5 research outputs found
Systemic Loss and Gain of Chromatin Architecture throughout Zebrafish Development
Summary: The spatial organization of chromosomes is critical in establishing gene expression programs. We generated in situ Hi-C maps throughout zebrafish development to gain insight into higher-order chromatin organization and dynamics. Zebrafish chromosomes segregate in active and inactive chromatin (A/B compartments), which are further organized into topologically associating domains (TADs). Zebrafish A/B compartments and TADs have genomic features similar to those of their mammalian counterparts, including evolutionary conservation and enrichment of CTCF binding sites at TAD borders. At the earliest time point, when there is no zygotic transcription, the genome is highly structured. After zygotic genome activation (ZGA), the genome loses structural features, which are re-established throughout early development. Despite the absence of structural features, we see clustering of super-enhancers in the 3D genome. Our results provide insight into vertebrate genome organization and demonstrate that the developing zebrafish embryo is a powerful model system to study the dynamics of nuclear organization. : How do developing zebrafish embryos organize their genome? Kaaij et al. show that, early in development, when there is no transcription, the genome is highly structured; however, when the zygotic genome is activated, this organization is lost. Later in development, the genome again adopts a structured organization
Hen1 is required for oocyte development and piRNA stability in zebrafish
Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2′-O-methyl modification at their 3′ end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3′–5′ exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing
The inactive X chromosome adopts a unique three-dimensional conformation that is dependent on Xist RNA
Three-dimensional topology of DNA in the cell nucleus provides a level of transcription regulation beyond the sequence of the linear DNA. To study the relationship between the transcriptional activity and the spatial environment of a gene, we used allele-specific chromosome conformation capture-on-chip (4C) technology to produce high-resolution topology maps of the active and inactive X chromosomes in female cells. We found that loci on the active X form multiple long-range interactions, with spatial segregation of active and inactive chromatin. On the inactive X, silenced loci lack preferred interactions, suggesting a unique random organization inside the inactive territory. However, escapees, among which is Xist, are engaged in long-range contacts with each other, enabling identification of novel escapees. Deletion of Xist results in partial refolding of the inactive X into a conformation resembling the active X without affecting gene silencing or DNA methylation. Our data point to a role for Xist RNA in shaping the conformation of the inactive X chromosome at least partially independent of transcription
Tdrd1 acts as a molecular scaffold for Piwi proteins and piRNA targets in zebrafish
Piwi proteins function in an RNAi-like pathway that silences transposons. Piwi-associated RNAs, also known as piRNAs, act as a guide to identify Piwi targets. The tudor domain-containing protein Tdrd1 has been linked to this pathway but its function has thus far remained unclear. We show that zebrafish Tdrd1 is required for efficient Piwi-pathway activity and proper nuage formation. Furthermore, we find that Tdrd1 binds both zebrafish Piwi proteins, Ziwi and Zili, and reveals sequence specificity in the interaction between Tdrd1 tudor domains and symmetrically dimethylated arginines (sDMAs) in Zili. Finally, we show that Tdrd1 complexes contain piRNAs and RNA molecules that are longer than piRNAs. We name these longer transcripts Tdrd1-associated transcripts (TATs). TATs likely represent cleaved Piwi pathway targets and may serve as piRNA biogenesis intermediates. Altogether, our data suggest that Tdrd1 acts as a molecular scaffold for Piwi proteins, bound through specific tudor domain–sDMA interactions, piRNAs and piRNA targets