Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures

Abstract

Enhancers are important regulators of gene expression in eukaryotes. Enhancers function independently of their distance and orientation to the promoters of target genes. Thus, enhancers have been difficult to identify. Only a few enhancers, especially distant intergenic enhancers, have been identified in plants. We developed an enhancer prediction system based exclusively on the DNase I hypersensitive sites (DHSs) in the Arabidopsis thaliana genome. A set of 10,044 DHSs located in intergenic regions, which are away from any gene promoters, were predicted to be putative enhancers. We examined the functions of 14 predicted enhancers using the b-glucuronidase gene reporter. Ten of the 14 (71%) candidates were validated by the reporter assay. We also designed 10 constructs using intergenic sequences that are not associated with DHSs, and none of these constructs showed enhancer activities in reporter assays. In addition, the tissue specificity of the putative enhancers can be precisely predicted based on DNase I hypersensitivity data sets developed from different plant tissues. These results suggest that the open chromatin signature-based enhancer prediction system developed in Arabidopsis may serve as a universal system for enhancer identification in plants.

Nascent RNA sequencing reveals distinct features in plant transcription

“…H3K4me3, H3K27ac, and H3K36me3 peaked in proximity to the +1 nucleosome and H3K4me2 slightly downstream (23,25). DNase hypersensitivity overlapped with the promoter region, and H3K4me1 and H3K36me2 increase along the body of the gene (18,23,25), as expected (30,31) (Fig. 2 E).…”

“…Therefore, given the absence of NELF, potential eRNAs may not have provided the same selective advantages in plants. In contrast, however, Zhu et al (25) predicted over 10,000 plant enhancers based on chromatin signatures in leaves and flowers. Without tissue-matched GRO-seq data for these predicted enhancers or targeted disruption, it is difficult to validate their in vivo role or potential for enhancer transcription.…”

“…Very little GRO-seq, RNA-seq, or enhancer-associated chromatin marks (H3K9/27ac) were found in Arabidopsis and maize compared with humans, and both plants lack the distinctive bidirectional transcription common at mammalian enhancers (Fig. 1F) (8,12,(18)(19)(20)(21)(22)(23)(24)(25). Given these data, it appears that if plants have enhancer elements, they rarely, if at all, produce transcripts and therefore differ from mammalian enhancers.…”

“…In total, 2,467 putative intergenic enhancers were identified in Arabidopsis and 4,665 in maize compared with 21,847 in the human lung fibroblast IMR-90 cell line. Each site was sorted based on their GRO-seq signals, and heat maps were generated for ±1 kb from the intergenic site of (19)], and heat maps were generated ±1 kb from intergenic sites for signal from DNase-seq, GROseq, RNA-seq, H3K4me3, H3K9/27ac, and input in Arabidopsis, maize, and IMR-90 cells (8,12,(18)(19)(20)(21)(22)(23)(24)(25). Sites are sorted based on the total GRO-seq signal observed within 400 bp of the intergenic peak.…”

Towards genome-wide prediction and characterization of enhancers in plants

“…However, for true validation of an enhancer, biochemical assays are needed to establish functional activity. We were able to identify and validate leaf-and flower-specific enhancers by utilizing a subset of intergenic DHSs in A. thaliana [83]. The sequence of each predicted enhancer was cloned into a GUS-based reporter vector.…”