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. 2013 May;25(5):1086-95.
doi: 10.1016/j.cellsig.2013.02.006. Epub 2013 Feb 14.

Long non-coding RNA ANRIL (CDKN2B-AS) is induced by the ATM-E2F1 signaling pathway

Guohui Wan  1 , Rohit Mathur, Xiaoxiao Hu, Yunhua Liu, Xinna Zhang, Guang Peng, Xiongbin Lu
Affiliations

Long non-coding RNA ANRIL (CDKN2B-AS) is induced by the ATM-E2F1 signaling pathway

Guohui Wan et al. Cell Signal. 2013 May.

Abstract

The maintenance of genome integrity is essential for the proper function and survival of all organisms. Human cells have evolved prompt and efficient DNA damage response to eliminate the detrimental effects of DNA lesions. The DNA damage response involves a complex network of processes that detect and repair DNA damage, in which long non-coding RNAs (lncRNAs), a new class of regulatory RNAs, may play important roles. Recent studies have identified a large number of lncRNAs in mammalian transcriptomes. However, little is known about the regulation and function of lncRNAs in the DNA damage response. In the present study, we demonstrate that one specific lncRNA, ANRIL, is transcriptionally up-regulated by the transcription factor E2F1 in an ATM-dependent manner following DNA damage, and elevated levels of ANRIL suppress the expression of INK4a, INK4b and ARF at the late-stage of DNA damage response, allowing the cell to return to normal at the completion of the DNA repair.

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Figures

Figure 1
Figure 1. ANRIL is induced after DNA damage
(A) Relative expression levels of 15 representative lncRNAs after DNA damage. GM0637 cells were treated with NCS (500 ng/ml), and cells were harvested at indicated time points for microarray analysis. Green or red color on the heat map indicates a decrease or increase of lncRNA level, and numbers correspond to fold change comparing to the control samples with mock treatment. (B) Schematic diagram showing the ANRIL gene and its transcript in the INK4B-ARF-INK4A locus. (C) ANRIL is induced in the DDR in human U2OS cells. Level of p21 was used as a positive control for the activation of the DDR. RNA samples were analyzed by quantitative RT-PCR, and error bars represent the mean ± SD. (D) Protein levels of p53 and p21 in the DDR. E) Induction of ANRIL is independent of DNA damage types. U2OS cells were treated with neocarzinostatin, etoposide and bleomycin respectively.
Figure 2
Figure 2. Induction of ANRIL is dependent on ATM, but not p53
ANRIL is induced in an ATM-dependent manner in human U2OS cells (A) and HCT116 p53+/+ cells (B) after DNA damage. The cells were infected by lentiviruses expressing control shRNA or ATM-shRNA. ATM knockdown efficiency was confirmed by western blot. (C) Silence of p53 has no significant effect on the DNA damage-induced ANRIL. HCT116 p53+/+ cells were infected by lentiviruses expressing control shRNA or p53 shRNA. p53 knockdown efficiency was confirmed by western blot. (D) Reintroduction of p53 into HCT115p53-/- cells has no effect on the DNA damage-induced ANRIL. Expression of p53 was confirmed by western blot.
Figure 3
Figure 3. E2F1 transcriptionally activates ANRIL in the DDR
(A) RNA stability of ANRIL is not affected by DNA damage. U2OS Cells were pretreated with Actomycin D prior to NCS treatment. Levels of ANRIL were measured by quantitative RT-PCR. (B) Schematic representation of the ANRIL promoter. One putative E2F1 binding element is shown in the indicated regions. Arrows stands for DNA-ChIP primers. (C) Activity of the ANRIL promoter is up-regulated by E2F1 after DNA damage. HCT116 p53+/+ cells were infected by lentiviruses expressing control or E2F1 shRNA. 72 h after infection, the cells were transfected with pGL3-basic vector or ANRIL promoter-driven firefly luciferase expression vector, and Renilla luciferase expression vector. The cells were treated with NCS (500 ng/ml) 24 h after transfection and then harvested 16 h after treatment. Firefly luciferase activity was measured and normalized to the activity of Renilla luciferase. (D) Enrichment of E2F1 in the ANRIL promoter after DNA damage. Cells were pretreated with DMSO or ATM inhibitor for 2 h prior to NCS treatment. E2F1 was immunoprecipitated and the promoter region containing E2F1-binding sequences were quantified by RT-PCR. Control IgG immunoprecipitation was used as controls. (E) Induction of E2F1 after DNA damage is dependent on ATM. Cells were pretreated with DMSO or ATM inhibitor prior to NCS treatment. Phosphorylated p53 at Ser15 was used as indicator for the ATM activity to validate the inhibitory effects by ATM inhibitor.
Figure 4
Figure 4. Expression of genes in the INK4B-ARF-INK4A locus is regulated by ANRIL
(A) Generation of stable HCT116 p53+/+ cells with ANRIL overexpression (OE) or knockdown (KD). Cells were infected with lentiviruses expressing ANRIL or its shRNA. Single colonies were selected and verified. RNA levels were measured by quantitative RT-PCR. (B) RNA levels of p15, p16 and p14 in HCT116 p53+/+ cells with altered levels of ANRIL. (C) RNA levels of p15, p16 and p14 in the DNA damage response. Cells were treated with 500 ng/ml NCS, harvested at indicated time points, subject to RNA analysis using quantitative RT-PCR. (D) Protein levels of p15, p16 and p14 in the DNA damage response. Cells were treated and harvested as above and proteins were analyzed by western blot.
Figure 5
Figure 5. Effects of ANRIL on cell proliferation, apoptosis and drug sensitivity
(A) ANRIL promotes cell proliferation. HCT116 p53+/+ cells with altered levels of ANRIL were seeded at 1,000 cells/well, cultured up to six days, and counted at indicated time points. (B) Cell cycle analysis of the above ANRIL-silenced and -overexpressing cells. Cells were treated with NCS (500 ng/ml) to induce cell cycle checkpoints. Cells were harvested at 0, 4, 8 and 24 h after treatment, fixed and stained with propidium iodide, and analyzed by flow cytometry. (C) Apoptosis analysis of the ANRIL-silenced or - overexpressing cells. Cells were harvested at indicated time points after DNA damage. Apoptotic cells were quantified and analyzed by AnnexinV-7ADD staining and flow cytometry. (D) Viability analysis of the ANRIL-silenced or -overexpressing cells. Cells were treated with NCS for one day and cell viability was measured at indicated time points after treatment.
Figure 6
Figure 6. ANRIL promotes homologous recombination in cells
A) Schematic of the DR-GFP construct. The DR-GFP reporter for measuring HR contains two defective GFP gene (SceGFP is interrupted by an I-SceI site and iGFP has an early stop codon). HR repair of the double –strand breaks generated by I-SceI results in a functional GFP gene. (B) ANRIL knockdown inhibits HR frequencies. (C) Working model of ANRIL in the DNA damage response. Upon DNA damage, ATM-initiated signaling pathway is activated to induce p53 that promotes cell cycle arrest, apoptosis and senescence. ATM-induced E2F1 transcriptionally activates ANRIL, which consequently represses the expression of INK4 family members, and thus alleviate p53 and pRB signaling pathways at the late-stage of the DDR. The cell will eventually return to normal at the completion of the DDR.
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