The p53 gene is mutated in over half of all cancers, reflecting its critical role as a tumor suppressor. Although p53 is a transcriptional activator that induces myriad target genes, those p53-inducible genes most critical for tumor suppression remain elusive. Here, researchers at Stanford University School of Medicine leveraged p53 ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) and RNA-seq (RNA sequencing) data sets to identify new p53 target genes, focusing on the noncoding genome. They identified Neat1, a noncoding RNA (ncRNA) constituent of paraspeckles, as a p53 target gene broadly induced by mouse and human p53 in different cell types and by diverse stress signals. Using fibroblasts derived from Neat1-/- mice, they examined the functional role of Neat1 in the p53 pathway. They found that Neat1 is dispensable for cell cycle arrest and apoptosis in response to genotoxic stress. In sharp contrast, Neat1 plays a crucial role in suppressing transformation in response to oncogenic signals. Neat1 deficiency enhances transformation in oncogene-expressing fibroblasts and promotes the development of premalignant pancreatic intraepithelial neoplasias (PanINs) and cystic lesions in KrasG12D-expressing mice. Neat1 loss provokes global changes in gene expression, suggesting a mechanism by which its deficiency promotes neoplasia. Collectively, these findings identify Neat1 as a p53-regulated large intergenic ncRNA (lincRNA) with a key role in suppressing transformation and cancer initiation, providing fundamental new insight into p53-mediated tumor suppression.
Neat1 is a p53 target gene in mouse and human cells
(A) Experimental outline that led to the discovery of Neat1 as a p53 target gene. (B) ChIP-qPCR testing for p53 binding at a peak identified in Neat1 by ChIP-seq analysis together with Cdkn1a as a positive control. The percentage of immunoprecipitated DNA relative to input is indicated. p53−/− MEFs served as a negative control. (C) qRT–PCR analysis of Neat1 expression in wild-type and p53−/− MEFs treated with 0.2 µg/mL doxorubicin (left) or 20 J/m2 UV light (right) and collected at the indicated time points, normalized to β-actin. (D) qRT–PCR analysis of Neat1 expression in mouse ESCs treated with 0.2 µg/mL doxorubicin for the indicated times, normalized to β-actin. (E) Human p53 ChIP-seq profiles (Younger et al. 2015) in primary human fibroblasts reveal a strong p53-binding site in the promoter of NEAT1. The top track shows the p53 ChIP sample, with the carat indicating the “called” peak as determined by DNANexus. The bottom track shows ChIP-seq input reads. The numbers in parentheses indicate the numbers of base pairs in individual half-sites matching the consensus sequence. (F) qRT–PCR analysis of NEAT1 and CDKN1A in primary human fibroblasts expressing shGFP or shp53 8 h after initiating doxorubicin treatment, normalized to β-ACTIN. (G) Northern blot of doxorubicin-treated (for 24 h) primary human fibroblasts transfected with a scrambled siRNA (siNT), sip53, or either of two different siRNAs against NEAT1 (siN1 and siN2). The numbers below the blots correspond to the expression levels normalized to the RPLPO loading control. (H, left) NEAT1 expression levels by qRT–PCR in two different human ESCs treated with Nutlin3a for 2 d, normalized to β-ACTIN. (Right) NEAT1 expression levels by qRT–PCR in human ESCs expressing shGFP or shp53 and left untreated or treated with doxorubicin for the indicated times, normalized to β-ACTIN. (I) NEAT1 expression levels by qRT–PCR in human A549 lung cancer cells treated with Nutlin3a for 1 or 2 d, normalized to β-ACTIN. (J) qRT–PCR analysis of NEAT1 and CDKN1A in wild-type and p53-null HCT116 cells treated with 0.2 µg/mL doxorubicin for different times, normalized to β-ACTIN. (K) Northern blot of wild-type and p53-null HCT116 cells after doxorubicin treatment for different lengths of time. RPLPO serves as a loading control. The numbers below the blots correspond to the expression levels normalized to RPLPO. Error bars represent ±SD. (*) P ≤ 0.05; (***) P ≤ 0.001; (n.s.) nonsignificant, based on the two-tailed unpaired Student’s t-test.