Long ncRNA expression associates with tissue-specific enhancers

Abstract

Long non-coding RNAs (ncRNA) have recently been demonstrated to be expressed from a subset of enhancers and to be required for the distant regulation of gene expression. Several approaches to predict enhancers have been developed based on various chromatin marks and occupancy of enhancer-binding proteins. Despite the rapid advances in the field, no consensus how to define tissue specific enhancers yet exists. Here, we identify 2,695 long ncRNAs annotated by ENCODE (corresponding to 28% of all ENCODE annotated long ncRNAs) that overlap tissue-specific enhancers. We use a recently developed algorithm to predict tissue-specific enhancers, PreSTIGE, that is based on the H3K4me1 mark and tissue specific expression of mRNAs. The expression of the long ncRNAs overlapping enhancers is significantly higher when the enhancer is predicted as active in a specific cell line, suggesting a general interdependency of active enhancers and expression of long ncRNAs. This dependency is not identified using previous enhancer prediction algorithms that do not account for expression of their downstream targets. The predicted enhancers that overlap annotated long ncRNAs generally have a lower ratio of H3K4me1 to H3K4me3, suggesting that enhancers expressing long ncRNAs might be associated with specific epigenetic marks. In conclusion, we demonstrate the tissue-specific predictive power of PreSTIGE and provide evidence for thousands of long ncRNAs that are expressed from active tissue-specific enhancers, suggesting a particularly important functional relationship between long ncRNAs and enhancer activity in determining tissue-specific gene expression.

Keywords: Enhancer; activating long non-coding RNA; enhancer prediction; long non-coding RNA; tissue-specific enhancer.

Figure 1.

Overlap of long ncRNAs with predicted tissue-specific enhancers. (A) Overview of the modified PreSTIGE enhancer prediction used in this study. PreSTIGE predicts enhancers by first finding PCGs with tissue-specific increased expression. In the tissue in which the PCG has an increased expression and within the specified domain size surrounding the TSS of the PCG (200kb) PreSTIGE predicts enhancers based on the presence of cell type specific H3K4me1 domains. (B) Example of a long ncRNA that is specifically expressed in HeLa and overlaps a predicted HeLa specific enhancer. Black bars labeled HeLa enhancers show predicted enhancers at 2 sites in the locus. Also shown is the ENCODE annotated isoforms of long ncRNAs and PCGs. ENCODE data for selected representative cell lines are shown for H3K4me1 and RNA sequencing data deposited in the UCSC genome browser. (C) The number of PreSTIGE predicted cell type specific enhancers in 11 different cell lines. (D) The number of annotated long ncRNAs that overlap a tissue-specific enhancer predicted by PreSTIGE. (E) The number of cell lines in which a given long ncRNA overlaps a predicted enhancer.

Figure 2.

Long ncRNA expression correlates with predicted tissue-specific enhancers. In (A–D) are shown expression values for all long ncRNAs overlapping a predicted enhancer in (A) GM12878, (B) H1ES, (C) HSMM, and (D) NHEK. For each long ncRNA the relative expression compared to the average expression across all cell lines is shown as bar-plots (upper panels) or as heatmaps (lower panels). Heatmaps are normalized for each transcript such that blue shows the lowest expression and red shows the highest expression. Statistical analysis is done using Mann-Whitney-Wilcoxon test. a P-value < 2.2e-16, b P-value 7.9e-15.

Figure 3.

Comparison of PreSTIGE predicted enhancers to previous methods. (A) Venn diagram showing the overlap between enhancers in HeLa predicted by using H3K4me1 and H3K4me3 profiles compared to HeLa tissue-specific enhancers predicted by PreSTIGE. (B) Quantification of H3K4me3 at enhancers predicted by overlapping or not overlapping PreSTIGE predicted enhancers, respectively. *** P-value < 2.2e-16. (C) The ratio of H3K4me1 to H3K4me3 for PreSTIGE predicted enhancers overlapping or not overlapping a long ncRNA, respectively. *** P-value 0.00039. (D and E) As in Figure 2. Shown are average relative expression values across 11 cell lines for all long ncRNAs overlapping predicted enhancers in HeLa cells as bar-plots (upper panels) or heatmaps (lower panels). (D) Expression at enhancers predicted by the modified PreSTIGE method. a P-value < 2.2e-16, b P-value 9.0e-10. (E) Expression at enhancers predicted by Heintzman et al., 2009. c P-value 9.5e-07, d P-value 3.9e-05, e P-value 6.4e-06 and f P-value 1.6e-06. Statistical analyses were done using Mann-Whitney-Wilcoxon test.

Figure 4.

PreSTIGE prediction of enhancers overlapping activating ncRNAs. A tissue-specific enhancer is predicted in K562 cells that overlap previously identified activating ncRNAs ncRNA-a3 and ncRNA-a4 shown to target TAL1 and CMPK1, respectively. Predicted enhancers are shown as black boxes (K562 enhancers). The ENCODE annotated transcripts are shown for discontinuous regions of the locus. See scale bar and coordinates. K562 PolII ChIA-PET data are from Li et al. as deposited in the UCSC genome browser, and show interacting regions as experimentally determined. Additionally, H3K4me1 and RNA sequencing data from ENCODE are shown for representative cell lines.