Periodic Reporting for period 1 - DIRECT-DSB (Dissecting the role of transcription in meiotic DNA double-strand breaks patterning)
Período documentado: 2022-05-01 hasta 2024-04-30
Meiosis is a unique cell division process that generates gametes, and defects in meiosis often lead to infertility or birth defects, such as Down syndrome. Successful meiosis requires the formation of hundreds of DNA double-strand breaks (DSBs) at specific chromatin sites. These DSB patterns are believed to be regulated by certain chromatin features established during transcription. However, because transcription is vital for cellular function, studying its influence on DSB patterning during meiosis in conventional model organisms is challenging.
Tetrahymena thermophila, a unicellular protist with two nuclei, provides a solution. Since all its protein-coding mRNAs are generated from its somatic nucleus, transcription can be halted in its meiotic nucleus without disrupting meiotic progression. Hence, the DIRECT-DSB project was initiated to exploit this unique feature, aiming to examine transcription's role in DSB patterning without the limitations faced in other model organisms. By characterizing and comparing DSB patterns and chromatin features in the presence and absence of transcription, a mutually exclusive relationship between DSB formation and transcription was uncovered. This finding provides a deeper understanding of how meiotic processes are regulated at the molecular level. It highlights the potential role of transcription in negatively influencing DSB patterns in Tetrahymena, which are crucial for the accurate segregation of chromosomes. Meanwhile, innovative techniques established during the implementation of this project facilitated using Tetrahymena as a model for the dissection of complex processes in ways that are not possible in other systems, potentially leading to broader discoveries in genetics and cell biology.
Tetrahymena thermophila, a unicellular protist with two nuclei, provides a solution. Since all its protein-coding mRNAs are generated from its somatic nucleus, transcription can be halted in its meiotic nucleus without disrupting meiotic progression. Hence, the DIRECT-DSB project was initiated to exploit this unique feature, aiming to examine transcription's role in DSB patterning without the limitations faced in other model organisms. By characterizing and comparing DSB patterns and chromatin features in the presence and absence of transcription, a mutually exclusive relationship between DSB formation and transcription was uncovered. This finding provides a deeper understanding of how meiotic processes are regulated at the molecular level. It highlights the potential role of transcription in negatively influencing DSB patterns in Tetrahymena, which are crucial for the accurate segregation of chromosomes. Meanwhile, innovative techniques established during the implementation of this project facilitated using Tetrahymena as a model for the dissection of complex processes in ways that are not possible in other systems, potentially leading to broader discoveries in genetics and cell biology.
The implementation of the project led to significant progress in accomplishing several critical tasks. First, we established a robust method for purifying meiotic nuclei. Second, using the purified nuclei, we generated the initial RNA Pol II occupancy map and chromatin accessibility map for meiotic chromatin. Third, we characterized the meiotic DSB sites in Tetrahymena cells, and revealed a mutually exclusive relationship between meiotic DSB formation and transcription through comparative genomic analysis. Fourth, we identified a previously uncharacterized protein as a novel partner of Spo11, essential for the induction of meiotic DSBs. Finally, we shared the key findings of this project at international conferences. These scientific and technical innovations achieved in this project provided new insights into the mechanisms governing meiosis and advanced technical capabilities in the field, allowing more precise studies of meiotic processes using Tetrahymena.
The scientific results of this project have advanced the state of the art in understanding the meiosis regulatory mechanism, from the perspective of the less explored model eukaryote Tetrahymena thermophila. Our findings, particularly in revealing the mutually exclusive relationship between ncRNA transcription and meiotic DSB formation, have expanded current understanding of the meiotic DSB formation regulatory mechanism and laid the groundwork for future investigations. By further pushing the boundaries of knowledge and fostering new perspectives, our work promises to improve the understanding the impact of transcription-induced chromatin alterations on the meiotic DSB formation.