Comparative Profiling Examines Roles of DNA Regulatory Sequences and Accessible Chromatin during Cold Stress Response in Grasses

Physicalaccess to regulatoryDNA, including cis-regulatory sequences found within proximal promoters and distal enhancer elements, is a vital property of chromatin. In turn, their access is determined by nucleosome occupancy and posttranslational modification of histone proteins. A continuum of chromatin accessibility states from closed to permissive and open chromatin is a critical determinant of chromatin’s regulatorycapacity inmodulatinggeneexpression. Transcription factors (TFs) can dynamically regulate local access to DNA bymodulating nucleosome occupancy; in turn, the cell/ tissue-specific chromatin landscape affects TF binding. Recent identification of chromatin signatures of cis-regulatory elements across angiosperms provides a strong foundation for understanding TFs and their interaction with the chromatin environment in modulating gene expression (Lu et al. 2019). In new work, Han et al. (2020) attempt to dissect the dynamics of the chromatin landscape and regulatory elements in response to cold stress. The authors performed a comprehensive analysis of active regulatory DNA through DNase I– hypersensitive site (DHS)mappingofcontrol and cold-stressed samples from three different tissue types—root, stem, and leaves—in three grass species: Brachypodium distachyon, Setaria italica, and Sorghum bicolor. Incorporating multiple datasets such as DNase sequencing (DNAseq), RNA-seq, and Hi-C, the authors identified tissueand species-specific patterns of open chromatin in response to cold stress. Consistent expression of a GFP-reporter gene driven by predicted B. distachyon leaf DHSs in protoplast-based transient transformation provided experimental validation of promoter and enhancer functions of a subset of the identified DHSs. Many more highly expressed genes contained genic DHSs than moderateto-lowly expressed genes, and highly expressed genes on average had more DHSs in all three grass species. Genes with more than four DHSs tended to be tissue specific and correlated with a higher dynamic range of expression. Consistent with previous reports, including one with the same three species, the authors show low conservation of accessible chromatin (Maher et al., 2018; Burgess et al., 2019). However, recentwork fromLuet al. (2019) show that more than half of the accessible chromatin regions are conserved between closely related pairs of species, maizesorghum,andsoybean-phaseolus.Asignificant proportion of tissue-specific genes showed a correlation with tissue-specific DHS, as far as 50 kb, suggesting potential distal regulation of tissue-specific gene expression. Tissue-specific DHSs were shown to regulate the expression of tissue-specific genes. For example, cellulose synthase–like gene CSLD2 (Bradi1g50170) and its orthologs showed root-specific gene expression.Of the twoDHSsupstreamof the gene,onewassharedacross three tissues.At the same time, the other DHS was root specific (given their experimental resolution) and present in all three species, containing MYClike sequence motif, CATGTG, potentially targeted by NAC TFs affecting the differential expression of CSLD2 in roots (see figure). A significant proportion of cold-induced DHSs was adjacent to genes differentially expressed between control and cold-treated samples. Although they did not find any common cold-induced DHS sharedacross the threespeciesor the three tissue types in each species, cold-induced TF bindingmotifs showedhigher conservation. These conserved motifs suggest similar transcriptional regulation in different cell types controlled by common TFs across species in specific biological functions. Cold-induced DHSs were almost exclusively tissue specific, suggesting that conservationof cold stress response ingrasses is mostly tissue specific. The authors identified 17TFs sharedacross all three species in the three tissues tested, including known regulators of cold stress-responsive genes. Han et al. (2020) provide a dynamic view of accessible chromatin and transcriptional regulatory networks in response to an important environmental challenge.Accessible chromatin is variable in number and location across species (Maher etal.,2018),andacompleteunderstanding of core regulatory mechanisms behind complex traits such as stress response is not feasible merely by looking at accessible chromatin. However, despite low Tissue-Specific DHS and TF Binding Motif in the Tissue-Specific Gene CSLD2.