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

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

<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

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

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

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

Intramuscular triglyceride content of <i>vastus lateralis</i> for R225W and control subjects.

<p>Muscle sections were prepared and stained with OsO₄, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000903#s2" target="_blank">methods</a> section (A: control, B: R225W). C: quantification of IMTG via imaging software. Means +/− SD. n = 4, Student's <i>t</i>-test, *p = 0.032.</p

γ subunit isoforms showing the 4 tandem repeats of CBS domains responsible for AMP and ATP binding.

<p>Locations of the human R225W and the porcine R225Q mutations in the γ₃ subunit as well as the R302Q and R61Q mutations in the γ₂ long and short forms, respectively, are indicated. Figure adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000903#pone.0000903-Carling1" target="_blank">[12]</a>.</p

Characteristics of PRKAG3-R225W subjects and controls who underwent muscle biopsy.

<p>Blood chemistry measurements were conducted following an overnight fast. Means +/− SD. n = 5, Student's <i>t-</i>test, no significant differences between R225W and control subjects.</p