Quantitative Cytogenetics Reveals Molecular Stoichiometry and Longitudinal Organization of Meiotic Chromosome Axes and Loops

Abstract

During meiosis, chromosomes adopt a specialized organization involving assembly of a cohesin-based axis along their lengths, with DNA loops emanating from this axis. We applied novel, quantitative and widely applicable cytogenetic strategies to elucidate the molecular bases of this organization using C. elegans. Analyses of WT chromosomes and de novo circular mini-chromosomes revealed that meiosis-specific HORMA-domain proteins assemble into cohorts in defined numbers and co-organize the axis together with two functionally-distinct cohesin complexes (REC-8 and COH-3/4) in defined stoichiometry. We further found that REC-8 cohesins, which load during S phase and mediate sister chromatid cohesion, usually occur as individual complexes, supporting a model wherein sister cohesion is mediated locally by a single cohesin ring. REC-8 complexes are interspersed in an alternating pattern with cohorts of axis-organizing COH-3/4 complexes (averaging three per cohort), which are insufficient to confer cohesion but can bind to individual chromatids, suggesting a mechanism to enable formation of asymmetric sister chromatid loops. Indeed, immuno-FISH assays demonstrate frequent asymmetry in genomic content between the loops formed on sister chromatids. We discuss how features of chromosome axis/loop architecture inferred from our data can help to explain enigmatic, yet essential, aspects of the meiotic program.

Phosphoregulation of HORMA domain protein HIM-3 promotes asymmetric synaptonemal complex disassembly in meiotic prophase inC. elegans

“…We also found a slight (approximately 1.2 times increase, n=6 gonads) but significant increase of panHIM-3 intensity on short arms after partitioning, suggesting a net addition of HIM-3 proteins to the SC toward the end of pachytene ( Figure 3B ). This is consistent with the previous observation that HIM-3 and HTP-1/2 continue to accumulate on the SC throughout pachytene and diplotene [33] . In nuclei with partitioned HIM-3phos, panHIM-3 staining levels did not differ between short and long arms, suggesting that the chromosome-wide increase in panHIM-3 levels does not derive from preferential addition of HIM-3 proteins to short arms but rather global addition along the entire SC ( Figure 3C ).…”

“…A previous study has shown that HTP-1/2 is able to bind either to HIM-3 or HTP-3's closure motifs [24] . A more recent study has shown that on average two HTP-1/2 molecules and three HIM-3 molecules are present at every HTP-3 molecule, with substantial variation between HTP-3 molecules [33] . This indicates that closure motifs are rarely if ever occupied to their theoretical maximum; instead, many must remain unbound.…”

“…HTP-2 proteins were purified and FP assays were conducted as described in [24] . [33,66] . right , Interactions between the HIM-3 closure motif (magenta) and the HTP-2 (grey) HORMA domain shown in PDB structure 4TZL [24] .…”

Cohesin in space and time: architecture and oligomerizationin vivo

“…The existence of cohesin oligomers is consistent with the in vitro detection of mammalian cohesin dimers, the co-immunoprecipitation of differentially tagged cohesins isolated from mammalian cells, and functional cooperation between mutated yeast cohesins in vivo (Eng et al, 2015;Kim et al, 2019;Srinivasan et al, 2018;Zhang et al, 2008) . A recent in vivo imaging study of worms suggests that clusters (~4) of cohesins containing COH-3/COH-4 bind at distinct loci on meiotic chromosomes (Woglar et al, 2020) . These clusters and the proximity of the head and hinges of COH-3/COH-4 cohesins is also consistent with the oligomerization of cohesins in the butterfly conformation (Köhler et al, 2017;Woglar et al, 2020) .…”

“…A recent in vivo imaging study of worms suggests that clusters (~4) of cohesins containing COH-3/COH-4 bind at distinct loci on meiotic chromosomes (Woglar et al, 2020) . These clusters and the proximity of the head and hinges of COH-3/COH-4 cohesins is also consistent with the oligomerization of cohesins in the butterfly conformation (Köhler et al, 2017;Woglar et al, 2020) . Thus, oligomers of cohesin butterflies may be conserved in eukaryotes.…”