In Vivo Characterization of Linc-p21 Reveals Functional cis-Regulatory DNA Elements

Abstract

The Linc-p21 locus, encoding a long non-coding RNA, plays an important role in p53 signaling, cell-cycle regulation, and tumor suppression. However, despite extensive study, confusion exists regarding its mechanism of action: is activity driven by the transcript acting in trans, in cis, or by an underlying functional enhancer? Here, using a knockout mouse model and a massively parallel enhancer assay, we delineate the functional elements at this locus. We observe that, even in tissues with no detectable Linc-p21 transcript, deletion of the locus significantly affects local gene expression, including of the cell-cycle regulator Cdkn1a. To characterize this RNA-independent regulatory effect, we systematically interrogated the underlying DNA sequence for enhancer activity at nucleotide resolution and confirmed the existence of multiple enhancer elements. Together, these data suggest that, in vivo, the cis-regulatory effects mediated by Linc-p21, in the presence or absence of transcription, are due to DNA enhancer elements.

Figure 1. Linc-p21 in vivo deletion overview

(A) Linc-p21/Cdkn1a locus on mouse chromosome 17. Asterisks indicate known p53 binding sites. (B) Dendrogram showing the type and number of samples sequenced and the Jenson-Shanon distance, a measure of total transcriptome similarity, between their expression profiles. Wildtype shown in black, knockout shown in grey. (C) Average Linc-p21 expression profile in each tissue. Error bars represent 95% confidence interval. (D) Heatmap depicting expression of genes significantly differentially expressed in every tissue. Log₂ fold-change was calculated using the average FPKMs for all replicates (KO/WT). See also Figure S1.

Figure 2. Linc-p21 transcript is not required for local gene regulation

(A) Summary of local transcriptional changes upon Linc-p21 deletion (+/− 2Mb of Linc-p21). Dots represent average log₂ fold change across all 6 tissues and error bars represent standard error. Red indicates significant differential expression in at least one tissue. (B-E) Expression of genes in local region (+/− 2Mb of Linc-p21) for E14.5 hindlimbs (B), heart (C), liver (D), and adult brain (E). In each plot, the Y-axis represents the log₂ FPKM fold change and genes marked in red were significantly differentially expressed. The p value represents the probability that this number of genes would be differentially expressed within a region of this size. Insets show Linc-p21 and lacZ expression (FPKM) in wildtype (black) and knockout (gray). See also Figure S2.

Figure 3. In vivo evidence of enhancer activity in the Linc-p21 locus

(A) Correlation of Cdkn1a expression with Linc-p21 transcript expression. X-axis represents Linc-p21 expression and Y-axis represents Cdkn1a expression in each wildtype replicate. Color indicates the tissue of origin, and the linear regression line is shown. (B) Average change in Cdkn1a expression in knockout tissues (1-KO/WT, bars, left y-axis) plotted against average wildtype Linc-p21 expression levels (red dots, right y-axis); error bars represent standard error. (C) Publically available chromatin interaction data between Linc-p21 and Cdkn1a from HiCap (plotted as black lines between capture probes and gray distal regions). Publically available histone mark and transcription factor binding data at this locus. All panels are from embryonic or adult heart tissue, except CEBPB and MyoD binding which are from C2C12 cells. See also Figure S3.

Figure 4. MPRA of the entire Linc-p21 locus reveals enhancer activity

(A) Experimental design for luciferase reporter assay using an intergenic control and regions from the Linc-p21 promoter and exon 1 (“exon”), intron 1 (“intron”), exon with mutated p53 binding sequence (“exon -p53”) and intron with p53 binding sequence (“intron+p53”). Each region was cloned into a luciferase reporter construct. P53 motif indicated in red. (B) Relative luminescence for each construct, normalized against the signal from exon fragment and averaged across triplicate samples. P values were calculated using unpaired one-tailed t-tests. Error bars represent standard error of the mean. (C) Massively Parallel Reporter Assay (MPRA) experimental design: oligos were synthesized, subcloned into a minimal backbone, opened by enzymatic digestion and re-ligated with a GFP cDNA insert. Pooled constructs were transfected into C2C12 cells in triplicate and libraries were made from GFP+ RNA. (D) Coverage of final pooled GFP+ vector library across the Linc-p21 locus and promoter (assessed by high throughput sequencing). (E) MPRA signal across the Linc-p21 locus. Y-axis represents the log₂ ratio of normalized RNA to control signal per base (averaged in 500bp sliding windows every 50bp). Significance (p<0.01) is calculated by comparing this signal to 1000 random shuffles of the input data. Significant peaks are shown in red. Inlayed tracks are CEBPB and MyoD ChIP-seq signals from UCSC genome browser. See also Figure S4.