Deep sequencing of DNase-I treated chromatin (DNase-seq) can be used to identify DNase I-hypersensitive sites (DHSs) and facilitates genome-scale mining of de novo cis-regulatory DNA elements. Here, we adapted DNase-seq to generate genome-wide maps of DHSs using control and cold-treated leaf, stem, and root tissues of three widely studied grass species: Brachypodium distachyon, foxtail millet (Setaria italica), and sorghum (Sorghum bicolor). Functional validation demonstrated that 12 of 15 DHSs drove reporter gene expression in transiently transgenic B. distachyon protoplasts. DHSs under both normal and cold treatment substantially differed among tissues and species. Intriguingly, the putative DHS-derived transcription factors (TFs) are largely colocated among tissues and species and include 17 ubiquitous motifs covering all grass taxa and all tissues examined in this study. This feature allowed us to reconstruct a regulatory network that responds to cold stress. Ethylene-responsive TFs SHINE3, ERF2, and ERF9 occurred frequently in cold feedback loops in the tissues examined, pointing to their possible roles in the regulatory network. Overall, we provide experimental annotation of 322,713 DHSs and 93 derived cold-response TF binding motifs in multiple grasses, which could serve as a valuable resource for elucidating the transcriptional networks that function in the cold-stress response and other physiological processes.

DNase-I 处理的染色质的深度测序 (DNase-seq) 可用于识别 DNase I 超敏感位点 (DHS)，并促进基因组规模的从头顺式调控DNA 元件的挖掘。在这里，我们采用 DNase-seq 技术，使用三种广泛研究的草种的对照和冷处理的叶、茎和根组织来生成 DHS 的全基因组图谱：二穗短柄草、谷子 ( Setaria italica ) 和高粱 ( Sorghum bicolor) ）。功能验证表明，15 个 DHS 中有 12 个在瞬时转基因二穗 B.原生质体中驱动报告基因表达。正常处理和冷处理下的 DHS 在组织和物种之间存在显着差异。有趣的是，假定的 DHS 衍生转录因子 (TF) 主要在组织和物种之间共存，并包括 17 个普遍存在的基序，涵盖本研究中检查的所有草类群和所有组织。这一特征使我们能够重建响应冷应激的调节网络。乙烯响应性转录因子 SHINE3、ERF2 和 ERF9 频繁出现在所检查组织的冷反馈回路中，表明它们在调控网络中可能发挥作用。总体而言，我们提供了多种草中 322,713 个 DHS 和 93 个衍生的冷反应 TF 结合基序的实验注释，这可以作为阐明在冷应激反应和其他生理过程中发挥作用的转录网络的宝贵资源。