A SUMO-ubiquitin relay recruits proteasomes to chromosome axes to regulate meiotic recombination

Abstract

Meiosis produces haploid gametes through a succession of chromosomal events, including pairing, synapsis, and recombination. Mechanisms that orchestrate these events remain poorly understood. We found that the SUMO (small ubiquitin-like modifier)-modification and ubiquitin-proteasome systems regulate the major events of meiotic prophase in mouse. Interdependent localization of SUMO, ubiquitin, and proteasomes along chromosome axes was mediated largely by RNF212 and HEI10, two E3 ligases that are also essential for crossover recombination. RNF212-dependent SUMO conjugation effected a checkpointlike process that stalls recombination by rendering the turnover of a subset of recombination factors dependent on HEI10-mediated ubiquitylation. We propose that SUMO conjugation establishes a precondition for designating crossover sites via selective protein stabilization. Thus, meiotic chromosome axes are hubs for regulated proteolysis via SUMO-dependent control of the ubiquitin-proteasome system.

Fig. 1. SUMO, ubiquitin, and proteasomes decorate the axes of meiotic prophase chromosomes

(A) Spermatocyte nuclei immunostained for axis marker SYCP3 and SUMO2/3, ubiquitin, or proteasomes. Bottom row shows single chromosomes imaged by SIM. Arrows indicate sex bodies; arrowheads highlight centromeric heterochromatin. In the SUMO2/3 stained SIM image, the arrow, arrowhead, and bar highlight supra-axial, axial, and SC central-region localizations, respectively. Scale bars: 10 μm, wide-field; 1 μm, SIM. (B) Quantification of immunostaining foci. L, leptonema; LZ, late zygonema; EP, early pachynema; and MP/LP, mid/late pachynema. Counts from MP and LP nuclei were pooled, as numbers remained stable over these stages. Ubiquitin foci in LP could not be accurately quantified because general chromatin staining obscured axis-specific foci. Error bars show means ± SD.

Fig. 2. Interrelated functions of the SMS and UPS in meiotic prophase

(A) Pachytene-like spermatocyte nuclei containing SYCP3 aggregates (top) or synapsis defects (bottom). Arrows indicate structures magnified in adjacent panels. (B and C) Quantification of SYCP3 aggregates (B) and synapsis defects (C). (D) Progression of spermatocytes after culture with or without inhibitors. (E) Spermatocyte nuclei immunostained for SYCP3 (red) and SUMO2/3, ubiquitin, or proteasomes (green) after chemical inhibition. (F) Quantification of experiments shown in (E). Error bars in (B), (C), and (D) show means ± SEM. Error bars in (G) show means ± SD. ***P ≤ 0.001, two-tailed Mann-Whitney test. Abbreviations as in Fig. 1. Scale bars: 10 μm, full nuclei; 1 μm, magnified structures in (A).

Fig. 3. The SMS and UPS regulate chromosomal dynamics of recombination factors

(A, C, and E) Spermatocyte nuclei immunostained for SYCP3 (red) and MEI4, RAD51, or MER3 (green) following chemical inhibition. (B, D, and F) Quantification of immunostaining foci. Abbreviations as in Fig. 1. Arrows indicate sex chromosomes. Error bars show means ± SD. Scale bars = 10 μm.

Fig. 4. RNF212 and HEI10 define an axis-associated SUMO-ubiquitin-proteasome relay that regulates turnover of recombination factors

(A, C, and E) Spermatocyte nuclei from indicated strains immunostained for SYCP3 (red) and either SUMO2/3, ubiquitin, or proteasomes (green). See fig. S8 for SUMO1 staining. (B, D, and F) Quantification of immunostaining foci. (G, I, and K) Spermatocyte nuclei immunostained for SYCP3 (red) and MEI4, RAD51, or MER3 (green). (H, J, and L) Quantification of immunostaining foci. EZ, early zygonema; LZ, late zygonema; MP, mid-pachynema. Arrows indicate sex chromosomes. Error bars show means ± SD. *P ≤ 0.05, ***P ≤ 0.001, two-tailed Mann-Whitney test. Scale bars, 10 μm.