Combining ATAC-seq with nuclei sorting for discovery of cis-regulatory regions in plant genomes

Abstract

Chromatin structure plays a pivotal role in facilitating proper control of gene expression. Transcription factor (TF) binding of cis-elements is often associated with accessible chromatin regions. Therefore, the ability to identify these accessible regions throughout plant genomes will advance understanding of the relationship between TF binding, chromatin status and the regulation of gene expression. Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) is a recently developed technique used to map open chromatin zones in animal genomes. However, in plants, the existence of cell walls, subcellular organelles and the lack of stable cell lines have prevented routine application of this technique. Here, we describe an assay combining ATAC-seq with fluorescence-activated nuclei sorting (FANS) to identify and map open chromatin and TF-binding sites in plant genomes. FANS-ATAC-seq compares favorably with published DNaseI sequencing (DNase-seq) results and it requires less than 50 000 nuclei for accurate identification of accessible genomic regions.Summary: Application of ATAC-seq to sorted nuclei identifies accessible regions genome-wide.

Cell Type-specific Genome Scans of DNA Methylation Diversity Indicate an Important Role for Transposable Elements

“…Their increased JSD suggests that inaccessible regions tend to harbor more methylation polymorphisms than accessible regions. This is confirmed by a negative correlation of JSD with DNAse1- [30] and ATAC-seq [31] signal levels ( Fig. 3b, see Fig.…”

Nuclear Transcriptomes at High Resolution Using Retooled INTACT

“…INTACT and other methods for isolation of nuclei vary in their advantages and disadvantages. INTACT has similar applications as FANS, used to assay nuclear poly(A) + RNA, endoreduplication (Zhang et al, 2008), and regions of open chromatin by assay for transposase accessible chromatin-sequencing (Lu et al, 2017;Bajic et al, 2018). INTACT constructs can be driven by nearconstitutive, cell-type-and region-specific promoters, enabling assay of subpopulation of nuclei of multicellular organs (Deal and Henikoff, 2010).…”

Chromatin accessibility changes betweenArabidopsisstem cells and mesophyll cells illuminate cell type-specific transcription factor networks

“…All reads that aligned to the organellar genomes were subsequently omitted from downstream analyses. More than 15 million reads per replicate passed the quality filtering stage of analysis ( Figure S1a), which is more than sufficient to successfully identify accessible chromatin regions in Arabidopsis, as has been recently demonstrated (Lu et al, 2016). The processed alignment files were compared using principal component analysis (PCA) (Ramirez et al, 2016).…”

“…One substantial drawback of this powerful technique, however, is the requirement for large numbers of nuclei as a starting material. Recently, a simple and sensitive assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) has been described (Buenrostro et al, 2013(Buenrostro et al, , 2015, which requires a much smaller amount of input material (about 500-50 000 nuclei) (Lu et al, 2016). In this method, a hyperactive Tn5 transposase loaded with sequencing adapters acts to simultaneously fragment and tag a genome with these adapters.…”

“…In this method, a hyperactive Tn5 transposase loaded with sequencing adapters acts to simultaneously fragment and tag a genome with these adapters. Mapping of the transposase-hypersensitive sites allows for detection of highly accessible chromatin regions and subsequent identification of TF-binding sites within these regions (Lu et al, 2016).…”