Tissue-Specific RNA Expression Marks Distant-Acting Developmental Enhancers

<div><p>Short non-coding transcripts can be transcribed from distant-acting transcriptional enhancer loci, but the prevalence of such enhancer RNAs (eRNAs) within the transcriptome, and the association of eRNA expression with tissue-specific enhancer activity <i>in vivo</i> remain poorly understood. Here, we investigated the expression dynamics of tissue-specific non-coding RNAs in embryonic mouse tissues <i>via</i> deep RNA sequencing. Overall, approximately 80% of validated <i>in vivo</i> enhancers show tissue-specific RNA expression that correlates with tissue-specific enhancer activity. Globally, we identified thousands of tissue-specifically transcribed non-coding regions (TSTRs) displaying various genomic hallmarks of bona fide enhancers. In transgenic mouse reporter assays, over half of tested TSTRs functioned as enhancers with reproducible activity in the predicted tissue. Together, our results demonstrate that tissue-specific eRNA expression is a common feature of <i>in vivo</i> enhancers, as well as a major source of extragenic transcription, and that eRNA expression signatures can be used to predict tissue-specific enhancers independent of known epigenomic enhancer marks.</p></div

Top 10 GO biological processes enriched in genes nearby TSTRs (sorted by p-value).

<p>Top 10 GO biological processes enriched in genes nearby TSTRs (sorted by p-value).</p

Intergenic regions marked by tissue-specific RNA expression may represent regulatory enhancer elements.

<p>(A) Fraction of TSTRs or random control regions (all size normalized to 1 kb from center) that are under strong evolutionary constraint (30 vertebrate phastCons; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004610#s4" target="_blank"><b>Methods</b></a>). Error bars represent 95% binomial proportion confidence interval. (B) Heatmap of Pearson correlation coefficient between tissue-specificity of TSTRs and nearby genes (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004610#s4" target="_blank"><b>Methods</b></a>). Genes 1 to 5 indicates the first to the fifth closest genes to the corresponding TSTR regardless of strand. For comparison, correlation with random genes on the same chromosome as the TSTR is shown. (C and D) Heatmap of p300 binding and H3K27ac signal within a −25 kb to +25 kb window surrounding the center of all heart TSTRs (C) or all limb TSTRs (D). Each line represents a single TSTR for individual tissues, and color scale indicates the normalized signal from individual ChIP-Seq experiment (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004610#s4" target="_blank"><b>Methods</b></a>).</p

Tissue-specific eRNA expression at a subset of tissue-specific <i>in vivo</i> enhancers.

<p>(A) The expression of eRNAs was quantified by RT-PCR for 8 randomly selected known limb enhancers in three tissues. (B) Tissue-specific eRNA expression from 7 known forebrain-specific enhancers. The expression of eRNAs were quantified by RT-PCR for 7 randomly selected forebrain enhancers in three tissues. Results from triplicate experiments were plotted (forebrain: blue; heart: red; limb: green). Error bars represent SEM. Representative LacZ-stained embryos at E11.5 from transgenic assays for individual elements are shown at the bottom. Arrowheads indicate reproducible LacZ staining patters in limb (green) or forebrain (blue).</p

Global eRNA expression profiles.

<p>(A) Tissue- and strand-specific eRNA expression around a known heart enhancer (hs1670). Scales corresponding to read count are shown on the left. Genomic region cloned for the transgenic reporter assay is indicated by the green bar. Representative LacZ-stained embryos at E11.5 from transgenic assays for element hs1670 are shown at the bottom. Red arrowheads indicate reproducible LacZ staining pattern in heart. (B) Differential eRNA expression at known heart- or limb-specific enhancers correlates with the tissue-specificity of <i>in vivo</i> enhancer activities. Log2-transformed expression fold-changes of eRNAs arising from heart- (red) or limb-specific (cyan) enhancers are plotted against their associated p-value for each fold change (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004610#s4" target="_blank"><b>Methods</b></a>). (C–F) Cumulative strand-specific eRNA expression across candidate enhancers in a 10 kb window centered on p300 (C/D) or H3K27ac (E/F) ChIP-Seq peaks from the respective tissue. Sequencing reads mapped to forward strand (red in heart, blue in limb) or reverse strand (pink in heart, cyan in limb) are displayed separately.</p

<i>De novo</i> identification of tissue-specifically transcribed regions.

<p>Dot plot showing all TSTRs identified by total RNA-Seq from heart (A) and limb (B) E11.5 tissues. Cyan and red dots indicate limb- or heart-specific TSTRs (p<0.01). Grey dots indicate RNA peaks without significant expression differences between the two tissues. RPKM<2⁻⁹ were arbitrarily set to 2⁻⁹ for visualization purposes (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004610#s4" target="_blank"><b>Methods</b></a>). A total of 22 candidate TSTRs were selected from heart (C) or limb (D) TSTRs. Tissue-specific RNA expression were quantified by RT-PCR by using total RNA samples from heart or limb tissues at E11.5 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004610#s4" target="_blank"><b>Methods</b></a>). Error bars represent SEM.</p

Transgenic characterization of TSTRs for tissue-specific enhancer activity.

<p>For each tested element, lateral views of whole-mount LacZ-stained embryos at E11.5 are shown in top left panels and transverse sections through heart or limb regions are shown in the top right panels. Arrowheads indicate reproducible LacZ staining pattern in heart (red) or limb (blue). Element ID and reproducibility of expression patterns are indicated at the bottom of the images. Strand-specific eRNA coverage of the tested regions in heart (red) or limb (blue) is shown in the bottom panels. Scales corresponding to read count are shown on the left of the coverage. Genomic regions cloned for the transgenic assay are indicated by green bars. (A) Enhancer element mm1052 with activity in both atrial and ventricular regions. (B) Enhancer element mm1018 shows activity in the right and left atrium. (C) Enhancer element 1054 with activity exclusively in the right and left ventricle. (D) Enhancer element mm1064 is active in the anterior domains of both forelimb and hindlimb, and only transverse section of forelimb is shown as an example. RA: right atrium; LA: left atrium; RV: right ventricle; LV: left ventricle; RFL; right forelimb; LFL: left forelimb. Transgenic results of all tested elements are available through the Vista Enhancer Browser (<a href="http://enhancer.lbl.gov" target="_blank">http://enhancer.lbl.gov</a>).</p

Limb-Enhancer Genie: An accessible resource of accurate enhancer predictions in the developing limb

<div><p>Epigenomic mapping of enhancer-associated chromatin modifications facilitates the genome-wide discovery of tissue-specific enhancers <i>in vivo</i>. However, reliance on single chromatin marks leads to high rates of false-positive predictions. More sophisticated, integrative methods have been described, but commonly suffer from limited accessibility to the resulting predictions and reduced biological interpretability. Here we present the <u>L</u>imb-<u>E</u>nhancer <u>G</u>enie (LEG), a collection of highly accurate, genome-wide predictions of enhancers in the developing limb, available through a user-friendly online interface. We predict limb enhancers using a combination of >50 published limb-specific datasets and clusters of evolutionarily conserved transcription factor binding sites, taking advantage of the patterns observed at previously <i>in vivo</i> validated elements. By combining different statistical models, our approach outperforms current state-of-the-art methods and provides interpretable measures of feature importance. Our results indicate that including a previously unappreciated score that quantifies tissue-specific nuclease accessibility significantly improves prediction performance. We demonstrate the utility of our approach through <i>in vivo</i> validation of newly predicted elements. Moreover, we describe general features that can guide the type of datasets to include when predicting tissue-specific enhancers genome-wide, while providing an accessible resource to the general biological community and facilitating the functional interpretation of genetic studies of limb malformations.</p></div

LEG predicts <i>bona fide</i> limb-enhancers genome-wide.

<p>(<b>A</b>) Overall enrichment scores (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005720#sec004" target="_blank">Methods</a>) for the indicated functional terms based on the proximity of the newly predicted elements to the genes annotated within each category (see also <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005720#pcbi.1005720.s007" target="_blank">S6 Fig</a>). (<b>B</b>) UCSC genome browser [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005720#pcbi.1005720.ref025" target="_blank">25</a>] snapshots showing the landscape at two previously <i>in vivo</i> validated limb-enhancers that were not part of the training set but were identified in the top 10,000 predictions. The region on the left is the ZRS (ZPA Regulatory Sequence), a known regulatory element for <i>Shh</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005720#pcbi.1005720.ref043" target="_blank">43</a>]; the one on the right is an intronic enhancer of <i>Tfap2a</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005720#pcbi.1005720.ref044" target="_blank">44</a>]. (<b>C</b>) UCSC genome browser snapshot of the <i>Hand2</i> gene locus. The probability of being a limb-enhancer (Ridge model) along with the top 5,000 predictions from both the Ridge Regression (RR) and the Sum Of Ranks (SOR) combined models are shown. The four elements tested for activity in the developing limbs are highlighted in boxes (green for those showing activity in the limbs at E11.5, red if negative). <i>LacZ</i> reporter staining (blue) indicates enhancer activities in the fore- and hindlimb mesenchyme at E11.5. One representative whole mount picture is reported for each tested element. Pictures of a representative forelimb and hindlimb are provided for the validated enhancers. Reproducibility is indicated in brackets below each whole mount picture, along with the corresponding VISTA identifier. The ranks for both combined scores (RR, SOR) are also reported. Scale bar, 100 μm.</p

LEG performances compared to two state-of-the-art approaches.

<p>LEG performances compared to two state-of-the-art approaches.</p