Bread wheat is an allohexaploid species with a 16-Gb genome that has large intergenic regions, which presents a big challenge for pinpointing regulatory elements and further revealing the transcriptional regulatory mechanisms. Chromatin profiling to characterize the combinatorial patterns of chromatin signatures is a powerful means to detect functional elements and clarify regulatory activities in human studies. In the present study, through comprehensive analyses of the open chromatin, DNA methylome, seven major chromatin marks, and transcriptomic data generated for seedlings of allohexaploid wheat, we detected distinct chromatin architectural features surrounding various functional elements, including genes, promoters, enhancer-like elements, and transposons. Thousands of new genic regions and cis-regulatory elements are identified based on the combinatorial pattern of chromatin features. Roughly 1.5% of the genome encodes a subset of active regulatory elements, including promoters and enhancer-like elements, which are characterized by a high degree of chromatin openness and histone acetylation, an abundance of CpG islands, and low DNA methylation levels. A comparison across sub-genomes reveals that evolutionary selection on gene regulation is targeted at the sequence and chromatin feature levels. The divergent enrichment of cis-elements between enhancer-like sequences and promoters implies these functional elements are targeted by different transcription factors. We herein present a systematic epigenomic map for the annotation of cis-regulatory elements in the bread wheat genome, which provides new insights into the connections between chromatin modifications and cis-regulatory activities in allohexaploid wheat.

Summary: Although polyploid plants have larger leaves than their diploid counterparts, the molecular mechanisms underlying this difference (or trait) remain elusive. Differentially expressed genes (DEGs) between triploid and full‐sib diploid poplar trees were identified from two transcriptomic data sets followed by a gene association study among DEGs to identify key leaf growth regulators. Yeast one‐hybrid system, electrophoretic mobility shift assay, and dual‐luciferase assay were employed to substantiate that PpnGRF5‐1 directly regulated PpnCKX1. The interactions between PpnGRF5‐1 and growth‐regulating factor (GRF)‐interacting factors (GIFs) were experimentally validated and a multilayered hierarchical regulatory network (ML‐hGRN)‐mediated by PpnGRF5‐1 was constructed with top‐down graphic Gaussian model (GGM) algorithm by combining RNA‐sequencing data from its overexpression lines and DAP‐sequencing data. PpnGRF5‐1 is a negative regulator of PpnCKX1. Overexpression of PpnGRF5‐1 in diploid transgenic lines resulted in larger leaves resembling those of triploids, and significantly increased zeatin and isopentenyladenine in the apical buds and third leaves. PpnGRF5‐1 also interacted with GIFs to increase its regulatory diversity and capacity. An ML‐hGRN‐mediated by PpnGRF5‐1 was obtained and could largely elucidate larger leaves. PpnGRF5‐1 and the ML‐hGRN‐mediated by PpnGRF5‐1 were underlying the leaf growth and development.

Wheat (Triticum aestivum) has a large allohexaploid genome. Subgenome-divergent regulation contributed to genome plasticity and the domestication of polyploid wheat. However, the specificity encoded in the wheat genome determining subgenome-divergent spatio-temporal regulation has been largely unexplored. The considerable size and complexity of the genome are major obstacles to dissecting the regulatory specificity. Here, we compared the epigenomes and transcriptomes from a large set of samples under diverse developmental and environmental conditions. Thousands of distal epigenetic regulatory elements (distal-epiREs) were specifically linked to their target promoters with coordinated epigenomic changes. We revealed that subgenome-divergent activity of homologous regulatory elements is affected by specific epigenetic signatures. Subgenome-divergent epiRE regulation of tissue specificity is associated with dynamic modulation of H3K27me3 mediated by Polycomb complex and demethylases. Furthermore, quantitative epigenomic approaches detected key stress responsive cis- and trans-acting factors validated by DNA Affinity Purification and sequencing, and demonstrated the coordinated interplay between epiRE sequence contexts, epigenetic factors, and transcription factors in regulating subgenome divergent transcriptional responses to external changes. Together, this study provides a wealth of resources for elucidating the epiRE regulomics and subgenome-divergent regulation in hexaploid wheat, and gives new clues for interpreting genetic and epigenetic interplay in regulating the benefits of polyploid wheat.

Genomic regions free of nucleosomes, which are hypersensitive to DNase I digestion, are known as DNase I hypersensitive sites (DHSs) and frequently contain cis-regulatory DNA elements. To investigate their prevalence and characteristics in maize (Zea mays), we developed high-resolution genome-wide DHS maps using a modified DNase-seq technique. Maize DHSs exhibit depletion of nucleosomes and low levels of DNA methylation and are enriched with conserved noncoding sequences (CNSs). We developed a protoplast-based transient transformation assay to assess the potential gene expression enhancer and/or promoter functions associated with DHSs, which showed that more than 80% of DHSs overlapping with CNSs showed an enhancer function. Strikingly, nearly 25% of maize DHSs were derived from transposable elements (TEs), including both class I and class II transposons. Interestingly, TE-derived DHSs (teDHSs) homologous to retrotransposons were enriched with sequences related to the intrinsic cis-regulatory elements within the long terminal repeats of retrotransposons. We demonstrate that more than 80% of teDHSs can drive transcription of a reporter gene in protoplast assays. These results reveal the widespread occurrence of TE-derived cis-regulatory sequences and suggest that teDHSs play a major role in transcriptional regulation in maize.

The success of common wheat as a global staple crop was largely attributed to its genomic diversity and redundancy due to the merge of different genomes, giving rise to the major question how subgenome-divergent and -convergent transcription is mediated and harmonized in a single cell. Here, we create a catalog of genome-wide transcription factor-binding sites (TFBSs) to assemble a common wheat regulatory network on an unprecedented scale. A significant proportion of subgenome-divergent TFBSs are derived from differential expansions of particular transposable elements (TEs) in diploid progenitors, which contribute to subgenome-divergent transcription. Whereas subgenome-convergent transcription is associated with balanced TF binding at loci derived from TE expansions before diploid divergence. These TFBSs have retained in parallel during evolution of each diploid, despite extensive unbalanced turnover of the flanking TEs. Thus, the differential evolutionary selection of paleo- and neo-TEs contribute to subgenome-convergent and -divergent regulation in common wheat, highlighting the influence of TE repertory plasticity on transcriptional plasticity in polyploid.

Background: Regulation of transcription depends on interactions betweencis-regulatory elements (CREs) and regulatory proteins. Active CREs are imbedded in open chromatin that are accessible to nucleases. Several techniques, including DNase-seq, which is based on nuclease DNase I, and ATAC-seq, which is based on transposase Tn5, have been widely used to identify genomic regions associated with open chromatin. These techniques have played a key role in dissecting the regulatory networks in gene expression in both animal and plant species.Results: We develop a technique, named MNase hypersensitivity sequencing (MH-seq), to identify genomic regions associated with open chromatin inArabidopsis thaliana. Genomic regions enriched with MH-seq reads are referred as MNase hypersensitive sites (MHSs). MHSs overlap with the majority (~ 90%) of the open chromatin identified previously by DNase-seq and ATAC-seq. Surprisingly, 22% MHSs are not covered by DNase-seq or ATAC-seq reads, which are referred to “specific MHSs” (sMHSs). sMHSs tend to be located away from promoters, and a substantial portion of sMHSs are derived from transposable elements. Most interestingly, genomic regions containing sMHSs are enriched with epigenetic marks, including H3K27me3 and DNA methylation. In addition, sMHSs show a number of distinct characteristics including association with transcriptional repressors. Thus, sMHSs span distinct classes of open chromatin that may not be accessible to DNase I or Tn5. We hypothesize that the small size of the MNase enzyme relative to DNase I or Tn5 allows its access to relatively more condensed chromatin domains.Conclusion: MNase can be used to identify open chromatin regions that are not accessible to DNase I or Tn5. Thus, MH-seq provides an important tool to identify and catalog all classes of open chromatin in plants.