Nuclear small RNA control of the germ to soma transition

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 DESCRIPTION (provided by applicant): The chromosomes contained in gametes are very different from the chromosomes contained in somatic cells just a few cell divisions following fertilization. Gametic chromosomes are transcriptionally inaccessible, while somatic chromosomes exhibit accessible chromatin in specific locations linked to actively transcribed genes. Establishment of these specific regions of transcriptionally permissive chromatin following fertilization is essential for early embryogenesis and beyond, but the neither the identities of the regulatory factors that directly participate in this process nor their molecular mechanisms of action are well understood. A candidate class of factors that directly mediate the process of sequence-specific establishment of somatic chromosome modifications are nuclear localized members of the Argonaute/Piwi family of proteins. These proteins associate with small RNAs, which enable them to specifically recognize and regulate complementary nucleic acid sequences by base pairing. Nuclear localized Piwi proteins are essential for repressing the mobilization of transposable DNA elements in the nuclei of germ cells of a wide variety of invertebrate and vertebrate species, but it is likely that they have additional function in gametes both before and after fertilization. Disentangling these functions from transposon repression is challenging in most experimental systems, which further complicates attaining a detailed understanding of their function. The ciliate Tetrahymena thermophila is a useful system for the study of nuclear small RNA functions during the germ to soma transition, as multiple, separate nuclear Piwi-guided pathways regulate transposon repression and other functions during sexual reproduction. The Tetrahymena Piwi protein Twi1p represses transposons by establishing repressive histone modifications, while the biological role and function of Twi8p is largely unknown. My work has demonstrated that Twi8p is essential for events that follow fertilization and that it is loaded with small RNAs derived from genomic loci that give rise to non coding RNAs, termed Twi8p-associated small RNA loci (TASLs). I propose that Twi8p guides the establishment of somatic chromosome modifications through TASLs during sexual reproduction. I will determine what these modifications are and what proteins establish them using the powerful molecular biology tools developed in Tetrahymena. I will also determine the biological role of the TASLs during sexual reproduction by specifically disrupting them in either the parental or zygotic genome and characterizing the molecular defects. By leveraging the unique tools and techniques established in Tetrahymena for small RNA gene regulation, I will contribute to our understanding of these conserved processes that are essential in invertebrates and vertebrates alike.