Using ATAC-seq and RNA-seq to increase resolution in GRN connectivity

Echinoderms have some of the most complete reconstructed developmental gene regulatory networks (GRN) of any embryo, accounting for the formation of most embryo tissues and organs. Yet, many nodes (genes and regulators) and their regulatory interactions are still to be uncovered. Traditionally, knockdown/knockout experiments are performed to determine regulator-gene interactions, which are individually validated by cis-regulatory analysis. Differential RNA-seq, combined with perturbation analysis, allows for genome-wide reconstruction of a GRN around given regulators; however, this level of resolution cannot determine direct interactions. ChiP-chip or ChIP-seq is better equipped for determining, genome-wide, whether binding of a given transcription factor (TF) to cis-regulatory elements occurs. Antibodies for the TFs of interest must be available, and if not, this presents a limiting factor. ATAC-seq identifies regions of open chromatin, that are typically trimethylated at H3K4, H3K36 and H3K79 (Kouzarides, 2007), for a given time point, condition, or tissue. This technology combined with RNA-seq and perturbation analysis provides high resolution of the possible functional interactions occurring during development. Additionally, ATAC-seq is less expensive than ChIP-seq, requires less starting material, and provides a global view of regulatory regions. This chapter provides detailed steps to identify potential regulatory relationships between the nodes of a GRN, given a well assembled genome, annotated with gene models, and ATAC-seq data combined with RNA-seq and knockdown experiments.