Over the course of more than two decades, numerous studies have meticulously explored the fundamental roles of the cohesin complex, unraveling its intricate functions in sister chromatid cohesion, DNA recombination and repair, gene expression regulation, telomere protection, and regulatory mechanisms in cell division processes. However, the detailed and multifaceted roles of individual subunits within the cohesin complex during meiosis remain poorly understood. During my PhD, I focused my attention on two principal subunits that complete the tripartite ring: the meiotic isoform of SMC1, namely SMC1β, and a kleisin subfamily protein, RAD21. While RAD21 is the sole kleisin during mitosis, it is accompanied by two other kleisin subfamily proteins, REC8 and RAD21L, during meiosis. From past research, it is known that SMC1β is crucial for telomere protection in both spermatocytes and oocytes. Interestingly, while several major phenotypes of SMC1β-deficient spermatocytes were rescued by SMC1α, telomere abnormalities were not. The expression of telomerase and shelterin components appeared usual in SMC1β-deficient spermatocytes. This study highlights SMC1β's role in safeguarding telomeres at chromosome ends from damage and abnormalities by regulating the expression of a long non-coding RNA transcribed from the subtelomeres of different chromosomes, known as TERRA (Telomeric repeat–containing RNA). TERRA comprises repetitive sequence motifs transcribed from the telomeric DNA strand that is complementary to the DNA sequence of the telomere itself. SMC1β
suppresses the expression of TERRA strongly in spermatocytes and mildly in oocytes, increasing the number of foci and intensity at the ends of spermatocyte chromosomes corresponding to visually elevated telomeric damage in the absence of SMC1β. This suggests the strong role of SMC1β in regulating TERRA at chromosome ends. TERRA, with a similar sequence to that of telomeres, has the potential to form RNA-DNA hybrids, often referred to as open R-loops, which may make telomeres more susceptible to damage. This study demonstrates that SMC1β-deficient mice exhibited increased staining for R-loops at both autosomal chromosome ends and sex chromosomes, which was mitigated upon treatment with RNase H endonuclease. In our recent publication, Biswas et al., 2023, it is confirmed that SMC1β helps maintain close chromatin at telomeric ends with support from ATAC sequencing and RNA sequencing data, thereby protecting the chromosome ends. One pertinent question that remains unanswered is what distinguishes SMC1β from SMC1α in maintaining these telomere functions. To address this, we generated a CRISPR-Cas9 mice strain with a deletion of the DNA binding domain at the carboxy terminal of SMC1β, which marks the major difference in sequence between SMC1β and SMC1α. While a reduced stability of these truncated proteins was observed at both RNA and protein levels in spermatocytes, yet it hints that the majority of SMC1β’s functions were abolished upon deletion of this C-tail. Interestingly, these mutant spermatocytes exhibit elevated telomere
abnormalities, albeit not to the extent seen in full knockouts. This suggests that the C-terminal tail, along with additional components, participates in protecting telomeres, considering that a minimal level of SMC1β protein is sufficient to maintain telomeres in germ cells. Another objective of my thesis is to emphasize the significance of RAD21 as a kleisin protein in female meiosis. While there is limited research on RAD21 in spermatocytes, it has been described to transiently appear during late prophase I of male meiosis. However, studies on RAD21 in oocytes are lacking. RAD21 exhibits an appearance on the chromosome axis during mid to late pachytene in embryonic oocytes before being depleted in the diplotene stage. As RAD21 is the only kleisin protein in somatic cells, generating a constitutive Rad21 knockout mouse would be lethal. Therefore, using the Cre-LOX system, conditional Rad21 knockout mouse models were created, where RAD21 was specifically eliminated in oocytes at various
stages of maturation. Early excision of Rad21 during the embryonic diplotene stage or in pups shortly after birth had a significant impact on ovary development and oocyte count with age, while oocyte sizes were reduced, indicating potential stress or onset of apoptosis. Conversely, late excision of Rad21 in activated germinal vesicle oocytes showed no notable differences in ovary size or oocyte number, and only mild differences in oocyte diameter, underscoring the significant role of RAD21 in the pre metaphase prophase stage. RAD21 does not actively participate in long-term arrest centromeric cohesin protection, chiasma maintenance, or DNA damage repair in heterozygous mice. However, young adult Rad21 conditional knockout mice with early excision exhibit a trend of delayed and less efficient oocyte maturation when exposed to DNA damage. These findings suggest a potential role of RAD21 in nonprogrammed DNA repair before metaphase I, which may ensure chromosome integrity after programmed recombination. Further investigation is necessary to study the mechanism of DNA repair by RAD21 through Homologous Recombination or End Joining pathways. In summary, these studies provide insights into the role of cohesin subunit SMC1β in telomere maintenance through TERRA regulation in spermatocytes, as well as the role of RAD21 in preserving ovarian reserve and oocyte health by potentially contributing to non-programmed DNA damage repair.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:92611 |
| Date | 26 July 2024 |
| Creators | Deb Mallik, Tanaya |
| Contributors | Jessberger, Rolf, Anastasiadis, Konstantinos, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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