利用 INTACT 分离的细胞核优化小麦幼苗根中的 ATAC-seq。

Optimization of ATAC-seq in wheat seedling roots using INTACT-isolated nuclei.

机构信息

University of California, Davis, CA, 95616, USA.

Howard Hughes Medical Institute, Chevy Chase, MD, 20815, USA.

出版信息

BMC Plant Biol. 2023 May 22;23(1):270. doi: 10.1186/s12870-023-04281-0.

DOI:10.1186/s12870-023-04281-0

PMID:37211599

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10201787/

Abstract

BACKGROUND

The genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however, cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification.

RESULTS

In this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks and cis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels.

CONCLUSIONS

We have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome.

摘要

背景

生物体基因组中的遗传信息组织在控制基因表达的基因和调控元件中。多个植物物种的基因组已经被测序，基因库已经被注释，然而，顺式调控元件的特征仍然较少，限制了我们对基因组功能的理解。这些元件作为招募正调控和负调控转录因子的开放平台，因此，染色质可及性是识别它们的重要特征。

结果

在这项工作中，我们在四倍体小麦中开发了一种 INTACT（特定细胞类型中标记核的分离）转基因系统，用于核纯化。然后，我们将 INTACT 系统与转座酶可及染色质测序（ATAC-seq）相结合，以鉴定小麦根尖样本中的开放染色质区域。我们的 ATAC-seq 结果显示，在基因间区和启动子区有大量开放染色质区域的富集，这是调控元件的预期特征，与在其他植物物种中获得的 ATAC-seq 结果相似。此外，根 ATAC-seq 峰与之前发表的小麦叶片原生质体的 ATAC-seq 数据有显著重叠，表明两个实验之间具有较高的重现性，以及根和叶组织中开放染色质区域的高度重叠。重要的是，我们观察到 ATAC-seq 峰与在小麦中已被功能验证的顺式调控元件之间存在重叠，并且归一化可及性与基因表达水平之间存在良好的相关性。

结论

我们已经在四倍体小麦中开发并验证了一种 INTACT 系统，该系统允许从根尖快速且高质量地纯化核。这些核成功地用于进行 ATAC-seq 实验，揭示了小麦基因组中的开放染色质区域，这将有助于鉴定顺式调控元件。这里提出的 INTACT 系统将有助于在其他组织、生长阶段和不同生长条件下开发 ATAC-seq 数据集，以生成小麦基因组中可及 DNA 区域的更完整图谱。