Brady, S. M. — Page 2 | Search bioRxiv

bioRxiv2017

Nuclear transcriptomes at high resolution using retooled INTACT

Isolated nuclei provide access to early steps in gene regulation involving chromatin as well as transcript production and processing. Here we describe transfer of the Isolation of Nuclei from TAgged specific Cell Types (INTACT) to the monocot rice (Oryza sativa L.). The purification of biotinylated nuclei was redesigned by replacing the outer nuclear envelope-targeting domain of the Nuclear Tagging Fusion (NTF) protein with an outer nuclear envelope-anchored domain. This modified NTF was combined with codon optimized E. coli BirA in a single T-DNA construct. We also developed inexpensive methods for INTACT, T-DNA insertion mapping and profiling of the complete nuclear transcriptome, including a rRNA degradation procedure that minimizes pre-rRNA transcripts. A high-resolution comparison of nuclear and steady-state poly (A)+ transcript populations of seedling root tips confirmed the capture of pre-mRNA and exposed distinctions in diversity and abundance of the nuclear and total transcriptomes. This retooled INTACT can enable high-resolution monitoring of the nuclear transcriptome and chromatin in specific cell-types of rice and other species.\n\nSummaryImproved technology and methodology for affinity purification of nuclei and analysis of nuclear transcriptomes, chromatin and other nuclear components.

plant biology↗

bioRxiv2017

Profiling of accessible chromatin regions across multiple plant species and cell types reveals common gene regulatory principles and new control modules

The transcriptional regulatory structure of plant genomes remains poorly defined relative to animals. It is unclear how many cis-regulatory elements exist, where these elements lie relative to promoters, and how these features are conserved across plant species. We employed the Assay for Transposase-Accessible Chromatin (ATAC-seq) in four plant species (Arabidopsis thaliana, Medicago truncatula, Solanum lycopersicum, and Oryza sativa) to delineate open chromatin regions and transcription factor (TF) binding sites across each genome. Despite 10-fold variation in intergenic space among species, the majority of open chromatin regions lie within 3 kb upstream of a transcription start site in all species. We find a common set of four TFs that appear to regulate conserved gene sets in the root tips of all four species, suggesting that TF-gene networks are generally conserved. Comparative ATAC-seq profiling of Arabidopsis root hair and non-hair cell types revealed extensive similarity as well as many cell type-specific differences. Analyzing TF binding sites in differentially accessible regions identified a MYB-driven regulatory module unique to the hair cell, which appears to control both cell fate regulators and abiotic stress responses. Our analyses revealed common regulatory principles among species and shed light on the mechanisms producing cell type-specific transcriptomes during development.

plant biology↗