A 42 kb region on human chromosome 9p21 encodes for three distinct tumor suppressors, p16INK4A, p14ARF and p15INK4B, and is altered in an estimated 30–40% of human tumors. The expression of the INK4A-ARF-INK4B gene cluster is silenced by polycomb during normal cell growth and is activated by oncogenic insults and during aging. How the polycomb is recruited to repress this gene cluster is unclear. Here, we show that expression of oncogenic Ras, which stimulates the expression of p15INK4B and p16INK4A, but not p14ARF, inhibits the expression of ANRIL (antisense non-coding RNA in the I NK4 locus), a 3.8 kb-long non-coding RNA expressed in the opposite direction from INK4A-ARF-INK4B. We show that the p15INK4B locus is bound by SUZ12, a component of polycomb repression complex 2 (PRC2), and is H3K27-trimethylated. Notably, depletion of ANRIL disrupts the SUZ12 binding to the p15INK4B locus, increases the expression of p15INK4B, but not p16INK4A or p14ARF, and inhibits cellular proliferation. Finally, RNA immunoprecipitation demonstrates that ANRIL binds to SUZ12 in vivo. Collectively, these results suggest a model in which ANRIL binds to and recruits PRC2 to repress the expression of p15INK4B locus.
The INK4A-ARF-INK4B gene 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 cancer. Genetic analyses in mice with mutations targeting each gene individually support the tumor suppression function for all three genes (Serrano et al., 1996; Kamijo et al., 1997; Krimpenfort et al., 2001, 2007; Sharpless et al., 2001). Of these three tumor suppressors encoded by this gene cluster, both p15INK4B and p16INK4A function as the inhibitors of CDK4 and CDK6, and thereby retain the growth suppressive activity of RB family proteins, whereas ARF, expressed from a distinct promoter and an alternative reading frame of INK4A, 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 (Zindy et al., 1997; Krishnamurthy et al., 2004; Gil and Peters, 2006), suggesting a common cellular mechanism and potentially coordinated regulation of these three genes—to 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 (Jacobs et al., 1999; Itahana et al., 2003; Bracken et al., 2007; Kotake et al., 2007; Kia et al., 2008; Agger et al., 2009). The polycomb proteins 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 the Hox gene cluster both in fly and human cells (Wang et al., 2004; Cao et al., 2005). A critical issue for better understanding the regulation of the INK4A-ARF-INK4B gene cluster is 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 include RepA, a 1.6-kb lncRNA involved in X-chromosome inactivation (Zhao et al., 2008), Kcnq1ot1, a 91.5 kb lncRNA required for 10 paternally imprinted genes on 11p15 (Pandey et al., 2008; Terranova et al., 2008; Zhao et al., 2008), and HOTAIR, a 2.2-kb ncRNA involved in the repression of the HOX loci and promoting cancer metastasis (Rinn et al., 2007; Gupta et al., 2010). These findings led us to explore the possibility that ANRIL (antisense non-coding RNA in the I NK4 locus), a 3834 bp transcript whose transcription is initiated from the INK4A-ARF-INK4B gene cluster (Figure 1a), may be involved in the repression of INK4A, ARF and/or INK4B. ANRIL contains 19 exons, a polyadenylation site in the last exon and spans over 126 kb of genomic sequence that is deleted in the melanoma–neural system tumor syndrome family analyzed by previous study (Pasmant et al., 2007). Exon 1 of ANRIL locates between the promoter of ARF and INK4B, 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 of ANRIL resulted in a significantly increased expression of both p16INK4A and p15INK4B in several organs and tissues, but had no effect on other neighboring genes (Visel et al., 2010), providing a genetic evidence for the negative regulation of p16INK4A and p15INK4B by the ncRNA sequences expressed in this region. In this report, we show that p15INK4B locus is repressed by PRC2 proteins and that ANRIL is required for the recruitment of PRC2 to and repression of the p15INK4B locus.
Results and discussion
We first determined the expression of ANRIL in response to two well-characterized oncoproteins, E7 and Ras, which are known to affect the expression of p16INK4A, ARF and/or p15INK4B. Although E7 inactivates RB family proteins and potently activates the expression of p16INK4A and to a lesser extent p14ARF (Kotake et al., 2007, 2009), oncogenic Ras induces the expression of p15INK4B (Malumbres et al., 2000) (Figures 1b and c). We established stable cell lines expressing either E7 or oncogenic RasG12V and determined the expression of these tumor suppressor genes as well as ANRIL. As expected, both p16INK4A and p14ARF mRNAs, but not p15INK4B, were substantially increased in WI38/E7 cells, whereas in WI38/RasG12V cells, p15INK4B and p16INK4A, but not p14ARF, was stimulated by more than 12-fold (p15INK4B) and 2-fold (p16INK4A ) (Figures 1b and c). The induction of p16INK4A by RasG12V in WI38 cells is somewhat lower than that seen in some other normal fibroblast such as MEFs or IMR-90. The reason for this is not clear, but may relate to the difference between species or cell lines. The expression of ANRIL was readily detectable in WI38 cells, was not significantly affected by the expression of E7 and was notably reduced by the expression of H-RasG12V (Figure 1c). These data indicate that stimulation of p15INK4B by oncogenic Ras in cultured normal human cells is associated with a decrease of ANRIL expression, suggesting a potential negative regulation of p15INK4B by ANRIL.
We then designed a retroviral vector encoding a short hairpin RNA that specifically targets the exon1 region of ANRIL. Infection of WI38 cells with ANRIL short hairpin RNA retroviruses efficiently reduced ANRIL level (Figure 2a). Quantitative reverse transcriptase PCR analysis showed that silencing ANRIL resulted in an increase of p15INK4B expression by nearly 8-fold, but had minimal effect on the expression of p16INK4A (increased by 1.8-fold) or p14ARF (reduced by half, Figure 2b). Similar result was also reported very recently by Yap et al. that silencing ANRIL by a different method using antisense DNA increases the p16INK4A expression fourfold in another normal human diploid fibroblasts line, IMR-90 cells (Yap et al., 2010). Associated with p15INK4B increase, ANRIL silencing also resulted in decreased cell growth (Figure 2c) and an increase of cells stained positively for senescence-associated β-galactosidase activity, an indicator of cell senescence (Figure 2d). These results indicate that ANRIL is involved in the selective repression of p15INK4B transcription and prevention of cellular senescence. To search for the mechanism underlying the repression of p15INK4B by ANRIL, we examined the role of PRC2 in p15INK4B regulation. Transduction of WI38 cells with a retroviral vector encoding an short hairpin RNA targeting the catalytic subunit of PRC2 histone methyltransferase, EZH2, efficiently reduced EZH2 level (Figure 3a). Associated with EZH2 decrease is a substantial increase of p15INK4B mRNA by eightfold and p16INK4A mRNA by threfold, but no detectable effect on p14ARF mRNA level (Figure 3b). Associated with increase of p15INK4B and p16INK4A, EZH2 knockdown decreased cell growth (Figure 3c) and induced cell senescence (Figure 3d). To further confirm this, we transfected WI38 cells with small interfering RNA silencing SUZ12, another component of PRC2, and found that silencing SUZ12 (Figure 3e) also increased the expression of both p15INK4B as well as p16INK4A expression (Figure 3f). These results indicate that PRC2 is involved in the p15INK4B repression and cellular senescence.
That both ANRIL and PRC2 repress p15INK4B expression led us to determine whether ANRIL is required for PRC2 recruitment to p15INK4B locus. We first carried out a chromatin immunoprecipitation assay to test whether PRC2 directly binds to the p15INK4B locus. This assay demonstrated that the p15INK4B, as well as p16INK4A, locus is bound by SUZ12 and is trimethylated at lys27 of histone H3 (Figure 4a). Chromatin immunoprecipitation–quantitative PCR assay showed that silencing ANRIL causes substantial loss of SUZ12 occupancy on the p15INK4B locus (Figure 4c), although the steady state level of the SUZ12 protein is unchanged (Figure 4b). Finally, we examined whether ANRIL binds directly to SUZ12 by RNA immunoprecipitation assay and found that the SUZ12 immunocomplex, but not the immunocomplexes of immunoglobulin G or another negative control YY1, substantially enriched ANRIL RNA compared with U1 RNA, a non-specific RNA that is expressed in the cells at high levels (Figure 4d). These results demonstrate that ANRIL can bind directly and specifically to SUZ12.
In this report, we showed that mRNA-like ncRNA, ANRIL, and the PRC2 histone methyltransferase complex are involved in the repression of p15INK4B transcription. We further demonstrated that ANRIL binds to SUZ12, a PRC2 component, and is required for the SUZ12 occupancy on the p15INK4B locus. Loss of ANRIL or PRC2 showed the same effects on cellular proliferation and both cause premature senescence, supporting a mechanistic link between PRC2 and ANRIL. The results presented herein provide new insight into the epigenetic control of p15INK4B locus by PRC2 and lncRNA. The detailed biochemical mechanisms underlying the function of ANRIL in the PRC2 recruitment to the p15INK4B locus remain to be determined including, in particular, the interaction between ANRIL and PRC2. The other ncRNA, Xist, directly associates with EZH2 through RepA, a motif comprising 7.5 tandem repeats of a 28-nucleotide sequence that folds into two conserved stem-loop structures (Wutz et al., 2002; Zhao et al., 2008). Although we could not find such a distinctive motif on ANRIL, we showed the direct binding between ANRIL and PRC2. A recent study utilized high-magnification RNA–DNA FISH assay to demonstrate that large ncRNA, Air, interacts with the chromatin of cis-linked target gene promoter and recruits the H3K9 histone methyltransferase to repress the gene expression (Nagano et al., 2008). Based on that, ANRIL may directly interact with the p15INK4B promoter chromatin to recruit PRC2. Very recently, Yap et al. reported that CBX7, an H3k27me3-recognizing component of PRC1, can bind directly to both ANRIL and H3K27me3 via its chromodomain, and both interactions are required for CBX7 to repress the INK4A and INK4B loci (Yap et al., 2010). In the present study, we demonstrated that ANRIL is also involved in the recruitment of another polycomb complex, PRC2, to another gene, p15INK4B, in the INK4A-ARF-INK4B region. Our study thus broadens the scope of ANRIL function. Given that ANRIL encodes an almost 4 kb ncRNA that binds at least two different polycomb proteins, it will be important to determine the responsible regions in ANRIL for the association with each polycomb protein.
Recent genome-wide association studies have linked common single-nucleotide polymorphisms in 9p21 region that is independently associated with both type II diabetes and coronary heart disease (Helgadottir et al., 2007; McPherson et al., 2007; Saxena et al., 2007; Scott et al., 2007). As the ANRIL genomic region spans the risk-associated single-nucleotide polymorphisms (Broadbent et al., 2008), expression of ANRIL might be affected by the unknown causative genetic variant. Therefore, it is possible that the deregulation of ANRIL by such risk-associated single-nucleotide polymorphisms changes the expression level of p15INK4B and/or other target genes, leading to the development of type II diabetes or coronary heart disease in humans.
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We thank Howard Chang for the insightful discussion during this study, Yaxue Zeng and Matt Smith for helpful discussion and reading the manuscript, Mika Matsumoto, Michiyo Hakamata and Harumi Shiratori for technical assistance. This study is supported by grants from the Ministry of Education, Science, Sports, Culture, and Technology of Japan (YK, MK, SS and KK) and NIH grant CA68377 (YX).
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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Kotake, Y., Nakagawa, T., Kitagawa, K. et al. Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15INK4B tumor suppressor gene. Oncogene 30, 1956–1962 (2011) doi:10.1038/onc.2010.568
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