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. 2014 Oct 23;10(10):e1004638.
doi: 10.1371/journal.pgen.1004638. eCollection 2014 Oct.

Protein Phosphatase 4 Promotes Chromosome Pairing and Synapsis, and Contributes to Maintaining Crossover Competence With Increasing Age

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Free PMC article

Protein Phosphatase 4 Promotes Chromosome Pairing and Synapsis, and Contributes to Maintaining Crossover Competence With Increasing Age

Aya Sato-Carlton et al. PLoS Genet. .
Free PMC article

Abstract

Prior to the meiotic divisions, dynamic chromosome reorganizations including pairing, synapsis, and recombination of maternal and paternal chromosome pairs must occur in a highly regulated fashion during meiotic prophase. How chromosomes identify each other's homology and exclusively pair and synapse with their homologous partners, while rejecting illegitimate synapsis with non-homologous chromosomes, remains obscure. In addition, how the levels of recombination initiation and crossover formation are regulated so that sufficient, but not deleterious, levels of DNA breaks are made and processed into crossovers is not understood well. We show that in Caenorhabditis elegans, the highly conserved Serine/Threonine protein phosphatase PP4 homolog, PPH-4.1, is required independently to carry out four separate functions involving meiotic chromosome dynamics: (1) synapsis-independent chromosome pairing, (2) restriction of synapsis to homologous chromosomes, (3) programmed DNA double-strand break initiation, and (4) crossover formation. Using quantitative imaging of mutant strains, including super-resolution (3D-SIM) microscopy of chromosomes and the synaptonemal complex, we show that independently-arising defects in each of these processes in the absence of PPH-4.1 activity ultimately lead to meiotic nondisjunction and embryonic lethality. Interestingly, we find that defects in double-strand break initiation and crossover formation, but not pairing or synapsis, become even more severe in the germlines of older mutant animals, indicating an increased dependence on PPH-4.1 with increasing maternal age. Our results demonstrate that PPH-4.1 plays multiple, independent roles in meiotic prophase chromosome dynamics and maintaining meiotic competence in aging germlines. PP4's high degree of conservation suggests it may be a universal regulator of meiotic prophase chromosome dynamics.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Mutations in the pph-4.1 gene lead to loss of chiasmata.
(A) Schematics of the pph-4.1 gene, deletion allele, and transgenes constructed in this study. (B) Age-dependent failure to create chiasmata at meiosis. The number of DAPI-staining bodies are shown as percentages of the indicated number of late prophase oocytes scored for each genotype. Image insets show a wild-type nucleus (left) and a tm1598 mutant nucleus (right) at 24 h post-L4.
Figure 2
Figure 2. Autosomal pairing is diminished in pph-4.1 mutants.
(A) Schematic showing hermaphrodite gonads divided into 5 equally-sized zones for scoring. (B) FISH images demonstrate paired 5S rDNA sites in wild-type (left; arrowheads indicate paired foci) and unpaired sites in pph-4.1 mutants (right; arrowheads indicate unpaired foci) at pachytene. (C) quantitation of pairing for chromosome V (left) and X (right) shown as the percent of nuclei with paired signals in each zone. Error bars show standard deviation. Six gonads were scored for each genotype. The total number of nuclei scored for zone 1,2,3,4,5 respectively was as follows: WT 24 h pL4: 293, 337, 409, 410, 222; wt 72 h pL4: 283, 322, 333, 314, 184; pph-4.1 mutant 24 h pL4: 237, 333, 303, 297, 269; pph-4.1 mutant 72 h pL4: 318, 340, 393, 347, 305.
Figure 3
Figure 3. Canonical SC structure but reduced synapsis-independent pairing in pph-4.1 mutants.
(A) 3D-SIM images of pachytene nuclei immunostained for axial element HTP-3 (violet in merged image) and central element SYP-1 (green). Boxed insets at 5x higher magnification demonstrate position of SYP-1 between parallel tracks of HTP-3. (B) quantitation of SC-independent pairing of 5S rDNA loci in syp-2 and syp-2; pph-4.1 mutants. The percent of nuclei with paired foci in each of 5 zones (see Figure 2) is shown; error bars show SD. Six gonads were scored for each genotype. The total number of nuclei scored for zones 1–5 was as follows: syp-2 single mutant: 294, 322, 427, 417, 249; syp-2; pph-4.1 double mutant: 268, 281, 245, 295, 251.
Figure 4
Figure 4. Multiple synaptic aberrations are found in pph-4.1 mutants.
(A) 3D-SIM image of synapsed chromosomes in a wild-type nucleus. Top row shows maximum-intensity projections of image data in multiple channels; bottom row shows computer-aided traces of the six paired chromosomes. Correspondences between computer model (left) and straightened chromosomes (right) shown by colored dots. (B) A wild-type nucleus stained for SYP-1 and ZIM-3 showing two ZIM-3 foci at the synapsed PC ends of chromosomes I and IV. (C) 3D-SIM image of a pph-4.1 nucleus shown in maximum-intensity projection of the entire nucleus (leftmost image, color) and a subset of Z sections (individual grayscale channels) highlighting a nonhomologously synapsed quartet of chromosomes, each making one or two switches of pairing partner. Computer traces (left) show seven individual strands, indicating two chromosomes likely undergoing foldback synapsis in the same nucleus. (D) pph-4.1 nucleus stained for SYP-1 and ZIM-3 shows three synapsed foci, indicating non-homologous synapsis. (E) Highlighted examples of aberrant synapsis in two pph-4.1 nuclei. HTP-3, SYP-1, and HIM-8 are shown to highlight axial elements, central elements, and the X chromosome. Straightened chromosome images are starred to correspond to individual chromosomes in the 3D traces. All chromosome configurations shown in schematic are inferred from straightened chromosome lengths and the requirement that 12 individual chromosomes are involved.
Figure 5
Figure 5. DSB initiation is perturbed in an age-dependent manner in pph-4.1 mutants.
(A) Wild-type and pph-4.1 nuclei shown with DAPI staining in magenta and α-RAD-51 staining in green. Top, γ-irradiation at 10Gy restores RAD-51 staining to pph-4.1 nuclei. Bottom, quantitation of RAD-51 focus formation in wild-type and mutant animals. RAD-51 focus numbers are depicted as a box plot, with box indicating mean and quartiles. Significance was assessed via the Mann-Whitney test. Three gonads were scored for each condition; the numbers of nuclei scored in zones 1–7 are as follows: for wild-type, 316, 312, 256, 252, 231, 198, 120; for pph-4.1, 231, 244, 237, 245, 231, 205, 136. (B) Quantitation of RAD-51 foci with increasing maternal age. Numbers of foci in each of 7 zones are depicted with box plots as in (A). Top, focus numbers compared between rad-54 and rad-54; pph-4.1 animals at 24 h post-L4. Bottom, comparison at 72 h post-L4. Asterisks indicate significant differences due to loss of pph-4.1; diamonds between top and bottom graphs show significance due to age; comparisons were performed via the Mann-Whitney test. Three gonads were scored for each condition; the numbers of nuclei scored in zones 1–7 are as follows: for rad-54 24 h, 252, 313, 443, 397, 311, 236, 111; for rad-54 72 h, 237, 321, 397, 467, 395, 268, 64; for rad-54; pph-4.1 24 h, 288, 288, 306, 359, 300, 232, 70; for rad-54; pph-4.1 72 h, 255, 230, 262, 251, 229, 218, 118.
Figure 6
Figure 6. COSA-1 foci are reduced, and γ-irradiation does not rescue bivalent formation in pph-4.1 mutants.
(A) COSA-1 staining suggests reduced COs in the pph-4.1 mutant. Top, meiotic cells at pachytene in WT (left) and pph-4.1 (right). Bottom, quantitation of COSA-1 focus number. p value of chi-square test is shown. Scale bar, 2 µm. (B) Estimation of age-dependent probabilities of COSA-1 sites to successfully mature into COs. Inset graphs show the sum of the squared differences between the DAPI body-inferred and the COSA-1 focus-inferred chiasma numbers as a function of varying Psuccess. The minimum value (used to generate the bar graph adjustments) is indicated with an arrow. (C) A frequency histogram (normalized to 100%) shows DAPI body numbers for pph-4.1 control (blue bars) and irradiated (green bars) nuclei. R, Spearman's rank correlation coefficient. (D) γ-irradiation restored chiasmata on all 6 chromosome pairs in spo-11(me44) gonads (top) but did not produce additional chiasmata in pph-4.1 gonads (bottom). Scale bar, 5 µm.
Figure 7
Figure 7. Transition zone nucleus numbers are abnormal, and SUN-1 phosphorylation is extended, in pph-4.1 mutants.
(A) Comparison of transition zone length (left) and SUN-1:Ser8p length (right) in wild-type and pph-4.1 mutant worms at 24 h, 48 h, and 72 h post-L4. Percentage of gonad length showing transition zone nucleus morphology based on DAPI staining (left) or showing SUN-1:Ser8p staining (right) is normalized to the gonad length from the transition zone until cellularization. Error bars show SEM; n = 8 gonads scored for all conditions. (B) Representative gonad images of wild-type and pph-4.1 mutant worms are shown at 24 h and 72 h post-L4. A single gonad arm is shown in each subpanel: grayscale images show DAPI staining of nuclei (magenta in merged image) and SUN-1:Ser8p immunostaining (green); color images show the channels merged in magenta and green. Color-coded lines through gonads demarcate the beginning and end of the transition zone (scored by DAPI morphology) or of SUN-1:Ser8p staining, and the beginning of oocyte cellularization, as used for quantitation in Figure 7A.

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