P-045 Deciphering meiotic chromatin organization by SCP3

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Abstract Study question What’s the pattern of genome-wide chromatin occupancy of SCP3 during meiotic prophase, and how its interaction with chromatin is regulated? Summary answer We document SCP3 enriched at active chromatin regions with largely inherited from leptotene to pachytene stage, which facilitated by transcription and condensate assembly in spermatocytes. What is known already Meiosis is a critical step during the process of gametogenesis to form haploid cells which finally develop into functional gametes. The synaptonemal complex (SC) is a meiosis-specific protein assembly which bridges homologous chromosomes to establish the architecture of meiotic chromosome. Genetic ablation of genes encoding SC components impedes meiosis and gametogenesis in mice and mutation of these genes is frequently found in infertile patients. SCP3 localization changes with chromosome morphology in autosomes and is involved in maintaining the association of sex chromosomes. Male mice with null mutation of SCP3 failed to form SCs, resulting in developmental arrest during meiotic prophase. Study design, size, duration This study included male mice at post-natal day 10 (P10), 4 weeks or adult (8 weeks or older) were maintained on C57BL/6J background. Spermatocytes were performed modified CUT&Tag using antibodies. ChIP-seq data was analyzed together with WGBS and ATAC-seq data. Participants/materials, setting, methods Spermatocytes were obtained by a discontinuous BSA density gradient. Cell pellet was resuspended in culture medium (MEM with 10% FBS) at a concentration of 105 cells/ml and cultured at 37 °C in a 5% CO2 incubator. Spermatocytes were cultured with or without 1,6-hexanediol or α-amanitin. Spermatocytes with or without drug treatment were collected at indicated time points for further examination and then modified CUT&Tag and co-IP using anti-SCP3 and anti-POL II antibodies. Main results and the role of chance Chromatin occupancy of SCP3 in leptotene and pachytene spermatocytes, SCP3-Core sites retained upon treatment of 1,6-hexanediol which dissolves SCP3 condensate, and SCP1 occupancy at meiotic chromatin in pachytene spermatocytes. When we determined inter-peak distances and width of peaks, we observe that: (1) SCP3 occupancy in leptotene stage had greater inter-peak distances and narrower peak width than SCP3 occupancy in pachytene stage, demonstrating gradual accumulated SCP3 at chromatin during meiotic progression; (2) SCP3-Core sites have broader peak width than SCP3-NonCore sites, in agreement with stability of SCP3 occupancy at SCP3-Core sites; (3) SCP1 occupancy had greater inter-peak distances and narrower peak width than SCP3 occupancy in pachytene stage, supporting that SCP1 peaks are a subset of SCP3 peaks and SCP1’s major function in connecting LE and CE respectively. Generally, at pachytene-stage meiosis, SCP3 mainly occupies chromatin regions including transcriptionally active sites, Deu and Alu elements, while SCP1 mainly co-localizes with SCP3 at a subset of regions with open chromatin and high cohesin enrichment to connect meiotic chromosome axes with CE. In this way, a local active chromatin environment is created at meiotic chromosome axes, facilitating homologous chromosome recombination. Limitations, reasons for caution Our test was not successful to examine SCP3 occupancy with as few as less than 1000 spermatocytes using our protocol. It is improvement of efficiency of tagmentation or development of more efficient antibodies against SCP3 will be helpful for clinical sample detection. Wider implications of the findings SCP3 occupancy was largely inherited from leptotene to pachytene stage, facilitated by transcription and condensate assembly, and enriched at specific SINE repeats. Further exploration using knockout mouse models of meiotic axial proteins and epigenetic modifiers will provide valuable information on meiotic chromatin architecture and crosstalk among meiotic chromatin events. Trial registration number N/A