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Awakening of zygotic transcription in animal embryos relies on maternal pioneer transcription factors. The interplay of global and specific functions of these proteins remains poorly understood. Here, we analyze chromatin accessibility and time-resolved transcription in single and double mutant zebrafish embryos lacking pluripotency factors Pou5f3 and Sox19b. We show that two factors modify chromatin in a largely independent manner. We distinguish four types of direct enhancers by differential requirements for Pou5f3 or Sox19b. We demonstrate that changes in chromatin accessibility of enhancers underlie the changes in zygotic expression repertoire in the double mutants. Pou5f3 or Sox19b promote chromatin accessibility of enhancers linked to the genes involved in gastrulation and ventral fate specification. The genes regulating mesendodermal and dorsal fates are primed for activation independently of Pou5f3 and Sox19b. Strikingly, simultaneous loss of Pou5f3 and Sox19b leads to premature expression of genes, involved in regulation of organogenesis and differentiation.

The zebrafish embryo is mostly transcriptionally quiescent during the first 10 cell cycles, until the main wave of Zygotic Genome Activation (ZGA) occurs, accompanied by fast chromatin remodeling. At ZGA, homologs of mammalian stem cell transcription factors (TFs) Pou5f3, Nanog and Sox19b bind to thousands of developmental enhancers to initiate transcription. So far, how these TFs influence chromatin dynamics at ZGA has remained unresolved. To address this question, we analyzed nucleosome positions in wild-type and Maternal-Zygotic (MZ) mutants for pou5f3 and nanog by MNase-seq. We show that 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 nucleosome destabilization-depletion 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 destabilize nucleosomes on HNAR centers genome-wide. In the second step, post-ZGA, Nanog, Pou5f3 and SoxB1 maintain open chromatin state on the subset of HNARs, acting synergistically. Nanog binds to the HNAR center, while the Pou5f3 stabilizes the flanks. The HNAR model will provide a useful tool for genome regulatory studies in the variety of biological systems.

Nanog has been implicated in establishment of pluripotency in mammals and in zygotic genome activation in zebrafish. In this study we characterize the development of maternal and zygotic MZnanog null mutant zebrafish embryos. Without functional Nanog, epiboly is severely affected, embryo axes do not form and massive cell death starts at the end of gastrulation. We show that three independent defects in MZnanog mutants contribute to epiboly failure: yolk microtubule organization required for epiboly is abnormal, maternal mRNA fails to degrade due to the absence of miR-430 and actin structure of the yolk syncytial layer does not form properly. We further demonstrate that the cell death in MZnanog embryos is cell-autonomous. Nanog is necessary for correct spatial expression of the ventral specifying genes bmp2b, vox, and vent, and neural transcription factor her3. It is also required for the correctly timed activation of endoderm genes and for the degradation of maternal eomesa mRNA via miR-430. Our findings suggest that maternal Nanog coordinates several gene regulatory networks that shape the embryo during gastrulation.