Abstract
Early embryonic development in metazoans is characterised by extensive changes in gene expression and chromatin remodelling at all levels. In most metazoans, zygotic genomes are transcriptionally repressed for a species-dependent length of time. Embryonic developmentis temporarily reliant on maternal factors,until the genome becomes actively transcribed during zygotic genome activation (ZGA).This transition period from maternal to zygotic control, is marked by extensivechromatin changes and the clearance of maternal products. The molecular mechanisms underlying this major event in embryogenesis are not fully understood.
A growing body of evidence suggests that the chromatin structure, the 3D architecture of the DNA in the nucleus, can regulate gene expression by mediating physical interactions of regulatory regions to their target genes over long genomic distances. Two highly conserved proteins, CCCTC binding factor (CTCF) and cohesin have been found to play a central rolein facilitating long range DNA contacts, thereby regulating gene expression in a variety of cellular contexts.
In this thesis, I have sought to investigate the roles of cohesin and CTCF in regulating transcription in developing embryos of the zebrafish (Danio rerio) during the maternal to zygotic transition (MZT). Using a genomics approach, I found that depletion of cohesin subunit Rad21 delays ZGA without affecting cell cycle progression. In contrast, CTCF depletion has little effect onthe timing ofZGA, butcomplete abrogation is lethal. Genome-wide profiling of Rad21 binding reveals a change in distribution from pericentromeric satellite DNA sequences and few locations including the miR-430 locus (whose products are responsible for maternal transcript degradation), to gene regulatory elements as embryos progress through the MZT. After MZT, a subset of Rad21 binding occurs at genes dysregulated upon Rad21 depletion and overlaps pioneer factor Pou5f3, which activates early-expressed genes. Rad21 depletion disrupts the formation of nucleoli and RNA polymerase II foci, suggestive of global defects in chromosome architecture. I propose that Rad21/cohesin redistribution to active areas of the genome is key to the establishment of chromosome organization and the embryonic developmental program.
Rad21 binding profiles from pre-and post-gastrulation embryos were compared to identify stage-specific and constitutive binding sites. Stage-specific Rad21 binding sites are correlated with the timely activation of associated genes, but lack canonical enhancer marks. In contrast, constitutive sites are strongly enriched for active chromatin marks and show characteristics of topological domain boundaries with high enrichment of divergent CTCF binding sites and well-positioned nucleosomes.