A Role for Mediator Complex Subunit MED13L in Rb&Sol;E2F-Induced

Oncogene (2012) 31, 4709 --4717 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE A role for Mediator complex subunit MED13L in Rb/E2F-induced growth arrest SP Angus and JR Nevins The Rb/E2F pathway is deregulated in virtually all human tumors. It is clear that, in addition to Rb itself, essential cofactors required for transcriptional repression and silencing of E2F target genes are mutated or lost in cancer. To identify novel cofactors required for Rb/E2F-mediated inhibition of cell proliferation, we performed a genome-wide short hairpin RNA screen. In addition to several known Rb cofactors, the screen identified components of the Mediator complex, a large multiprotein coactivator required for RNA polymerase II transcription. We show that the Mediator complex subunit MED13L is required for Rb/E2F control of cell growth, the complete repression of cell cycle target genes, and cell cycle inhibition. Oncogene (2012) 31, 4709--4717; doi:10.1038/onc.2011.622; published online 16 January 2012 Keywords: tumor suppressor; cell cycle; senescence INTRODUCTION Typically, the levels of CDKN2A are greatly increased, leading to The retinoblastoma tumor suppressor (Rb) is a critical negative the accumulation of active, hypophosphorylated Rb. Senescent regulator of cellular proliferation that is frequently inactivated in cells characteristically exhibit flat-cell morphology and are positive 20 human cancer.1,2 In its active form, Rb binds to the members of for senescence-associated beta-galactosidase (SA-b-gal) activity. 21 the E2F family of transcription factors and either suppresses their Additionally, Narita et al. described the formation of senescence- transactivation function or assembles an active repressor com- associated heterochromatic foci at E2F promoters during V12 plex.3 E2F family members are essential regulators of genes Ras -induced senescence. Overexpression of the adenoviral required for DNA replication and mitosis.4--6 The transcriptional E1A protein, which targets Rb and its related family members, repression functions of Rb/E2F complexes have been shown to bypassed the senescent phenotype, alleviated E2F target gene reflect the recruitment of transcriptional co-repressors.3,7 --12 In this repression, and blocked the formation of senescence-associated manner, multi-protein transcriptional repressor complexes are heterochromatic foci. The evidence for senescence as a relevant 15,22 --24 guided to specific genes via the E2F binding sites in target gene tumor-suppressive mechanism in vivo is compelling. promoters. Multiple independent investigators have observed senescence The ability of Rb to bind stably to E2F is regulated by markers in premalignant murine lesions induced by activated 25 --29 phosphorylation. In response to mitogenic stimuli in G0, cyclin oncogenes. Furthermore, human benign melanocytic nevi, D/CDK4/6 complexes initiate the phosphorylation of Rb and premalignant lesions in the development of melanoma, possess 29 disrupt Rb/E2F interactions.1,5,6 The alleviation of Rb-mediated multiple characteristics of a senescent phenotype. The loss of repression of E2F enables activated transcription of requisite cell cooperating factors required for transcriptional repression and cycle genes and passage through the G1 --S transition. Deregulated heterochromatin formation initiated by Rb/E2F complexes could Rb/E2F control of cell proliferation is a common feature of cancer represent a mechanism for the progression from benign neoplasia and is underscored by the multitude of observed lesions in human to more advanced malignancies. Here, we performed a genome- tumors.1,2,6 Loss of Rb, loss of the CDK4/6 inhibitor, CDKN2A, and wide short hairpin RNA (shRNA) screen to identify factors critical in amplification/overexpression of CDK4 or cyclin D are examples of the induction and maintenance of the Rb/E2F-induced growth disruptions of cell growth control seen in human tumors. arrest state. Importantly, the loss of key mediators of transcriptional repression and gene silencing has also been shown to confer resistance to Rb/E2F-mediated cell cycle arrest and have been observed in RESULTS human tumors.13,14 A screen for Rb/E2F cofactors that maintain cell cycle arrest Previous studies from multiple independent laboratories have and senescence induction clearly implicated the Rb/E2F pathway in the maintenance of a In an effort to identify essential cofactors in the maintenance of stable cell cycle arrest state and the senescent phenotype in Rb/E2F-mediated cellular senescence, an allele of the large pocket 15 human cells. Cellular senescence was initially observed as a domain of Rb (amino acids 379--928) that harbors mutations in 16 barrier limiting the proliferative capacity of cells in culture. In critical serine and threonine residues, and is thereby refractory cultured human cells, the erosion of telomeres at chromosome to CDK phosphorylation and inactivation (phosphorylation-site ends leads to a p53-dependent response that arrests cell cycle mutant-Rb, PSM-Rb) was expressed in the T98G glioblastoma cell 15,17,18 progression. In addition to telomere shortening, senescence line via retroviral transduction. The large pocket domain of Rb has has been shown to be induced by oncogenic signaling, been demonstrated to be the minimal domain required for E2F 15,19 non-telomeric DNA damage and other cellular stresses. binding and growth suppression, and for tumor suppression in Duke Institute for Genome Sciences & Policy, Duke University Medical Center, Durham, NC, USA. Correspondence: Dr JR Nevins, Duke Institute for Genome Sciences & Policy, Duke University Medical Center, Durham, NC 27710, USA. E-mail: [email protected] Received 19 June 2011; revised and accepted 29 November 2011; published online 16 January 2012 MED13L requirement for Rb/E2F function SP Angus and JR Nevins 4710 100 SA-β-gal 80 pQCXIH pQCXIH-PSM-Rb 60 40 20 BrdU positive (%) 0 pQCXIH pQCXIH- PSM-Rb shLuc shLuc shPool 7 days 19 days post-infection post-infection pQCXIH pQCXIH-PSM-Rb pQCXIH-empty pQCXIH-PSM-Rb Identify shRNA clones shLuc shLuc Figure 1. shRNA screen design: active Rb expression induces stable cell cycle arrest and senescent phenotype. (a) T98G glioblastoma cells expressing the murine ecotropic receptor (T98G-EcoR) were infected with empty retrovirus or retrovirus producing a PSM-Rb. Three days post selection in hygromycin, cells were labeled with BrdU for 16 h, fixed, and BrdU incorporation was detected by immunofluorescence. At least 200 cells were counted per condition. Bar graphs represent mean±s.d. from two independent experiments for PSM-Rb. (b) T98G-EcoR cells treated as in (a) were stained to detect SA-beta-galatosidase activity. (c) The shRNA screen was designed to identify cells resistant to PSM-Rb expression in the shPool populations as compared with the control, shLuc (luciferase). (d) T98G-EcoR cells stably producing shRNA targeting firefly luciferase were infected with empty retrovirus or retrovirus producing PSM-Rb. Either 7 days (empty retrovirus) or 19 days (PSM-Rb retrovirus) post selection in hygromycin, dishes were fixed in methanol, stained with crystal violet and images were captured. mouse models.30,31 Previous studies have demonstrated that this luciferase (shLuc) formed very few colonies upon PSM-Rb mutant form of Rb represses transcription of E2F target genes and introduction compared with control retrovirus (Figure 1d). strongly enforces cell cycle arrest.32,33 Indeed, cells infected with Following the infection of T98G-EcoR populations harboring PSM-Rb-producing retrovirus fail to undergo DNA replication, as pools of shRNA clones, cells were selected with hygromycin measured by 5-bromo-2-deoxyuridine (BrdU) incorporation when for 19--21 days. As shown in Figure 2a, the proliferation of shLuc compared with control cells (Figure 1a). Consistent with prior cells was inhibited, whereas colony outgrowth could be observed observations, chronic PSM-Rb expression elicited stable growth more readily in the pooled shRNA populations. Cells were arrest and characteristics of the senescent phenotype. After 7 days harvested from each shRNA pool population, and genomic of hygromycin selection, population of cells expressing PSM-Rb DNA was prepared. PCR was performed to amplify candidate display a flat-cell morphology and are positive for SA-b-gal activity shRNAs and the resultant fragments were cloned and sequenced. (Figure 1b). In all, 20--30 sequencing reactions were performed from Based on the stability of the arrest state induced by PSM-Rb each shRNA pool (Figure 2b) and 292 specific candidate gene expression, we designed a protocol for a genome-wide shRNA targets were identified by Human BLAT Search of the UCSC screen to identify factors required for the induction and Genome Browser (http://www.genome.ucsc.edu; Supplementary maintenance of the arrest phenotype (Figure 1c). The retroviral Table 1). vector, pQCXIH, produces PSM-Rb and the hygromycin-resistance gene on a bicistronic transcript, ensuring that hygromycin- resistant cells have not lost PSM-Rb expression. The use of the Validation of the screen: identification of E2F5 PSM-Rb allele was intended to reduce the complexity of hits from With the expectation that the candidate genes included false the screen by (1) eliminating factors that might affect Rb positives, we initially sought to validate genes known to function phosphorylation, thereby targeting components of the active in the Rb/E2F pathway. Confirming the success of the

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