State Key Laboratory for Crop Genetics and Germplasm Enhancement, JCIC-MCP, CIC-MCP, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China.
Department of Plant Biology, Michigan State University, East Lansing, MI, USA.
Methods Mol Biol. 2023;2594:29-43. doi: 10.1007/978-1-0716-2815-7_3.
Functional cis-regulatory elements (CREs) act as precise transcriptional switches for fine-tuning gene transcription. Identification of CREs is critical for understanding regulatory mechanisms of gene expression associated with various biological processes in eukaryotes. It is well known that CREs reside in open chromatin that exhibits hypersensitivity to enzyme cleavage and physical shearing. Currently, high-throughput methodologies, such as DNase-seq, ATAC-seq, and FAIRE-seq, have been widely applied in mapping open chromatin in various eukaryotic genomes. More recently, differential MNase (micrococcal nuclease) treatment has been successfully employed to map open chromatin in addition to profiling nucleosome landscape in both mammalian and plant species. We have developed a MNase hypersensitivity sequencing (MH-seq) technique in plants. The MH-seq procedure includes plant nuclei fixation and purification, differential treatments of purified nuclei with MNase, specific recovery of MNase-trimmed small DNA fragments within 20~100 bp in length, and MH-seq library construction followed by Illumina sequencing and data analysis. MH-seq has been successfully applied for global identification of open chromatin in both Arabidopsis thaliana and maize. It has been proven to be an attractive alternative for profiling open chromatin. Thus, MH-seq is expected to be valuable in probing chromatin accessibility on a genome-wide scale for other plants with sequenced genomes. Moreover, MHS data allow to implement footprinting assays to unveil binding sites of transcription factors.
功能顺式调控元件 (CREs) 作为精确的转录开关，可精细调节基因转录。鉴定 CREs 对于理解真核生物中与各种生物学过程相关的基因表达调控机制至关重要。众所周知，CREs 位于开放染色质中，该染色质对酶切割和物理剪切表现出超敏性。目前，高通量方法，如 DNase-seq、ATAC-seq 和 FAIRE-seq，已广泛应用于绘制各种真核生物基因组中的开放染色质图谱。最近，差异 MNase（微球菌核酸酶）处理已成功用于绘制开放染色质图谱，此外还可在哺乳动物和植物物种中分析核小体景观。我们在植物中开发了 MNase 超敏性测序 (MH-seq) 技术。MH-seq 程序包括植物细胞核固定和纯化、用 MNase 对纯化的细胞核进行差异处理、特异性回收长度在 20~100bp 之间的 MNase 修剪的小 DNA 片段、以及 MH-seq 文库构建，随后进行 Illumina 测序和数据分析。MH-seq 已成功应用于拟南芥和玉米中开放染色质的全局鉴定。事实证明，它是一种有吸引力的替代方法，可用于分析开放染色质。因此，MH-seq 有望在探测其他具有测序基因组的植物的全基因组范围内的染色质可及性方面具有重要价值。此外，MHS 数据可用于实施足迹测定法来揭示转录因子的结合位点。
