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Exploring the role of STAG3 in mammalian meiosisSuresh, Laya 06 August 2024 (has links)
In the intricate realm of biology, meiosis stands as the remarkable process responsible for generating genetically diverse haploid gametes from diploid cells. In 2000, Pezzi et al., identified STAG3 as a novel meiotic-specific synaptonemal-complex associated protein belonging to the highly conserved family of stromalin nuclear proteins. Later, over the years, research groups characterised the depletion phenotype of STAG3 in mice, where deficiency of STAG3 causes severe chromosomal defects and early meiotic arrest. These studies together collectively highlighted STAG3 as the most important meiotic cohesin. Traditionally, the role of the cohesin complex was understood as maintaining cohesion between chromatids during cell division. However, over the years, this perception has evolved significantly, expanding to include the regulation of dynamic chromosomal configurations during meiosis. With the realisation of STAG3's importance in meiotic progression, the next pressing question becomes: how does STAG3 coordinate this intricate process? This study sought to address that question by examining the STAG3 interactome in male germ cells, aiming to uncover novel pathways through which STAG3 contributes to maintaining meiotic progression. Through successful purification of the STAG3-REC8 complex, its ability to form functional complexes in-vitro was demonstrated. During my PhD thesis, I discovered links between STAG3 and DNA repair mechanisms beyond the well-known homologous recombination /non-homologous end joining pathway in meiotic recombination. By looking at the meiotic-specific protein interactome bound to the STAG3-REC8 complex through Mass Spectroscopic analysis, we identified STAG3 involvement in PARP-1-mediated repair of DNA double-strand breaks occurring outside of the programmed DSB repair during the zygotene stage of prophase I. PARP-1 is an ADP-ribose polymerase which acts as a first responder that detects DNA damage and facilitates the activation of the DNA repair pathway. STAG3 shows a preferential interaction with PARP-1 when spermatocytes are challenged with extensive DNA damage. Furthermore, the interaction of SMC3, another component of the cohesin complex, with PARP-1 during DNA damage suggests that STAG3, as part of the cohesin complex, contributes to DNA damage repair in spermatocytes. To gain deeper insights into the distinctive characteristics of STAG3, an extensive analysis of spermatogenesis in mice expressing a C-terminus truncated form of STAG3 was performed. The C-terminal region of STAG3 is not conserved among the stromalin family members, and hence it was speculated that this region might have unique functions to meiosis. Removal of the C-terminal end comprising 47 amino acids led to an early meiotic arrest, mirroring the phenotype in most cohesin subunit deletion mutants. The phenotype observed mimics the complete STAG3 depletion phenotype to some extent. The truncated STAG3 resulted in an
arrest at a late zygotene/early pachytene-like stage during meiotic prophase I. One of the most notable observations was the significant reduction in the length of the axial elements (AE) in
this mutant. Despite stable expression of and localisation of STAG3 to the axis, the axis length decreased by over 60%. This mutation compromised synaptonemal complex formation, leading to the early meiotic arrest. Although SYCP1 loads onto the axis and initiate synapsis, the shortened axial elements could not synapse, marked by HORMAD-1, a well-known asynapsis marker. The average number of SYCP3-marked stretches was 35 in this mutant. The increased number of AE and shortened axis length did not result from chromosome fragmentation because most chromosomes/axes had intact telomere and centromeric signals, validated by RAP1 and ACA foci, respectively. Centromeric and telomeric cohesion may be partially affected as some chromosome showed aberrant telomeric and centromeric defects. C terminal truncated STAG3 impairs synapsis between homologous chromosomes, but the sister chromatid cohesion remains largely unaffected. Also, this deletion did not affect the loading of the cohesin complex subunits onto the chromosome axis. The early meiotic arrest resulted in underdeveloped gonads, leading to infertility in otherwise healthy mice. Taken together, these results suggest novel roles for STAG3 in meiosis, and the meiotic-specific C terminal region of STAG3 is critical for proper meiotic progression in mice.
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