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.
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 MZ (maternal and zygotic 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 MZ mutants contribute to epiboly failure: yolk microtubule organization required for epiboly is abnormal, maternal mRNA fails to degrade owing 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 MZ embryos is cell-autonomous. Nanog is necessary for correct spatial expression of the ventral-specifying genes , and , and the neural transcription factor It is also required for the correctly timed activation of endoderm genes and for the degradation of maternal mRNA via miR-430 Our findings suggest that maternal Nanog coordinates several gene regulatory networks that shape the embryo during gastrulation.
SummaryZygotic genome activation (ZGA) in the development of flies, fish, frogs and mammals depends on pioneer-like transcription factors (TFs). Those TFs create open chromatin regions, promote histone acetylation on enhancers, and activate transcription. Here, we use the panel of single, double and triple mutants for zebrafish genome activators Pou5f3, Sox19b and Nanog, multi-omics and mathematical modeling to investigate the combinatorial mechanisms of genome activation. We show that Pou5f3 and Nanog act differently on synergistic and antagonistic enhancer types. Pou5f3 and Nanog both bind as pioneers on synergistic enhancers, promote histone acetylation and activate transcription. Antagonistic enhancers are activated by pioneer binding of one of these factors. The other TF binds as non-pioneer, competes with the activator and blocks all its effects, partially or completely. This activator-blocker mechanism mutually restricts widespread transcriptional activation by Pou5f3 and Nanog and prevents premature expression of late developmental regulators in the early embryo.Pou5f3 and Nanog compete as pioneer-like and non-pioneer factors on common genomic sites.Pioneer-like binding activates transcription, non-pioneer binding blocks transcription.Competition between Pou5f3 and Nanog establishes the order of gene expression.
PouV and SoxB1 family transcription factors (TFs) have emerged as master regulators of cell fate transitions. To investigate the genetic interactions between Pou5f3 and Sox19b in zebrafish embryos passing through Zygotic Genome Activation (ZGA), we combined time-resolved mutant transcription analysis using the novel tool RNA-sense, chromatin state and phenotypic assays. We distinguish four types of embryonic enhancers, differentially regulated by the two TFs. Pou5f3 is critical for activation of enhancer types 1 and 2, which are responsible for transcription of genes involved in gastrulation and ventral genes. Pou5f3 or Sox19b prevent premature activation of type 3 and 4 enhancers, which are responsible for transcription of organogenesis regulators, differentiation factors and dorsal genes. We also show that the balance between Sox19b and Pou5f3 is important for bulk ZGA timing. Our results uncover how independent activities of maternal Pou5f3 and Sox19b add up or antagonize to determine the early gene expression repertoire. Bullet points:• Pou5f3 and Sox19b bind independently to DNA • Disbalance between maternal Pou5f3 and Sox19b delays ZGA • Pou5f3 suppresses premature transcription of neural patterning genes, activated by SoxB1 factors • Pou5f3 and Sox19b synergistically suppress premature transcription of a wide range of differentiation genes • Pou5f3 and Sox19b restrict the dorsal organizer
Zygotic genome activation (ZGA) in the development of flies, fish, frogs and mammals depends on pioneer-like transcription factors (TFs). Those TFs create open chromatin regions, promote histone acetylation on enhancers, and activate transcription. Here, we use the panel of single, double and triple mutants for zebrafish genome activators Pou5f3, Sox19b and Nanog, multi-omics and mathematical modeling to investigate the combinatorial mechanisms of genome activation. We show that Pou5f3 and Nanog act differently on synergistic and antagonistic enhancer types. Pou5f3 and Nanog both bind as pioneers on synergistic enhancers, promote histone acetylation and activate transcription. Antagonistic enhancers are activated by pioneer binding of one of these factors. The other TF binds as non-pioneer, competes with the activator and blocks all its effects, partially or completely. This activator-blocker mechanism mutually restricts widespread transcriptional activation by Pou5f3 and Nanog and prevents premature expression of late developmental regulators in the early embryo
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