An inducible long noncoding RNA amplifies DNA damage signaling

Abstract

Long noncoding RNAs (lncRNAs) are prevalent genes with frequently precise regulation but mostly unknown functions. Here we demonstrate that lncRNAs guide the organismal DNA damage response. DNA damage activated transcription of the DINO (Damage Induced Noncoding) lncRNA via p53. DINO was required for p53-dependent gene expression, cell cycle arrest and apoptosis in response to DNA damage, and DINO expression was sufficient to activate damage signaling and cell cycle arrest in the absence of DNA damage. DINO bound to p53 protein and promoted its stabilization, mediating a p53 auto-amplification loop. Dino knockout or promoter inactivation in mice dampened p53 signaling and ameliorated acute radiation syndrome in vivo. Thus, inducible lncRNA can create a feedback loop with its cognate transcription factor to amplify cellular signaling networks.

Figure 1. DINO encodes a p53-dependent DNA damage-induced transcript

(a) Upper Panel: Transcription across the CDKN1A locus in human fibroblasts before or after 24 hours of doxorubicin (Dox) treatment, (Hung et al., 2011). Lower Panel: p53 ChIP-seq in untreated or doxorubicin treated U2OS cells. (b) Time course of DINO and CDKN1A expression in human fibroblasts after DNA damage. Below: p53 and histone H3 immunoblot (WB). Mean +/− s.d are shown, n = 3. (c) DINO expression and p53 immunoblot in fibroblasts treated with siCDKN1A or siTP53 24 hours prior to dox treatment. Mean +/− s.d. are shown, n = 3. (d) DINO expression and p53 immunoblot in p53^+/+ wildtype (WT) or p53^−/− HCT116 cells +/− Dox (16hr). Mean +/− s.d are shown, n = 3. See also Supplementary Figs. 1–2.

Figure 2. Loss of DINO abrogates DNA damage-induced gene regulation and cell cycle arrest

(a) DINO and CDKN1A expression in fibroblasts treated with the indicated siRNAs in the presence or absence of doxorubicin (24hr), as measured by qRT-PCR. Mean +/− s.d. are shown; *, P < 0.05 compared to siCTRL (students T-test), n = 3. (b) Columns 1 and 2: heatmap of genes > 2 fold changed in fibroblasts upon DNA damage with doxorubicin (26hr) by microarray analysis. Same genes were represented as DINO knockdown + Dox relative to control + Dox in columns 3–5. (c) Genome-wide chromatin accessibility at p53 binding elements in fibroblasts treated with doxorubicin and DINO knockdown as indicated (24hr), as measured by ATAC-seq. Mean +/− s.e.m are shown. * P = 1.2 × 10⁻⁴, Control ASO (n = 4), Control ASO + Dox (n = 4), DINO ASO (n = 6), DINO ASO + Dox (n = 7). (d) p53 ChIP-qPCR at indicated promoters in control or DINO knockdown fibroblasts treated with doxorubicin (24hr). Mean +/− s.d are shown, n = 3. (e) Cell cycle arrest following 26 hours of doxorubicin treatment in shControl and shDINO U2OS cells. See also Supplementary Fig. 3 and Supplementary Tables 1–2.

Figure 3. p53 is a DINO-binding protein

(a) Left: Summary of RNA chromatography with DINO retrieval of proteins from whole cell lysate of doxorubicin (24hr) treated fibroblasts. Right: RNA chromatography using 6 pmol RNA and 1 ug of each recombinant protein as indicated. (b) RNA IP of p53 in human fibroblasts followed by qRT-PCR of DINO, GAPDH and U6 snRNA. *, P < 0.05 compared to IgG control (students T-test). Mean +/− s.d. are shown, n = 3. (c) UV crosslinking and immunoprecipitation (CLIP) of p53-bound DINO RNA in doxorubicin (24hr) treated human fibroblasts. qRT-PCR identifies the DINO region bound by p53 in vivo. Location of primer pairs along the DINO transcript indicated in the diagram above. Bottom: Immunoblot of p53 retrieved by CLIP. Mean +/− s.d. are shown, n = 3. (d) p53 RIP of DINO with the indicated p53 constructs in p53^−/− H1299 cells. Mean +/− s.d. are shown, n = 3. See also Supplementary Fig. 4.

Figure 4. DINO stabilizes p53 and enhances regulation of its target genes

(a) Immunoblot of p53, p53 phospho-S9, and p21 in shControl (shSCR) and shDINO U2OS cells. Doxorubicin (12hr) was used at the following concentrations: 0, 0.05, 0.1, 0.2 ug/ml. (b) Human or mouse DINO expression stabilizes p53 in fibroblasts, as indicated by cycloheximide (CHX) chase analysis. p53 and p21 levels at indicated hours post cycloheximide addition. p53 densitometry values are indicated below. (c) p53 target gene expression in 293 cells with empty vector, DINO overexpression, doxorubicin treatment (16hr), or DINO + doxorubicin. Left: nCounter quantification. Right: confirmation using qRT-PCR. Mean +/− s.d. are shown; *, P < 0.05 (students T-test), n = 3. (d) Cell cycle analysis of BrdU/PI labeled 293 cells transfected with DINO. (e) p53 retrieval of wildtype DINO or a DINO mutant lacking the putative p53 binding site, measured by p53 CLIP-qRT-PCR. Mean +/− s.d. are shown, n = 3. (f) qRT-PCR for p53 target genes in 293 cells transfected with vector, full length DINO, or DINO deletion mutants DINOΔ323–405 or DINOΔ320–450 that lack the CLIP RNA fragment. Blue box indicates the primary p53 binding site identified by UV-CLIP-qPCR. Mean +/− s.d. are shown, n = 3. See also Supplementary Fig. 5.

Figure 5. Germline disruption of mouse Dino impairs the p53-dependent, DNA damage-induced apoptosis

(a) Design of the Dino^gfp allele. (b) DNA damage induces Dino and Cdkn1a expression in MEFs of indicated genotype 16 hours after 0.2 ug/mL doxorubicin, measure by qRT-PCR. Mean +/− s.d. are shown, n = 3. (c) qRT-PCR of DNA damage inducible, p53 responsive genes in MEFs of indicated genotype under conditions of physiologic oxygen (2%). Mean +/− s.d. are shown, n = 3. (d) Relative apoptotic and viable fractions of thymocytes of indicated genotypes 6 hours after 1 Gy irradiation. Mean +/− s.d. are shown, ** P < 0.01 (students T-test), n = 3. (e) Kaplan Meier survival curve of mice treated with a single fraction of lethal total body irradiation of 12 Gy. P < 0.01 (log-rank test for trend). See also Supplementary Fig. 6.

Figure 6. Dino regulates the DNA damage response in both a p21-dependent and p21-independent manner

(a) Design of Dino^lox allele. (b) Dino^lox/lox inactivates basal and DNA-damage inducible expression of Dino lncRNA. Dino expression in MEFs with indicated genotypes +/− 0.2ug/mL dox for 8 hours is measured by qRT-PCR. p53^−/− MEFs is shown for comparison. Mean +/− s.d. are shown, n = 3. (c) qRT-PCR of p53-dependent transcripts in MEFs of indicated genotype 8 hours after treatment with the indicated doses of doxorubicin. Mean +/− s.d. are shown, n = 3. (d) p53 protein abundance following DNA damage with dox. Top: Immunoblot. Bottom: Quantification of p53 and p21 protein abundance normalized to beta-tubulin. Summed area under the curve [AUC] shows 40% reduction of p53 protein accumulation in Dino^lox/lox cells. (e) Cell cycle analysis of doxorubicin-treated Dino^+/+ and Dino^lox/lox MEFs. Mean +/− s.d. are shown, n = 3. (f) Population doublings of serially passaged primary Dino^+/+ and Dino^lox/lox MEFs. See also Supplementary Fig. 7.

Figure 7

Model of DINO function in p53 signaling.