A network of transcription factors governs the dynamics of NODAL/Activin transcriptional responses.

SMAD2, an effector of the NODAL/Activin signalling pathway, regulates developmental processes by sensing distinct chromatin states and interacting with different transcriptional partners. However, the network of factors that controls SMAD2 chromatin binding and shapes its transcriptional programme over time is poorly characterised. Here, we combine ATAC-seq with computational footprinting to identify temporal changes in chromatin accessibility and transcription factor activity upon NODAL/Activin signalling. We show that SMAD2 binding induces chromatin opening genome wide. We discover footprints for FOXI3, FOXO3 and ZIC3 at the SMAD2-bound enhancers of the early response genes, Pmepa1 and Wnt3, respectively, and demonstrate their functionality. Finally, we determine a mechanism by which NODAL/Activin signalling induces delayed gene expression, by uncovering a self-enabling transcriptional cascade whereby activated SMADs, together with ZIC3, induce the expression of Wnt3. The resultant activated WNT pathway then acts together with the NODAL/Activin pathway to regulate expression of delayed target genes in prolonged NODAL/Activin signalling conditions. This article has an associated First Person interview with the first author of the paper.

Distinct modes of SMAD2 chromatin binding and remodeling shape the transcriptional response to NODAL/Activin signaling.

NODAL/Activin signaling orchestrates key processes during embryonic development via SMAD2. How SMAD2 activates programs of gene expression that are modulated over time however, is not known. Here we delineate the sequence of events that occur from SMAD2 binding to transcriptional activation, and the mechanisms underlying them. NODAL/Activin signaling induces dramatic chromatin landscape changes, and a dynamic transcriptional network regulated by SMAD2, acting via multiple mechanisms. Crucially we have discovered two modes of SMAD2 binding. SMAD2 can bind pre-acetylated nucleosome-depleted sites. However, it also binds to unacetylated, closed chromatin, independently of pioneer factors, where it induces nucleosome displacement and histone acetylation. For a subset of genes, this requires SMARCA4. We find that long term modulation of the transcriptional responses requires continued NODAL/Activin signaling. Thus SMAD2 binding does not linearly equate with transcriptional kinetics, and our data suggest that SMAD2 recruits multiple co-factors during sustained signaling to shape the downstream transcriptional program.