Meiotic Silencing is Regulated by Asynapsis Levels and Gel-like Phase Separation

Yiheng Peng¹, Shuangqi Wang¹, Royal Shrestha¹, Xing Tian¹, So Maezawa², Marcus Smolka³, Paula Cohen⁴, Satoshi Namekawa⁵, Huanyu Qiao^1, *

1.1. Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

< !2.  Department of Applied Biological Science, Tokyo University of Science, Noda-shi, Chiba-ken, Japan

< !3.  Department of Molecular Biology and Genetics, Cornell University, Ithaca, USA

< 4.    Department of Biomedical Sciences, Cornell University, Ithaca, USA

< !5. Department of Microbiology and Molecular Genetics, University of California – Davis, Davis, California, USA

Abstract Text: In mammals, meiotic silencing of unsynapsed chromatin (MSUC) occurs in both males and females and is driven by the DNA damage response (DDR) pathway. During normal male meiosis, MSUC is confined to the unsynapsed X and Y chromosomes, a process known as meiotic sex chromosome inactivation (MSCI), which is essential for spermatogenesis. Broader MSUC can occur in autosomes when asynapsis happens. However, variability in the silencing regions across cells makes it challenging to precisely define the location and extent of MSUC. To address this, we developed a novel approach called “digital chromosome banding” to accurately assess the meiotic silencing, including both MSCI and MSUC, at the single cell level. Using this technique, we identified two critical silencing steps in MSCI: one during the zygotene-to-early-pachytene transition and another during early-to-mid pachytene transition. The mechanism of second-step silencing associates with the formation of mature sex bodies, which achieve silencing through gel-like phase separation that creates a barrier to the diffusion of small molecules. Our analysis of three synapsis-defective mouse mutants (Spo11^-/-, Tdp43^-/-, and Nelfb^-/-) revealed that MSUC silencing levels correlates with the degree of asynapsis in each mutant. Notably, the X chromosome more frequently involved in silencing than autosomes. Although MSUC is proposed to form a pseudo sex body, its cytological characteristics resemble the first-step silencing of MSCI. Comparative analysis between sexes also uncovered notable sexual dimorphisms in meiotic silencing.