Long non-coding RNA ANRIL (CDKN2B-AS) is induced by the ATM-E2F1 signaling pathway
Highlights
► A large number of lncRNAs are induced after DNA damage. ► LncRNA ANRIL is induced after DNA damage in an ATM-dependent manner. ► Induction of ANRIL is independent of p53. ► E2F1 transcriptionally activates ANRIL. ► ANRIL inhibits the expression of INK4a, INK4b and ARF in the DNA damage response.
Introduction
Long non-coding RNAs (lncRNAs) are a new class of regulatory RNAs that are defined as transcribed RNA molecules ranging in length from 200 to 100,000 nucleotides and lacking protein-coding capacity. Recent high-throughput transcriptome analyses have identified a large number of lncRNAs in mammalian genomes. While only 1.5% of the genome is responsible for protein coding genes, a vast majority of non-coding regulatory elements are transcribed to noncoding RNAs, among which lncRNAs have been recognized for their essential role in controlling every level of the gene expression program in various physiological processes including development, differentiation and metabolism [1], [2]. Current reports have suggested that lncRNAs regulate gene expression by executing as signals, decoys, guides and scaffolds and acting as a repressor or activator to modulate the process of gene transcription and translation [3]. For instance, lncRNA PTENNP1, previously considered as a biologically nonfunctional RNA, acts as a decoy molecule that sequesters miRNAs to abrogate their functions in regulating target gene expression. In particular, the 3′ UTR of PTENP1 binds the same set of regulatory miRNA sequences that target the tumor suppressor PTEN. As a result, the miRNA inhibition of PTEN expression is attenuated and PTEN levels are increased [4]. Similar to normal protein-coding genes, transcription of lncRNAs is mediated by RNA polymerase II complex. However, it has not been well studied about how lncRNA transcription is regulated. A recent study by Huarte et al. identified a number of lncRNAs that are transcriptionally regulated by the central tumor suppressor p53. One of the p53-transactivated lncRNAs, lincRNA-p21 (long intergenic non-coding RNA-p21) serves as a key repressor in p53-dependent transcriptional responses by physically associating with hnRNP-K and modulating its localization [5]. Further study also revealed that lincRNA-p21 functions as a posttranscriptional inhibitor of translation by selectively binding JUNB and CTNNB1 mRNAs, thus repressing their translation [6]. In recent years, lncRNAs have been emerging as a critical layer in the regulation of the gene transcription program. However, the function of lncRNAs in the context of various physiological conditions it is yet to be understood.
The DNA damage response (DDR) is an important anti-cancer barrier to maintain genome integrity against intrinsic and extrinsic genotoxic stresses including ultraviolet light (UV), ionizing radiation (IR), chemo- and radio-therapeutic agents, oncogenic insults, and reactive oxygen species. The DDR involves a number of networks connecting tumor suppressor genes to DNA repair pathways, damage tolerance processes, cell-cycle checkpoints and apoptosis [7], [8]. The DDR is predominantly initiated by PI3K family proteins, ATM (ataxia-telangiectasia mutated), ATR (ATM and Rad3-related) and DNA-PKcs (DNA-dependent protein kinase, catalytic subunit), which control the activity and localization of various downstream proteins and orchestrate many events at transcriptional, post-transcriptional and post-translational levels [9], [10]. The ATM kinase is a key sensor in the DDR pathway that responds in particular to double-strand DNA breaks, the most severe genomic lesions. The ATM-mediated phosphorylation of downstream target proteins triggers a cascade of signals to activate cell cycle checkpoints and DNA repair [9]. In addition to canonical DNA damage signaling pathways, epigenetic alterations, such as altered DNA methylation status, histone modification patterns, chromatin remodeling, and non-coding RNA (in particular, microRNA and lncRNA) regulation have been shown to contribute as novel layers of regulation to the complexity of the DDR signaling network [11], [12], [13], [14].
Given the large quantity of lncRNAs in genomes, it is assumed that expression of lncRNAs may be regulated in the DDR and provide feedback effects on the DDR. Aberrant expression of individual lncRNAs has been reported in tumors of various tissue origins [15] and recent data revealed that lncRNA transcripts can modulate gene activity in response to DNA damage [16]. A long non-coding RNA, ANRIL (antisense non-coding RNA in the INK4 locus) was found to be involved in the repression the INK4B–ARF–INK4A locus [17], [18], [19]. The INK4B–ARF–INK4A locus spans around 35 kilobases on human chromosome 9p21 that includes three intimately linked tumor suppressor genes (INK4a, INK4b, and ARF) that trigger the anti-proliferative activities of both RB and p53. Loss of the INK4B–ARF–INK4A locus is the most frequent copy number alteration across tumors and cancer cell lines [20], [21]. ANRIL is transcribed in anti-sense direction with respect to the primary INK4 and ARF transcripts [22]. ANRIL was shown to be involved in epigenetic regulation of the INK4B–ARF–INK4A locus by direct binding to the INK4b transcript and recruiting the Polycomb Repressor Complex (PRC) to repress the transcription of genes at this locus [18], [19]. However, how ANRIL is regulated in response to genotoxic stress is largely unknown. In the present study, we investigated the expression of ANRIL and its roles in the DDR. We show that ANRIL is induced by E2F1 transcription factor in an ATM-dependent manner after DNA damage, and that elevated ANRIL suppresses the expression of INK4B–ARF–INK4A at the late-stage of DDR, forming a negative feedback loop to the DDR.
Section snippets
Cell lines and plasmids
GM0637 cell line is a human fibroblast line that expresses wildtype ATM. It was obtained from Coriell Cell Repositories. U2OS cell line (human osteosarcoma line) was obtained from the American Type Culture Collection (ATCC). HCT116 p53+/+ and HCT116 p53−/− cell lines were obtained from the Vogelstein laboratory at Johns Hopkins University. Cells were cultured following standard protocols described previously [23].
Human ANRIL cDNA clone L6ChoCKO-2-E10 with functional region was provided by the
ANRIL is induced in the DNA damage response
To examine the regulation of lncRNAs in the DDR, we assessed the genome-wide lncRNA expression profiles in human fibroblast GM0637 cells that have functional DDR [23]. The cells were treated with a radiomimetic drug, neocarzinostatin (NCS) that generates double strand breaks, and harvested at various time points (0, 4 and 8 h). A total of 10,602 human lncRNAs were examined using a specific lncRNA microarray containing oligo probes for these lncRNAs. As many as 1903 specific lncRNAs, representing
Discussion
Recent genome sequencing and transcriptome analyses demonstrate that transcription is not limited to the protein-coding genes. As a matter fact, a vast majority of transcripts are produced from those “junk” DNA regions. In addition to well-studied microRNAs, ribosomal RNAs, small nuclear RNAs, thousands of lncRNAs have been identified and this number has been increasing [1], [3]. While these lncRNAs have little or no protein-coding capacity, a major question needs to be addressed: how do they
Acknowledgements
We are very grateful to Drs. Yoshida and Kotake for providing the reagents. This work was supported by grants to X.L. from the National Institutes of Health (R01CA136549 and R03CA142605) and the American Cancer Society (119135-RSG-10-185-01-TBE).
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These authors contributed equally to this work.