Pint is a p53-regulated long intergenic non-coding RNA (lincRNA). (A) Schematic representation of the Pint genomic locus. Asterisks represent p53 response elements (p53REs). (B) Relative firefly luciferase expression driven by genomic sequences containing p53REs in p53-restored p53LSL/LSL (p53+/+) or p53LSL/LSL (p53-/-) cells treated with doxorubicin. Values were normalized by Renilla levels and are the mean ± standard deviation (SD) of three biological replicates. Asterisks represent significant differences determined by t-test relative to the same plasmid transfected in doxorubicin (DOX)-treated p53-/-. (C) Effect on Pint p53RE-1, p53RE-2, and p53RE-3, Cdkn1a p53RE, or an irrelevant region (control) of p53 chromatin immunoprecipitation (ChIP) enrichment in p53-restored p53LSL/LSL (p53+/+) or p53LSL/LSL (p53-/-) cells treated with doxorubicin (+DOX) or left untreated (-DOX). Enrichment values are relative to input and the mean ± SD of three biological replicates. Asterisks represent statistical significant differences from the control as determined by t-test (*P<0.05, **P<0.01). (D) (Top) p53 ChIP sequencing (ChIP-seq) peaks from mouse embryonic fibroblasts (MEFs) treated with doxorubicin . Positions of p53REs are indicated by red asterisks. (Bottom) Pint variants identified by 5' and 3' rapid amplification of cDNA ends (RACE) cloning. (E) Pint levels detected by quantitative real time RT-qPCR in p53-restored p53LSL/LSL (p53+/+) or p53LSL/LSL (p53-/-) cells treated with 150 nM doxorubicin (+DOX) or left untreated (-DOX) for the indicated time (values represent the mean ± SD of three biological replicates, and asterisks represent significant differences of Pint level at 48 hours relative to the doxorubicin-treated p53-/- cells). (F,G) Pint levels at different times after p53 restoration in (F) lung tumor (G) and sarcoma cell lines. Values are the mean ± SD of four replicates.
Marín-Béjar et al. Genome Biology 2013 14:R104 doi:10.1186/gb-2013-14-9-r104