Epigenetics: from covalent modifications to small RNAs

Abstract

The role of epigenetic modifications, for instance DNA methylation, in cell fate transition is tremendous. In this thesis we present two studies in which we present genome wide in vivo methylomes of the small intestinal (SI) stem cell, a close descendent and villus and of 4 stages during zebrafish development. Unexpectedly, no role for DNA methylation was observed in regulating gene expression by promoter methylation during differentiation of the small intestinal stem cell. Between the stem cell and the villus only 50 differentially methylated regions (DMRs) were observed which are strongly correlated with differentially expressed genes and are enriched for two epigenetic marks associated with regulatory elements, H3K4me1 and H3K27ac. In conclusion our data shows that DNA methylation is very stable upon differentiation of the SI stem cell, but that at a limited number of regulatory sequences DNA methylation levels does change. During zebrafish development we mainly focused on the interplay between DNA methylation and enhancers. Previously it has been shown in cell culture that enhancer activation coincides with a decrease in DNA methylation at that locus. Integration of DNA methylation data with the histone modification code specific for enhancers revealed a previously unidentified enhancer type. This enhancer type is characterized by a hypomethylated region (HMR). Interestingly this hypomethylated state does not seem to be a consequence of enhancer activation as it displays a low degree in which it coincidences with H3K27ac. Interestingly, this new enhancer type is highly enriched close to transcription factors (TFs) important for lineage commitment. 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 find that Tdrd1 binds both zebrafish Piwi proteins, Ziwi and Zili. Furthermore, we show that Tdrd1 associates with long RNA molecules. These Tdrd1 associated transcripts (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-pathway components, comprising Piwi proteins, their piRNA cofactors and their targets. Germ plasm is an evolutionary conserved structure that can induce germ cell fate upon early embryonic cells. We describe zebrafish Tdrd6a as a germ plasm component. We demonstrate two distinct molecular functions for Tdrd6a. First, it is involved in the Piwi-piRNA pathway and interacts with the Piwi protein Ziwi. In absence of Tdrd6a, the typical anti-sense strand-bias of piRNA populations is lost, potentially affecting transposon-defense in progeny. Second, our findings demonstrate that Tdrd6a is required for the proper assembly of germ plasm-related structures. Consistently, we demonstrate a strong effect of Tdrd6a on PGC formation in embryos derived from homozygous tdrd6a mutant mothers. We propose that Tdrd6a functions as a scaffold to recruit both Piwi-pathway components, as well as germline-specifying mRNAs in order to ensure the accurate specification and maintenance of germ cells in new generations.