ZYP1-mediated recruitment of PCH2 to the synaptonemal complex remodels the chromosome axis leading to crossover restriction

Abstract

Chromosome axis-associated HORMA domain proteins (HORMADs), e.g. ASY1 in Arabidopsis, are crucial for meiotic recombination. ASY1, as other HORMADs, is assembled on the axis at early meiosis and depleted when homologous chromosomes synapse. Puzzlingly, both processes are catalyzed by AAA+ ATPase PCH2 together with its cofactor COMET. Here, we show that the ASY1 remodeling complex is temporally and spatially differently assembled. While PCH2 and COMET appear to directly interact in the cytoplasm in early meiosis, PCH2 is recruited by the transverse filament protein ZYP1 and brought to the ASY1-bound COMET assuring the timely removal of ASY1 during chromosome synapsis. Since we found that the PCH2 homolog TRIP13 also binds to the ZYP1 homolog SYCP1 in mouse, we postulate that this mechanism is conserved among eukaryotes. Deleting the PCH2 binding site of ZYP1 led to a failure of ASY1 removal. Interestingly, the placement of one obligatory crossover per homologous chromosome pair, compromised by ZYP1 depletion, is largely restored in this separation-of-function zyp1 allele suggesting that crossover assurance is promoted by synapsis. In contrast, this zyp1 allele, similar to the zyp1 null mutant, showed elevated type I crossover numbers indicating that PCH2-mediated eviction of ASY1 from the axis restricts crossover formation.

Figure 1.

Recruitment of PCH2 to the synaptonemal complex is independent of the chromosome axis and its co-factor COMET. (A) A schematic representation of the structure of synapsing chromosomes and the key proteins involved. (B) Localization of PCH2:GFP in the male meiocytes of wild-type (WT), asy1, asy3, and comet mutant plants at pachytene or pachytene-like stages using confocal laser scanning microscope. Red arrowheads indicate the short stretches of PCH2 signal localizing at synapsed chromosomal regions. Bars: 10 μm. (C) Co-immunolocalization of PCH2 and ZYP1 in WT, asy1 and comet mutants at pachytene or pachytene-like stages and at late prophase I. Bars: 5 μm.

Figure 2.

Recruitment of PCH2 to the synaptonemal complex is dependent on ZYP1. (A) Schematic depiction of the structure of synapsing/co-aligning chromosomes in WT, zyp1a/b-1, and ZYP1B^Δ661–725 (zyp1a/b-1) mutant plants. The question marks represent the questions raised in the relevant mutants. (B) Localization patterns of PCH2:GFP together with ASY3:RFP in male meiocytes of WT and zyp1a/b-1 mutant plants at pachytene or pachytene-like stages. Bars: 10 μm. (C) Localization patterns of COMET:GFP together with ASY3:RFP in male meiocytes of WT and zyp1a/b-1 mutant plants at early prophase. Bars: 10 μm. (D) Localization pattern of PCH2:GFP together with ASY3:RFP in male meiocytes of WT and ZYP1B^Δ661–725 (zyp1a/b-1) mutant plants at pachytene-like stage. Bars: 10 μm.

Figure 3.

PCH2/TRIP13 interacts with ZYP1/SYCP1. (A) Schematic graph of Arabidopsis ZYP1B and mouse SYCP1 showing the central α-helical core domain flanked by the unstructured N- and C-terminal tails. The lines below indicate the truncations used in the yeast two-hybrid interaction assay. Red lines indicate the positive interaction and black line indicates the autoactivation. (B–D) Yeast two-hybrid assay of PCH2 and ZYP1 (B and C) or TRIP13 and SYCP1 (D) with different truncations. Red and black arrowheads indicate positive interaction and autoactivation, respectively.

Figure 4.

Absence of ZYP1 results in the deficient removal of ASY1 from the chromosome axis. (A) Schematic depiction of the structure of paired chromosomes in WT, zyp1a/b-1, and ZYP1B^Δ661–725 (zyp1a/b-1) mutant plants. (B–D) Co-immunolocalization of ASY1 with ZYP1 or ZYP1B^Δ661–725 in WT (B), zyp1a/b-1 (C) and ZYP1B^Δ661–725 (zyp1a/b-1) (D) mutant plants at different prophase I stages. Bars: 5 μm.

Figure 5.

ZYP1^△661–725 does not completely polymerize in zyp1 mutants. (A) Co-immunolocalization of ZYP1B^Δ661–725 and ASY1 in ZYP1B^Δ661–725 (zyp1a/b-1) mutant plants at pachytene-like stage. Bars: 5 μm. (B) Co-immunolocalization of ZYP1B^Δ661–725 and ASY1 in WT plants at pachytene. Bars: 5 μm.

Figure 6.

ZYP1 and ZYP1-mediated ASY1 removal regulate the formation of wild-type level of COs. (A) Scatter dot plot of the estimation of minimum chiasma number in WT, zyp1a/b-1, zyp1a/b-2, zyp1a/b-3 and ZYP1B^Δ661–725 (zyp1a/b-1) mutant plants. The statistical analysis was performed by one-way ANOVA with Tukey's correction for multiple comparison test. (B) Z-stack imaging of MLH1:GFP in WT, zyp1a/b-1 and ZYP1B^Δ661–725 (zyp1a/b-1) mutant plants at late prophase of male meiocytes. The upper panel shows one representative z-stack image for each genotype and lower panel depicts the maximum intensity projection (MIP) of z-stack images displaying all MLH1 foci from each meiocyte. Bars: 10 μm. (C) Scatter dot plot of the number of MLH1 foci in WT, zyp1a/b-1, zyp1a/b-2, and ZYP1B^Δ661–725 (zyp1a/b-1) mutant plants. The statistical analysis was performed by one-way ANOVA with Dunnett's correction for multiple comparison test. * and ** indicate significant differences at P < 0.05 and P < 0.01, respectively.

Figure 7.

Model for a conserved recruitment mechanism of PCH2 to the SC. (A) Hypothetical generic model of PCH2 binding to the transversal filament that combines information from different organisms and assumes the formation of a similar sized tetrahelical bundle formed by the antiparallel oriented C-terminal ends of two transverse filament dimers, as published for human SYCP1 (57), also occurs in Arabidopsis, mouse and yeast. Two hexameric PCH2 wheels might bind through probably their N-termini to the region of the tetrahelical bundle parallel to the chromosome axis. Alternatively, one hexameric PCH2 wheel might lay flat on top of the tetrahelical bundle. (B–D) The localization of PCH2 at the SC is possibly conserved between diverse organisms although the domains in the transverse filament proteins making the main contact with PCH2 have slightly shifted in evolution. The domains found to be relevant for correct PCH2 binding are indicated in magenta for Arabidopsis (this study), mouse (this study) and Yeast (37).