DDB 1-CUL 4 and MLL 1 Mediate Oncogene-Induced p 16 INK 4 a Activation

The induction of cellular senescence by oncogenic signals acts as a barrier to cellular transformation and is attained, in part, by the elevation of the p16 tumor suppressor gene. p16 expression is repressed epigenetically by Polycomb, but how p16 is induced is not known. We report here that the p16 locus is H3K4-methylated in highly expressing cells. H3K4 methyltransferase MLL1 directly binds to and is required, along with its core component RbBP5, for the induction of p16 by oncogenic Ras. We further show that damaged DNA binding protein DDB1 and CUL4, which assemble distinct E3 ubiquitin ligases by recruiting various WD40 proteins, act upstream of MLL1-mediated H3K4 methylation. We showed that CUL4A directly binds to p16 and that silencing DDB1 blocks Rasinduced p16 activation. Ras expression dissociates BMI1 from the p16 locus, whereas both CUL4 and MLL1 bind to the p16 locus similarly in both normal and oncogenic stimulated cells. These results suggest that DDB1-CUL4 and MLL1 complexes constitute a novel pathway that mediates p16 activation during oncogenic checkpoint response and is repressed by the polycomb repression complexes during normal growth of young cells. [Cancer Res 2009;69(5):1809–14] Introduction p16 encodes a specific inhibitor of the D-type cyclindependent kinases (CDK) Cdk4 and Cdk6 (CDK4/6) and is frequently mutated or inactivated in a wide range of human cancers (1, 2). Mice deficient for p16 are more prone to spontaneous cancers (3, 4) and have a reduced decline of stem and progenitor cell function during aging (5–7). In contrast to its well-established biochemical property as a CDK inhibitor and physiologic function in tumor suppression and cell aging, little is known about the molecular mechanism regulating the expression level of p16. INK4 proteins (p16 , p15 , p18 , and p19) are relatively stable and the primary regulation of INK4 is through transcriptional control. The expression of individual INK4 genes is distinctly regulated during development, in various adult tissues, and in response to different cellular conditions (1, 2). The expression of p16 is undetectable during embryogenesis and in young tissues (8, 9), due in large part to the transcriptional repression by the collaborative function of Polycomb repression complex (PRC) and RB family proteins (10–14). The expression of p16 increases markedly with aging in many tissues and contributes to an irreversible growth arrest, termed replicative senescence, in both differentiated and stem and progenitor cells during aging (5–7). Recently, hyperproliferative stimulation resulting from ectopic oncogene expression has been shown to cause similar cellular senescence, which is accompanied by the induction of p16 (15, 16). The molecular mechanism underlying oncogene-induced p16 activation is not known and is investigated in this study. Materials and Methods Antibodies and immunologic procedures. Antibodies to BMI1 (F6; Upstate), p16 (Ab-4; Neomarkers), MLL1 (BETHYL Laboratories, Inc.), RbBP5 (BETHYL Laboratories, Inc.), WDR5 (ab22512; abcam), H-Ras (OP23; Calbiochem), 3m-H3K4 (ab8580; abcam), 3m-H3K27 (Upstate), tubulin (DM1A; Neomarkers), and actin (C-11; Santa Cruz) were purchased commercially. Polyclonal antibodies to CUL4A (MADEAPRKGSFSALVGRTNG) and DDB1 (REKEFNKGPWKQENVE) were raised by immunizing rabbits with synthetic peptides conjugated to keyhole limpet hemocyanin. Rabbit polyclonal antibody to MLL1-C was kindly provided by Dr. Y. Dou (University of Michigan, Ann Arbor, MI). Cell culture, transfection, and retroviral transduction. The early passage normal human diploid fetal lung fibroblast WI38 cells were purchased from American Type Culture Collection. WI38 cells were cultured in MEM supplemented with 10% fetal bovine serum (FBS), and 293T cells was cultured in DMEM supplemented with 10% FBS. Cell transfection was performed using calcium phosphate buffer. WI38 cells were infected with a retrovirus expressing human papillomavirus oncoprotein E7 that binds to and functionally inactivate Rb family proteins. Retroviruses encoding shRNAs silencing MLL1, WDR5, RbBP5, Menin , and control GFP were constructed by ligating respective oligonucleotides (MLL1 , GAAGTCAGAGTGCGAAGTC; WDR5 , CACCTGTGAAGCCAAACTA; RbBP5 , GAGCCGAGATGGTCATAAA; Menin , GTCGCAAGTGCAGATGAAG; GFP, GCTACGTCCAGGAGCGCAC) into a pSuper-retro vector. pBabe H-Ras was kindly provided by Channing Der (University of North Carolina at Chapel Hill, Chapel Hill, NC). Retrovirus expressing shRNA silencing DDB1 has been described previously (17). The retroviral production and transduction were performed as previous described (13). Quantitative reverse transcription-PCR. Total RNA was extracted by RNeasy (Qiagen), and 1 Ag was used for cDNA synthesis primed with Oligo(dT)20 primers (Invitrogen). The cDNA was added to a quantitative reverse transcription-PCR mixture that contained 1 SYBR Green PCR master mix (Applied Biosystems) and 500 nmol/L gene-specific primers. Assays were performed in triplicate on a 7900 HT sequence detection system (Applied Biosystems). The PCR protocol comprised incubations at 50jC for 2 min and 95jC for 10 min, followed by 40 cycles, each consisting of 95jC for 15 s and 60jC for 1 min. The expression level of each gene was normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Specific PCR pairs used in this study have been described (13). Chromatin immunoprecipitation assay. Chromatin immunoprecipitation (ChIP) assays were performed as previous described (13). Briefly, 5 10 WI38 cells were treated with 1% formaldehyde for 10 min. The cross-linking was stopped by the addition of 0.125 mol/L glycine and incubated for 5 min. The cells were lysed with cell lysis buffer on ice Requests for reprints: Yue Xiong, University of North Carolina, 22-012 Lineberger Cancer Center, CB# 7295, Chapel Hill, NC 27599-7295. Phone: 919-962-2142; Fax: 919966-8799; E-mail: yxiong@email.unc. edu. I2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-2739 www.aacrjournals. org 1809 Cancer Res 2009; 69: (5). March 1, 2009 Research Article Research. on May 1, 2017. © 2009 American Association for Cancer cancerres.aacrjournals. org Downloaded from Published OnlineFirst February 10, 2009; DOI: 10.1158/0008-5472.CAN-08-2739