent studies have demonstrated that en- mation and tumorigenesis. Since the c-Jun N-terminal kinases (JNKs) hanced RNA pol III transcription is required for promoting onco- display both oncogenic and tumor suppressor properties, the roles of genic transformation (12, 13). Thus, understanding the molecular these proteins in regulating RNA pol III transcription were examined. pathways by which this transcription process is controlled may In both mouse and human cells, loss or reduction in JNK1 expression provide useful therapeutic approaches for cancer. represses RNA pol III transcription. In contrast, loss or reduction in The TATA binding protein (TBP) associates with specific JNK2 expression induces transcription. The JNKs coordinately regu- proteins, TBP-associated factors (TAFs), to form at least 3 late expression of all 3 TFIIIB subunits. While JNK1 positively regulates distinct complexes, SL1, TFIID, and TFIIIB, which specify the TBP expression, the RNA pol III-specific factors, Brf1 and Bdp1, JNK2 role of TBP in the expression of genes directed by RNA pol I, negatively regulates their expression. Brf1 is coregulated with TBP II, and III, respectively. TFIIIB and the multiprotein complex, through the JNK target, Elk-1. Reducing Elk-1 expression decreases TFIIIC, are required to form specific transcription initiation Brf1 expression. Decreasing JNK1 expression reduces Elk-1 occupancy complexes on RNA pol III-dependent promoters. TFIIIB, com- at the Brf1 promoter, while decreasing JNK2 expression enhances posed of TBP and the RNA pol III-specific factors, Brf1 and recruitment of Elk-1 to the Brf1 promoter. In contrast, regulation of Bdp1, is often the target of regulatory responses that give rise to Bdp1 occurs through JNK-mediated alterations in TBP expression. altered RNA pol III transcription activity. Phosphorylation of Altered TBP expression mimics the effect of reduced JNK1 or JNK2 Brf1 and Bdp1 has been shown to regulate the assembly or levels on Bdp1 expression. Decreasing JNK1 expression reduces the function of the TFIIIB complex (7, 14–16). TBP expression is occupancy of TBP at the Bdp1 promoter, while decreasing JNK2 up-regulated by oncogenic Ras signaling and required to medi- expression enhances recruitment of TBP to the Bdp1 promoter. ate Ras transforming function (17). Together, these results provide a molecular mechanism for regulating TBP expression is induced through the activation of the epider- RNA pol III transcription through the coordinate control of TFIIIB mal growth factor receptor (EGFR1), requiring the activation of subunit expression and elucidate opposing functions for the JNKs in Ras and all 3 classes of MAPKs (18). JNK1 induces TBP expression, regulating a large class of genes that dictate the biosynthetic capacity while JNK2 represses TBP expression (19). This is accomplished of cells. through the opposing functions of the JNKs to modulate the phosphorylation state of Elk-1 and its recruitment to the TBP c-jun N-terminal kinases ͉ Elk-1 ͉ TATA-binding protein promoter. The ability of the JNKs to regulate TBP expression determines the proliferation rates of mouse embryo fibroblasts he c-jun N-terminal kinases (JNKs) are members of the mito- (MEFs). TBP-mediated changes in MEF proliferation are due, at Tgen-activated protein kinase (MAPK) family. JNKs are acti- least in part, to its ability to regulate c-jun transcription. TBP vated in response to stress, proinflammatory stimuli, and mitogenic expression can also be induced through AP-1 via the recruitment factors (1, 2). Three distinct genes encode the JNKs. JNK1 and of c-jun/c-fos to the TBP promoter (20). How alterations in cellular JNK2 are ubiquitously expressed, while JNK3 is more selectively TBP concentrations might affect the expression of other genes expressed in brain, heart, and testis (3). In addition, alternative involved in growth and proliferation remains to be determined. splicing gives rise to at least 10 JNK isoforms. JNK1 and JNK2 have As TBP is a component required for the transcription of RNA been shown to phosphorylate transcription factors such as c-Jun, pol III-dependent genes, we explored the possibility that the JNKs ATF-2, c-Fos, p53, Elk-1, and c-Myc. While initial studies indicated might modulate the activity of this class of genes. Our studies reveal that JNK1 and JNK2 possess redundant functions, subsequent that the JNKs differentially regulate RNA pol III transcription. studies support the idea that these proteins also have distinct JNK1 induces, while JNK2 represses, RNA pol III transcription. functions. The JNKs can induce both apoptotic and proliferative Notably, the expression of all 3 TFIIIB components, TBP, Bdp1, responses, depending on the physiological context and the time and Brf1, are coordinately regulated by these JNKs. Regulation of course of activation (4). The roles of these JNKs in tumorigenesis Bdp1 expression is modulated through JNK-mediated changes in are controversial (3). Depending on the cell type and stimulus, the TBP expression and by altered recruitment of TBP to the Bdp1 JNKs have been shown to stimulate oncogenic transformation or promoter. In contrast, Brf1 expression is regulated via JNK- act as tumor suppressors. Identifying the downstream molecular events selectively modulated by JNK1 and JNK2 will importantly clarify their roles in determining the oncogenic state of cells. Author contributions: S.Z. and D.L.J. designed research; S.Z. performed research; S.Z. and RNA polymerase (pol) III-dependent transcription and the D.L.J. analyzed data; and S.Z. and D.L.J. wrote the paper. production of its major products, 5S rRNAs and tRNAs, is tightly The authors declare no conflict of interest. regulated, controlling the translational and growth capacity of cells. This article is a PNAS Direct Submission. RNA pol III transcription is induced by oncogenic proteins, such as 1To whom correspondence should be addressed. E-mail: [email protected]. Ras (5), c-myc (6), and PI3 kinase (7), and repressed by tumor This article contains supporting information online at www.pnas.org/cgi/content/full/ suppressors Rb (8, 9), p53 (10), and PTEN (7). Enhanced RNA pol 0904843106/DCSupplemental. 12682–12687 ͉ PNAS ͉ August 4, 2009 ͉ vol. 106 ͉ no. 31 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904843106 Downloaded by guest on September 25, 2021 e mediated recruitment of Elk-1 to the Brf1 promoter. Together, p - - y / / Jnk2 -/- A t 1- 2- B Jnk1 -/- d k k il n n these results support the idea that JNK1 acts to promote RNA pol J J W -- III transcription, while JNK2 negatively regulate this process, JNK1 ++ JNK2 -- ++ thereby differentially controlling the biosynthetic capacity of cells. 5.0 6 1.5 Results 4 1.0 JNK1 and JNK2 Have Opposing Roles in Regulating RNA pol III 2.5 2 0.5 gene transcription gene transcription Transcription. The potential for JNK1 and JNK2 to regulate RNA gene transcription Fold change in tRNA in change Fold Fold changein tRNA polymerase (pol) III-dependent transcription was first assessed tRNA in change Fold 0.0 0 0.0 Jnk1-/- Jnk2 -/- WT JNK1 -+ JNK2 - + using MEFs that were deficient for either Jnk1 or Jnk2. A tRNA Jnk1-/- Jnk2-/- Ϫ/Ϫ gene reporter was transiently expressed in wild-type, Jnk1 , and JNK1 +- JNK2 +- Ϫ Ϫ Jnk2 / cells, and the amount of transcription was measured by a HA-JNK1 HA-JNK2 ribonuclease protection assay (Fig. 1A). The RNA produced con- β-actin β-actin tains a 12-bp insert that does not permit processing of the precursor C wild type Jnk1 -/- Jnk2 -/- transcript and allows it to be distinguished from endogenous Anisomycin - + - + - + transcripts (10). Compared with tRNA gene transcription activity in the wild-type cells, cells lacking Jnk1 displayed a decrease in RNA 3 3 3 pol III transcription, whereas transcription was enhanced in cells lacking Jnk2. To determine whether this difference in transcription 2 2 2 was a result of JNK status, expression plasmids for JNK1 or JNK2 1 1 1 were used to re-express these proteins. Expression of JNK1 in the transcriptiongene Ϫ/Ϫ Fold change in tRNA Jnk1 cells enhanced RNA pol III transcription, whereas expres- 0 0 0 Ϫ/Ϫ sion of JNK2 in the Jnk2 cells decreased transcription (Fig. 1B). Anisomycin - + -+ -+ MEFs were treated with anisomycin, a potent inducer of the JNKs, e p /- /- D ty 1- 2- to determine the role of activated JNKs on tRNA gene transcription ld K K i N N W J J (Fig. 1C). Anisomycin induced transcription in both the wild-type Nuclear exract and Jnk2Ϫ/Ϫ cells, but no effect on transcription was observed in the Ϫ/Ϫ 3 wild type Jnk1 cells. This suggests that while anisomycin induces tran- Jnk1-/- scription through JNK1, JNK2 acts to repress transcription in the Jnk2-/- absence of anisomycin-induced stimulation. To examine whether 2 JNK-mediated effects on transcription could be reproduced in 1 vitro, nuclear extracts were prepared, and transcription reactions gene transcription were performed. Extracts prepared from Jnk1Ϫ/Ϫ cells displayed a tRNA in change Fold decreased capacity for transcription, whereas extracts from the 0 Ϫ/Ϫ Jnk2 cells exhibited an increased capacity for transcription, 5 5.0 wild type E Jnk1-/- compared to extracts prepared from the wild-type cells (Fig.