Meiotic sex chromosomes inactivation is dependent on chromosome integrity

Meiotic sex chromosomes inactivation (MSCI) is a conserved germline phenomenon in which sex chromosomes are silenced during gametogenesis. Although known for decades, very little is known about its induction mechanism. As part of our effort to study longRNA’s role in gametogenesis, we used CRISPR/Cas9 to delete the lincRNA linc-20 gene from the genome of Caenorhabditis elegans. This gene is transcribed from the X chromosome during early oogenesis when the X chromosome is silenced, and therefore was suspected to take a part in the silencing machinery. linc-20 mutants demonstrate several severe meiotic defects including low progeny and embryonic viability. DAPI staining revealed that most of mutants’ oocytes had chromosomal aggregates and fragments. Immunostaining for double strand break repair (DSBR) loci revealed perturbations in DSBR of meiocytes. Immunostaining and RNA-Seq revealed that X chromosomes silencing was significantly decreased in the mutants. Following the creation of more linc-20 mutants we found out that most strains don’t present these phenotypes. Nano-pore and Illumina sequencing as well as immunofluorescence analyses indicated that mutants with MSCI loss detected by reactivated X chromosome, have broken X chromosomes, probably due to aberrant CRISPR-Cas-9 activity. In one case the CRISPR activity cleaved and circularized half of the X, this circularized chromosome was propagated through multiple generations and was not silenced. We conclude that disrupting sex chromosome integrity can lead to loss of MSCI. We are currently studying if MSCI loss is present in known mutants with X chromosome translocations, and if this regulation is dependent on chromosome’s axis tension.