Distinct modes of SMAD2 chromatin binding and remodeling shape the transcriptional response to Nodal/Activin signaling [SMAD2 ChIP & RNA-seq]. Mus musculus

During development, key processes are orchestrated by Nodal/Activin signaling via SMAD2. Interplay between the SMADs, co-factors and chromatin determines cell-type specific responses, but the sequence of events underpinning SMAD2-mediated transcription is unknown. We performed RNA-and ChIP-sequencing for SMAD2, RNA Polymerase II and various histone modifications in different signaling states. Integration of these data reveals a dynamic transcriptional network downstream of Nodal/Activin signaling regulated by SMAD2 acting via multiple mechanisms. Upon ligand stimulation, SMAD2 can bind to pre-acetylated nucleosome-depleted sites, but also to unacetylated closed chromatin, where it induces nucleosome displacement and histone acetylation. Importantly, SMAD2 binding is highly dynamic and does not directly correlate with the transcriptional kinetics of target genes. Moreover, we show that SMAD2 initiates transcription by inducing RNA Polymerase II recruitment. We therefore define new paradigms for SMAD2-dependent transcription and provide a framework to understand how cells correctly execute gene expression programs in response to Nodal/Activin signaling. Overall design: RNA-sequencing, SMAD2 ChIP-sequencing, H3K9Ac, H3K27Ac, H3K9Me3, H3K27Me3, RNA Polymerase II (ser5P), RNA Polymerase II (ser2P) CHIP-sequencing performed at various time points following Activin/Nodal signal stimulation or inhibition in p19 mouse embryonic teratoma cells: signal-ihibited, 1 hr Activin, 4 hr Activin, 8 hr Activin, Untreated (chronically Nodal-signalling cells)