The Position and Complex Genomic Architecture of Plant T-DNA Insertions Revealed by 4SEE.

The integration of T-DNA in plant genomes is widely used for basic research and agriculture. The high heterogeneity in the number of integration events per genome, their configuration, and their impact on genome integrity highlight the critical need to detect the genomic locations of T-DNA insertions and their associated chromosomal rearrangements, and the great challenge in doing so. Here, we present 4SEE, a circular chromosome conformation capture (4C)-based method for robust, rapid, and cost-efficient detection of the entire scope of T-DNA locations. Moreover, by measuring the chromosomal architecture of the plant genome flanking the T-DNA insertions, 4SEE outlines their associated complex chromosomal aberrations. Applying 4SEE to a collection of confirmed T-DNA lines revealed previously unmapped T-DNA insertions and chromosomal rearrangements such as inversions and translocations. Uncovering such events in a feasible, robust, and cost-effective manner by 4SEE in any plant of interest has implications for accurate annotation and phenotypic characterization of T-DNA insertion mutants and transgene expression in basic science applications as well as for plant biotechnology.

Regulatory chromatin landscape in Arabidopsis thaliana roots uncovered by coupling INTACT and ATAC-seq.

BACKGROUND: There is a growing interest in the role of chromatin in acquiring and maintaining cell identity. Despite the ever-growing availability of genome-wide gene expression data, understanding how transcription programs are established and regulated to define cell identity remains a puzzle. An important mechanism of gene regulation is the binding of transcription factors (TFs) to specific DNA sequence motifs across the genome. However, these sequences are hindered by the packaging of DNA to chromatin. Thus, the accessibility of these loci for TF binding is highly regulated and determines where and when TFs bind. We present a workflow for measuring chromatin accessibility in Arabidopsis thaliana and define organ-specific regulatory sites and binding motifs of TFs at these sites. RESULTS: We coupled the recently described isolation of nuclei tagged in specific cell types (INTACT) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) as a genome-wide strategy to uncover accessible regulatory sites in Arabidopsis based on their accessibility to nuclease digestion. By applying this pipeline in Arabidopsis roots, we revealed 41,419 accessible sites, of which approximately half are found in gene promoters and contain the H3K4me3 active histone mark. The root-unique accessible sites from this group are enriched for root processes. Interestingly, most of the root-unique accessible sites are found in nongenic regions but are correlated with root-specific expression of distant genes. Importantly, these gene-distant sites are enriched for binding motifs of TFs important for root development as well as motifs for TFs that may play a role as novel transcriptional regulators in roots, suggesting that these accessible loci are functional novel gene-distant regulatory elements. CONCLUSIONS: By coupling INTACT with ATAC-seq methods, we present a feasible pipeline to profile accessible chromatin in plants. We also introduce a rapid measure of the experiment quality. We find that chromatin accessibility at promoter regions is strongly associated with transcription and active histone marks. However, root-specific chromatin accessibility is primarily found at intergenic regions, suggesting their predominance in defining organ identity possibly via long-range chromatin interactions. This workflow can be rapidly applied to study the regulatory landscape in other cell types, plant species and conditions.