Assaying Chromatin Accessibility Using ATAC-Seq in Invertebrate Chordate Embryos - PubMed (original) (raw)
Assaying Chromatin Accessibility Using ATAC-Seq in Invertebrate Chordate Embryos
Marta Silvia Magri et al. Front Cell Dev Biol. 2020.
Abstract
_Cis_-regulatory elements (CREs) are non-coding DNA regions involved in the spatio-temporal regulation of gene expression. Gene regulatory changes drive animal development and play major roles during evolution of animal body plans. Therefore, we believe that determining CREs at different developmental stages and across animal lineages is critical to understand how evolution operates through development. The Assay for Transposase-Accessible Chromatin followed by high-throughput sequencing (ATAC-seq) is a powerful technique for the study of CREs that takes advantage of Tn5 transposase activity. Starting from fewer than 105 cells, in a 1-day procedure, it is possible to detect, at a genome-wide level, CREs located in open chromatin regions with high resolution. Here, we describe a detailed step-by-step ATAC-seq protocol for invertebrate chordate marine embryos. We have successfully applied this technique to amphioxus and two species of tunicate embryos. We also show an easy workflow to analyze data generated with this technique. Moreover, we point out that this method and our bioinformatic pipeline are efficient to detect CREs associated with Wnt signaling pathway by simply using embryos treated with a drug that perturbs this pathway. This approach can be extended to other signaling pathways and also to embryo mutants for critical genes. Our results therefore demonstrate the power of ATAC-seq for the identification of CREs that play essential functions during animal development in a wide range of invertebrate or vertebrate animals.
Keywords: ATAC-seq; amphioxus; cis-regulatory elements; development; evolution; invertebrate chordates; tunicates.
Copyright © 2020 Magri, Jiménez-Gancedo, Bertrand, Madgwick, Escrivà, Lemaire and Gómez-Skarmeta.
Figures
FIGURE 1
(A) ATAC-seq cartoon reaction. The Tn5 transposase (dark pink) inserts two sequencing adapters (yellow and blue) only in accessible regions, between nucleosomes and binding sites for proteins (green), such as transcription factors (TF). Promoter regions (P) and transcription start sites (TSS) are also considered open chromatin regions. Tn5 generates sequencing fragments that can be amplified by PCR. (B) ATAC-seq tracks in the six3 region from chordates embryos at late gastrula stage. ATAC-seq tracks are marked in orange while the gene model is represented in blue. Mapped sequenced reads, <130 bp, identify discrete open chromatin regions mostly upstream the body gene region.
FIGURE 2
ATAC-seq data and analysis of Ciona embryos at the 112-cell stage after Wnt/β-catenin signaling pathway was altered. (A) Snapshot from the UCSC Browser of Ciona genome showing the ATAC-seq signal around the nkx2.1 gene. The gene model is represented in blue. ATAC-seq track of embryos treated with a GSK3 inhibitor is shown in orange whereas ATAC-seq track of wt embryos is in black. Gray bars mark called peaks, whereas black bars indicate peaks that are significantly higher in treated embryos. (B) Most relevant GO results generated by PANTHER. In orange: GO term bars related to genes associated with chromatin regions significantly more accessible in embryos where the activity of Wnt pathway was increased. In brown: GO term bars related to genes associated with more accessible chromatin regions in wt embryos. (C) HOMER results for peaks differentially represented between wt and the perturbed condition. In the orange box: known motif enrichment results of peaks significantly more represented when Wnt/β-catenin signaling pathway was increased. In the brown box: known motif enrichment results of peaks significantly more represented in wt embryos.
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