Pou5f3, SoxB1 and Nanog remodel chromatin on High Nucleosome Affinity Regions at Zygotic Genome Activation.

Purpose: Find out the consequences of Pou5f3/Oct4 and Nanog binding to the nucleosome distribution before and after Zygotic Genome Activation Methods: MNase-seq of the wild-type(WT), Pou5f3 (MZspg) and Nanog (MZnanog) mutant zebrafish embryos at 512-cell stage and dome stage. Results: Nanog, Sox19b and Pou5f3 bind to the High Nucleosome Affinity Regions (HNARs). HNARs are spanning over 600 bp, featuring high in vivo and predicted in vitro nucleosome occupancy and high predicted propeller twist DNA shape value. We suggest a two-step model, where the same intrinsic DNA properties of HNAR promote both high nucleosome occupancy and differential binding of TFs. In the first step, already prior to ZGA, Pou5f3 and Nanog reduce nucleosome occupancy on HNARs genome-wide. In the second step, Nanog and Pou5f3/SoxB1 complex maintain open chromatin state on the subset of HNARs, acting synergistically. Nanog binds to the HNAR center, while the Pou5f3/SoxB1 complex stabilizes the flanks. Genome-wide nucleosome occupancy in 512-cell and dome staged wild-type (WT), MZspg (Pou5f3) and MZnanog (Nanog) zebrafish mutant embryos was assayed by MNase treatment followed by deep sequencing (MNase-seq), using Illumina HiSeq 2500.