Genetic Interactions Between the Meiosis-Specific Cohesin Components, STAG3, REC8, and RAD21L

Abstract

Cohesin is an essential structural component of chromosomes that ensures accurate chromosome segregation during mitosis and meiosis. Previous studies have shown that there are cohesin complexes specific to meiosis, required to mediate homologous chromosome pairing, synapsis, recombination, and segregation. Meiosis-specific cohesin complexes consist of two structural maintenance of chromosomes proteins (SMC1α/SMC1β and SMC3), an α-kleisin protein (RAD21, RAD21L, or REC8), and a stromal antigen protein (STAG1, 2, or 3). STAG3 is exclusively expressed during meiosis, and is the predominant STAG protein component of cohesin complexes in primary spermatocytes from mouse, interacting directly with each α-kleisin subunit. REC8 and RAD21L are also meiosis-specific cohesin components. Stag3 mutant spermatocytes arrest in early prophase ("zygotene-like" stage), displaying failed homolog synapsis and persistent DNA damage, as a result of unstable loading of cohesin onto the chromosome axes. Interestingly, Rec8, Rad21L double mutants resulted in an earlier "leptotene-like" arrest, accompanied by complete absence of STAG3 loading. To assess genetic interactions between STAG3 and α-kleisin subunits RAD21L and REC8, our lab generated Stag3, Rad21L, and Stag3, Rec8 double knockout mice, and compared them to the Rec8, Rad21L double mutant. These double mutants are phenotypically distinct from one another, and more severe than each single knockout mutant with regards to chromosome axis formation, cohesin loading, and sister chromatid cohesion. The Stag3, Rad21L, and Stag3, Rec8 double mutants both progress further into prophase I than the Rec8, Rad21L double mutant. Our genetic analysis demonstrates that cohesins containing STAG3 and REC8 are the main complex required for centromeric cohesion, and RAD21L cohesins are required for normal clustering of pericentromeric heterochromatin. Furthermore, the STAG3/REC8 and STAG3/RAD21L cohesins are the primary cohesins required for axis formation.

Keywords: SMC; STAG3; cohesin; meiosis; synapsis.

Figure 1

Combining the Stag3 mutation with Rec8 or Rad21l mutations result in decreased axis length and increased axis number. (A) Example chromatin spread preparations from purified testicular germ cells of control, Rad21l, Rec8, Stag3 single mutants and the three possible double mutant combinations aged 15 d postpartum. Chromatin spreads were immunolabeled using antibodies against the SC lateral element protein SYCP3 (red), and the transverse filament of the central region of the SC SYCP1 (green). Zygotene and pachytene stages are depicted for control, and typical examples for each mutant are given. (B) Scatter dot-plot graph of the average SYCP3 length per spermatocyte chromatin spread. (C) Scatter dot-plot graph of the number of SYCP3 linear stretches per spermatocyte chromatin spread. Mean and SD of the columns of each graph are represented by the black bars, and P values are given for indicated comparisons (Mann–Whitney, two-tailed), significant differences were defined when the P value was < 0.05, otherwise it was considered not significant. Experiments were performed using four separate littermate pairs of mutant and control mice. Scale bars = 10 µm. Images in (A), and data in (B) and (C) are of spermatocytes carrying the Stag3^OV mutant allele, but similar phenotypes were observed for spermatocytes with the Stag3^JAX mutant allele (Figure S3).

Figure 2

STAG3 maintains centromere cohesion, which is primarily mediated by REC8-containing cohesin complexes. (A) Example chromatin spread preparations from purified testicular germ cells of control, Rad21l, Rec8, Stag3 single mutants, and the three possible double mutant combinations aged 15 d postpartum. Chromatin spreads were immunolabeled using antibodies against the SC lateral element protein SYCP3 (red), and the CEN anti-centromere autoantibody (green). Zygotene and pachytene stages are depicted for control, and typical examples for each mutant are given. (B) Scatter dot-plot graph of the average number of centromere signals per spermatocyte chromatin spread. Mean and SD of the columns of each graph are represented by the black bars, and P values are given for indicated comparisons (Mann–Whitney, two-tailed), significant differences were defined when the P value was < 0.05, otherwise it was considered not significant. Experiments were performed using four separate littermate pairs of mutant and control mice. Scale bars = 10 µm. Images in (A), and data in (B) and (C) are of spermatocytes carrying the Stag3^OV mutant allele, but similar phenotypes were observed for spermatocytes with the Stag3^JAX mutant allele (Figure S4).

Figure 3

RAD21L and REC8 cohesins are differentially required for regulating pericentromeric heterochromatin clustering. (A) Example chromatin spread preparations from purified testicular germ cells of control, Rad21l, Rec8, Stag3 single mutants, and the three possible double mutant combinations aged 15 d postpartum. Chromatin spreads were stained with DAPI (turquoise, DNA), and immunolabeled using antibodies against the SC lateral element protein SYCP3 (red). Zygotene and pachytene stages are depicted for control, and typical examples for each mutant are given. (B) Scatter dot-plot graph of the average number of pericentromeric heterochomatin signals per spermatocyte chromatin spread. Mean and SD of the columns of each graph are represented by the black bars and P values are given for indicated comparisons (Mann–Whitney, two-tailed), significant differences were defined when the P value was < 0.05, otherwise it was considered not significant. Experiments were performed using four separate littermate pairs of mutant and control mice. Scale bars = 10 µm.

Figure 4

STAG3 is required for stable localization of RAD21L and REC8 cohesins at chromosome axes. (A–C) Example chromatin spread preparations from purified testicular germ cells of control, Rad21l, Rec8, Stag3 single mutants, and the three possible double mutant combinations aged 15 d postpartum. Chromatin spreads were immunolabeled using antibodies against the SC lateral element protein SYCP3 (red) and either RAD21 (A), or RAD21L (B), or REC8 (C), all of which are shown in green. Zygotene and pachytene stages are depicted for control, and typical examples for each mutant are given. The numbers within each chromatin spread represent the average Manders’ colocalization/overlap coefficient for each cohesin component within the SYCP3 axes (N = 25 chromatin spreads per strain). Scale bars = 10 µm.

Figure 5

STAG3 is required for stable axial localization of meiosis-specific cohesins, but not the mitotic cohesins. (A–C) Example chromatin spread preparations from purified testicular germ cells of control, Rad21l, Rec8, Stag3 single mutants, and the three possible double mutant combinations aged 15 d postpartum. Chromatin spreads were immunolabeled using antibodies against the SC lateral element protein SYCP3 (red), and either SMC3 (A), or SMC1α (B), or SMC1β (C), all of which are shown in green. Zygotene and pachytene stages are depicted for control, and typical examples for each mutant are given. The numbers within each chromatin spread represent the average Manders’ colocalization/overlap coefficient for each cohesin component within the SYCP3 axes (N = 25 chromatin spreads per strain). Scale bars = 10 µm.