Contamination of WI38 cells withANRILshort hairpin RNA retroviruses efficiently reducedANRILlevel (Physique 2a). increases the expression ofp15INK4B, but notp16INK4Aorp14ARF, and inhibits cellular proliferation. Finally, RNA immunoprecipitation demonstrates thatANRILbinds to SUZ12in vivo. Collectively, these results suggest a model in whichANRILbinds to and recruits PRC2 to repress the expression Banoxantrone D12 ofp15INK4Blocus. Keywords:long non-coding RNA,ANRIL, polycomb, p15INK4B, p16INK4A, ARF == Introduction == TheINK4A-ARF-INK4Bgene cluster occupies a 42 kb stretch on the human chromosome 9p21 and is homozygously deleted or expression silenced in a wide range of human cancers with an estimated frequency of ~40% (Sherr, 1998;Kim and Sharpless, 2006), representing one of the most frequently altered genes in human malignancy. Genetic analyses in mice with mutations targeting each gene individually support the tumor suppression function for all those three genes (Serranoet al., 1996;Kamijoet al., 1997;Krimpenfortet al., 2001,2007;Sharplesset al., 2001). Of these three tumor suppressors encoded by this gene cluster, both p15INK4Band p16INK4Afunction as theinhibitors of CDK4and CDK6, and thereby retain the growth suppressive activity of RB family proteins, whereas ARF, expressed from a distinct promoter and analternativereadingframe ofINK4A, binds to and inhibits the activity of oncoprotein MDM2 and thus stabilizes and activates p53. Clustering three distinct growth and tumor suppressors in such a small region may aid the coordinated integration of different growth insults into two major tumor suppression pathways in mammalian cells, one mediated by RB and the other by p53 (Gil and Peters, 2006). All the three genes are expressed at a barely detectable low level in young and normal cells, and are accumulated during cell aging or in cells insulted by hyperproliferative oncogenic stimuli (Zindyet al., 1997;Krishnamurthyet al., 2004;Gil and Peters, 2006), suggesting a common cellular mechanism and potentially coordinated regulation of these three genesto maintain aged cells in senescence and to arrest oncogenic-insulted cells from proliferation. The repression of this gene cluster involves polycomb proteins and histone H3 lysine27 (H3K27) trimethylation (Jacobset al., 1999;Itahanaet al., 2003;Brackenet al., 2007;Kotakeet al., 2007;Kiaet al., 2008;Aggeret al., 2009). The polycomb proteins Banoxantrone D12 form multimeric protein complexes, PRC (polycomb repression complex)-1 and -2 that are involved in the heritable stable repression of genes through histone modification. Previous genetic and biochemical studies suggest a hierarchical recruitment model where PRC2-mediated H3K27 methylation is required for recruitment of PRC1, which causes H2A-K119 ubiquitination on theHoxgene cluster both in travel and human cells (Wanget al., 2004;Caoet al., 2005). A critical issue for better understanding the regulation of theINK4A-ARF-INK4Bgene cluster is usually how PRC1 and PRC2 are recruited to this region. Several long non-coding RNAs (lncRNAs) have recently been reported to have a direct role in recruiting PRC2 complexes to specific loci and repress gene expression. These includeRepA, a 1.6-kb lncRNA involved in X-chromosome inactivation (Zhaoet al., 2008),Kcnq1ot1, a 91.5 kb lncRNA required for 10 paternally imprinted genes on 11p15 (Pandeyet al., 2008;Terranovaet al., 2008;Zhaoet al., Banoxantrone D12 2008), andHOTAIR, a 2.2-kb ncRNA involved in the repression of theHOXloci and promoting cancer metastasis (Rinnet al., 2007;Guptaet al., 2010). These findings led us to explore the possibility thatANRIL(antisensenon-codingRNA in theINK4locus), a 3834 bp transcript whose Banoxantrone D12 transcription is initiated from theINK4A-ARF-INK4Bgene cluster (Physique 1a), may be involved in the repression ofINK4A, ARFand/orINK4B.ANRILcontains 19 exons, a polyadenylation site in the last exon and spans over 126 kb of genomic sequence that is deleted in the melanomaneural system tumor syndrome family analyzed by previous study (Pasmantet al., 2007). Exon 1 ofANRILlocates between the promoter ofARFandINK4B, and is transcribed in the direction opposite from that of p15INK4B. Deletion of a 70-kb sequence in mouse chromosome 4 synteny to a 58-kb non-coding region in human chromosome 9p21 that includes seven exons ofANRILresulted in a significantly increased expression of bothp16INK4Aandp15INK4Bin several organs and tissues, but had no effect on other neighboring genes (Viselet al., 2010), providing a genetic evidence for the unfavorable regulation ofp16INK4Aandp15INK4Bby the ncRNA sequences expressed in this region. In this report, we show thatp15INK4Blocus is usually repressed by PRC2 proteins and thatANRILis required for the recruitment of PRC2 to and repression of thep15INK4Blocus. == Physique 1. Oncogenic Ras inhibits the expression ofANRILand stimulatesp15INK4Bexpression. == (a) Schematic representation ofINK4locus. The arrows indicate the direction of transcription of each gene. The black boxes indicate the exons ofINK4andANRILgenes, respectively. (b) WI38 Rabbit Polyclonal to Cofilin cells were infected with vacant (Mock) or E7-expressing retroviruses and selected by 400 g/ml G418 treatment as described inKotakeet al.(2007). (Left panel) The levels ofp15INK4B,p16INK4Aandp14ARFmRNA were determined by quantitative reverse transcriptase PCR, and results are expressed relative Banoxantrone D12 to the corresponding values for WI38/Mock cells. The mean values and s.d.s were calculated from triplicates of a representative experiment. The specific PCR pairs forp15INK4B,p16INK4A,ARFandGAPDHare described inKotakeet al.(2007). (Right panel) The expression ofANRILwas determined by reverse transcriptase PCR.