Dynamics of enhancer activity and gene expression after Zygotic Genome Activation in zebrafish.

• Applicant: Dr. Daria Onichtchouk
• Subject Area: Developmental Biology ; Evolutionary Cell and Developmental Biology (Zoology)
• Term: from 2015 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 274453961

Research on indefinitely pluripotent in-vitro cell cultures, derived from mammalian early embryos, revealed the central role of transcription factors Pou5f1/Oct4, SoxB1 and Nanog in the genetic control of the pluripotency state. We and others have demonstrated that zebrafish homologs of the “pluripotency transcription factors (TFs)”: Pou5f3, SoxB1 and Nanog play central roles in zebrafish Zygotic Gene Activation. The mechanistic connections between chromatin state, TF binding and gene expression are still not completely understood. The zebrafish model system offers unique experimental opportunities to resolve these basic questions by combining mutant analyses with functional genomics. During the last three years we established mutant lines for Nanog and SoxB1 and the double mutant lines Pou5f3/SoxB1, Pou5f3/Nanog and SoxB1/Nanog. All single and double maternal-zygotic mutants survive at least three hours after ZGA, which makes it possible to investigate the gene expression dynamics and connect it to the time-dependent and genotype-dependent changes of accessibility of enhancers. In order to uncover the dynamics of regulatory networks controlling gene expression, it is essential to obtain information about gene transcription and changing chromatin status for different time points for each gene regulatory region. To achieve this goal we will collect and analyse time-resolved RNA-seq and ATAC-seq data from the single and double mutants. We will further investigate if the binding of each “Pluripotency TFs” to its cognate motifs in the genome is facilitated or impeded by the other two factors. For this purpose we will perform ChIP-seq analyses in the mutant embryos for the other two “Pluripotency TFs”. We will use these data to build regulatory models for all zygotic genes that are expressed within the embryo at pre-gastrulation stages. Further, we aim to characterise the differentiation potential of double mutant cells using transplantation assays. As we have previously demonstrated, zebrafish post-ZGA cells closely resemble mammalian ES cells by their chromatin landscape, high transcriptional activity, and also by the set of expressed genes. Therefore we expect that our results will shed light on the organizing principles of pluripotency GRNs in vertebrates and would be of use for the rational design of cell reprogramming approaches.

• DFG Programme: Research Grants