PIWI-mediated Control of Tissue-specific Transposons is Essential for Somatic Cell Differentiation

Date of Award

Fall 10-1-2021

Document Type


Degree Name

Doctor of Philosophy (PhD)


Molecular, Cellular, and Developmental Biology

First Advisor

van Wolfswinkel, Josephina


All eukaryote genomes lie under the constant threat of transposon invasion. About 45% of the human genome is derived from transposon elements. Although the vast majority of these transposons are inactive, they still lead to a mutagenesis rate of around 1 new insertion per 20 births. Multiple mechanisms have been evolved to silence transposons, among them is the PIWI-interacting RNA (piRNA) pathway. piRNAs rely strictly on PIWI proteins for their biogenesis and function, which includes target RNA cleavage and chromatin state alterations. PIWI and piRNAs are most well known for their transposon repression function in the germline, where PIWI depletion often lead to transposon upregulation, DNA damage, meiosis arrest and sterility. PIWI is also present in the stem cells of multiple regenerative species, but its function in the stem cell context is less well understood. Here we use the freshwater flatworm planarian Schmidtea mediterranea to study the function of PIWI proteins in adult pluripotent stem cells. RNAi-mediated knockdown of the planarian PIWI gene smedwi-2 causes tissue turnover defects, regeneration arrest, and 100% lethality, but the survival and proliferation of stem cells are not affected at early stages of RNAi. Instead, tissue-specific transposon upregulation is observed during the process of cell differentiation. Mechanistically, we find that repetitive sequences are the major target of SMEDWI-2, and SMEDWI-2 directly represses these loci through induction of H3K9me3 modification. The threat of transposon activation continues during differentiation, and SMEDWI-2 together with the associating piRNAs are faithfully inherited from the stem cells to the differentiating cells to counteract such threats. We further demonstrate that lineage specific changes in chromatin states during normal differentiation allow for the opening of certain transposon copies, which more readily activate in their specific lineages unless repressed by SMEDWI-2. We also observe insufficient opening of certain coding genes, including lineage-specific transcription factors, upon smedwi-2 depletion. These genes are not targeted by piRNAs, and are potentially affected through changes in their surrounding chromatin environment. Accordingly, we observe defective differentiation of the epidermal and the intestinal lineages, which is very likely the cause for tissue organization defects and eventual lethality of smedwi-2(RNAi) animals. Finally, we find that smedwi-2 deficient stem cells already demonstrate transposon derepression on the chromatin level, but transposon RNA expression is kept in check by the other two stem cell-specific PIWI proteins, SMEDWI-1 and SMEDWI-3. Together, these three PIWI proteins provide a double layer of transposon protection in planarian stem cells. Overall, we showed that SMEDWI-2 is essential for transposon repression and proper stem cell differentiation. By studying PIWI proteins in an adult pluripotent stem cell system, we reveal unexpected tissue-specificity in PIWI targeting, and demonstrate how PIWI proteins cope with the differential regulatory demand of tissues with a generic pool of piRNAs. We also find that the chromatin changes during differentiation naturally place planarian cells at risk of transposon upregulation, and that repression of repetitive regions may be an intrinsic part of the normal chromatin transitions in the differentiation program. Our study reveals a unique role of PIWI proteins in safeguarding the epigenome of somatic stem and differentiated cells, and may lend new insights to PIWI studies in other biological contexts, particularly those involving major chromatin shifts.

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