Enhancer transcription detected in the nascent transcriptomic landscape of bread wheat

Abstract

The precise spatiotemporal gene expression is orchestrated by enhancers that lack general sequence features and thus are difficult to be computationally identified. By nascent RNA sequencing combined with epigenome profiling, we detect active transcription of enhancers from the complex bread wheat genome. We find that genes associated with transcriptional enhancers are expressed at significantly higher levels, and enhancer RNA is more precise and robust in predicting enhancer activity compared to chromatin features. We demonstrate that sub-genome-biased enhancer transcription could drive sub-genome-biased gene expression. This study highlights enhancer transcription as a hallmark in regulating gene expression in wheat.

Keywords: Bread wheat; Enhancer; Nascent RNA; Subgenome-bias; eRNA.

Fig. 1

Unstable intergenic transcripts captured by GRO-seq and pNET-seq and eRNA characterization. a GRO-seq and pNET-seq reads aligned to different genomic regions. TSS1K indicates the 1 kb upstream region of the gene transcription start site. TES1K indicates the 1 kb downstream region of the transcription end site/polyadenylation site. b Browser shot of a protein-coding transcript and an intergenic transcript, the number of reads aligned to forward (+) and reverse (−) genomic strands are separately displayed. c Expression levels of nascent/steady-state transcripts from protein-coding and intergenic regions. d Read densities of pNET-seq, DHS and ChIP-seq of Pol II, H3K9ac, H3K4me3, H3K36me3, H3K27ac, and H3K4me1 around the intergenic and genic TCs (± 3 kb). All intergenic and genic TCs were ranked in a descending order of pNET-seq signals (±250 bp around the 5′ end), and chromatin features were plotted around the 5′ end of each intergenic TC. e pNET-seq signals around the intergenic TCs. Intergenic TCs were divided into ten equal parts based on the decreasing level of pNET-seq signals (± 250 bp). f Read densities of DHS and ChIP-seq of H3K9ac, H3K4me3, H3K36me3, H3K27ac, and H3K4me1 around each of the ten parts of intergenic TCs. g Intergenic TCs counts within different chromatin states defined in wheat genome [1]. h Chromatin signatures of transcribed and untranscribed enhancer examples. i Boxplots showing the expression levels of unidirectional and bidirectional enhancers determined by pNET-seq. j A heatmap displays the expression levels of unidirectional and bidirectional enhancers in primary (sense) and/or secondary (antisense) orientations (± 3 kb)

Fig. 2

A putative role of enhancer transcription in regulating subgenome-divergent gene expression and conservation of eRNA regions. a–d Boxplots showing transcript levels of target genes. a Genes associated with transcribed enhancer(s) display higher expression levels. b The enhancer transcriptional level positively correlates with its target gene expression. c The number of associated transcribed enhancers positively correlates with its target gene expression. d Genes targeted by unidirectional or bidirectional enhancers show no significant differences in expression levels. e The enhancer activity (relative intensity) determined in wheat protoplasts of transcribed and untranscribed enhancer candidates. f Correlations between the enhancer activity and the nascent RNA-seq, DHS, and ChIP-seq read density in predicted enhancer regions. g Schematic illustration of the biased expression homeolog genes targeted by enhancers with different transcription levels. Histone modification correlations and physical interactions between enhancers and putative target genes were counted. h Snapshot of a triad (PR10) with subgenome-biased expression and eRNAs. i Subgenome biased transcribed enhancers are enriched with unbalanced expressed homoeologs between each two subgenome pairs (from pNET-seq data). Each dot represents a gene pair in which one homeolog is associated with at least one transcribed enhancer in the same chromosome, while the other homeolog is not. The X-axis and Y-axis coordinates represent the expression levels of the two homeolog genes respectively. Red dots represent gene pairs in which the expression level of a homeolog gene associated with transcribed enhancer(s) was significantly higher than that of the other one not associated with transcribed enhancer; while the grey dots represent a homeolog gene associated with transcribed enhancer(s) was equally or lower expressed than the other one. “A w/ transcribed enhancer, B w/o transcribed enhancer” means homeolog A is associated with transcribed enhancer(s), but homeolog B is not. The odds ratio (OR) is a ratio of two sets of odds to measure the degree to which homeolog gene expression is correlated with its association with enhancer(s). See detailed analyses and interpretation in Additional file 1: Figure S15. j Merge all homoeolog pairs in i showed that the dominantly expressed homoeologs essentially associated with transcribed enhancers. k, l Nucleotide diversity distribution of enhancers with eRNA and random intergenic regions across the genome (k) and within A, B, and D subgenomes (l)