24 research outputs found
Phosphoregulation of HORMA domain protein HIM-3 promotes asymmetric synaptonemal complex disassembly in meiotic prophase in Caenorhabditis elegans
正常な精子・卵子の形成メカニズムを解明 --染色体の分離に重要なタンパク質の発見--. 京都大学プレスリリース. 2020-12-04.In the two cell divisions of meiosis, diploid genomes are reduced into complementary haploid sets through the discrete, two-step removal of chromosome cohesion, a task carried out in most eukaryotes by protecting cohesion at the centromere until the second division. In eukaryotes without defined centromeres, however, alternative strategies have been innovated. The best-understood of these is found in the nematode Caenorhabditis elegans: after the single off-center crossover divides the chromosome into two segments, or arms, several chromosome-associated proteins or post-translational modifications become specifically partitioned to either the shorter or longer arm, where they promote the correct timing of cohesion loss through as-yet unknown mechanisms. Here, we investigate the meiotic axis HORMA-domain protein HIM-3 and show that it becomes phosphorylated at its C-terminus, within the conserved “closure motif” region bound by the related HORMA-domain proteins HTP-1 and HTP-2. Binding of HTP-2 is abrogated by phosphorylation of the closure motif in in vitro assays, strongly suggesting that in vivo phosphorylation of HIM-3 likely modulates the hierarchical structure of the chromosome axis. Phosphorylation of HIM-3 only occurs on synapsed chromosomes, and similarly to other previously-described phosphorylated proteins of the synaptonemal complex, becomes restricted to the short arm after designation of crossover sites. Regulation of HIM-3 phosphorylation status is required for timely disassembly of synaptonemal complex central elements from the long arm, and is also required for proper timing of HTP-1 and HTP-2 dissociation from the short arm. Phosphorylation of HIM-3 thus plays a role in establishing the identity of short and long arms, thereby contributing to the robustness of the two-step chromosome segregation
Phosphoregulation of HORMA domain protein HIM-3 promotes asymmetric synaptonemal complex disassembly in meiotic prophase in Caenorhabditis elegans.
Protein phosphatase 4 promotes chromosome pairing and synapsis, and contributes to maintaining crossover competence with increasing age.
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
Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity
生殖細胞におけるDNA切断制御の解明 --よい塩梅にDNAを切断する仕組み--. 京都大学プレスリリース. 2022-07-21.Breaking DNA Goldilocks-style. 京都大学プレスリリース. 2022-09-08.In the first meiotic cell division, proper segregation of chromosomes in most organisms depends on chiasmata, exchanges of continuity between homologous chromosomes that originate from the repair of programmed double-strand breaks (DSBs) catalyzed by the Spo11 endonuclease. Since DSBs can lead to irreparable damage in germ cells, while chromosomes lacking DSBs also lack chiasmata, the number of DSBs must be carefully regulated to be neither too high nor too low. Here, we show that in Caenorhabditis elegans, meiotic DSB levels are controlled by the phosphoregulation of DSB-1, a homolog of the yeast Spo11 cofactor Rec114, by the opposing activities of PP4[PPH-4.1] phosphatase and ATR[ATL-1] kinase. Increased DSB-1 phosphorylation in pph-4.1 mutants correlates with reduction in DSB formation, while prevention of DSB-1 phosphorylation drastically increases the number of meiotic DSBs both in pph-4.1 mutants as well as in the wild type background. C. elegans and its close relatives also possess a diverged paralog of DSB-1, called DSB-2, and loss of dsb-2 is known to reduce DSB formation in oocytes with increasing age. We show that the proportion of the phosphorylated, and thus inactivated, form of DSB-1 increases with age and upon loss of DSB-2, while non-phosphorylatable DSB-1 rescues the age-dependent decrease in DSBs in dsb-2 mutants. These results suggest that DSB-2 evolved in part to compensate for the inactivation of DSB-1 through phosphorylation, to maintain levels of DSBs in older animals. Our work shows that PP4[PPH-4.1], ATR[ATL-1], and DSB-2 act in concert with DSB-1 to promote optimal DSB levels throughout the reproductive lifespan
Multiple synaptic aberrations are found in <i>pph-4.1</i> mutants.
<p>(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 <i>pph-4.1</i> 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) <i>pph-4.1</i> nucleus stained for SYP-1 and ZIM-3 shows three synapsed foci, indicating non-homologous synapsis. (E) Highlighted examples of aberrant synapsis in two <i>pph-4.1</i> 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.</p
Autosomal pairing is diminished in <i>pph-4.1</i> mutants.
<p>(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 <i>pph-4.1</i> 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; <i>pph-4.1</i> mutant 24 h pL4: 237, 333, 303, 297, 269; <i>pph-4.1</i> mutant 72 h pL4: 318, 340, 393, 347, 305.</p
Canonical SC structure but reduced synapsis-independent pairing in <i>pph-4.1</i> mutants.
<p>(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 <i>syp-2</i> and <i>syp-2; pph-4.1</i> mutants. The percent of nuclei with paired foci in each of 5 zones (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004638#pgen-1004638-g002" target="_blank">Figure 2</a>) 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: <i>syp-2</i> single mutant: 294, 322, 427, 417, 249; <i>syp-2; pph-4.1</i> double mutant: 268, 281, 245, 295, 251.</p
DSB initiation is perturbed in an age-dependent manner in <i>pph-4.1</i> mutants.
<p>(A) Wild-type and <i>pph-4.1</i> nuclei shown with DAPI staining in magenta and α-RAD-51 staining in green. <i>Top</i>, γ-irradiation at 10Gy restores RAD-51 staining to <i>pph-4.1</i> nuclei. <i>Bottom</i>, 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 <i>pph-4.1</i>, 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). <i>Top</i>, focus numbers compared between <i>rad-54</i> and <i>rad-54; pph-4.1</i> animals at 24 h post-L4. <i>Bottom</i>, comparison at 72 h post-L4. Asterisks indicate significant differences due to loss of <i>pph-4.1</i>; 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 <i>rad-54</i> 24 h, 252, 313, 443, 397, 311, 236, 111; for <i>rad-54</i> 72 h, 237, 321, 397, 467, 395, 268, 64; for <i>rad-54; pph-4.1</i> 24 h, 288, 288, 306, 359, 300, 232, 70; for <i>rad-54; pph-4.1</i> 72 h, 255, 230, 262, 251, 229, 218, 118.</p
Mutations in the <i>pph-4.1</i> gene lead to loss of chiasmata.
<p>(A) Schematics of the <i>pph-4.1</i> 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 <i>tm1598</i> mutant nucleus (right) at 24 h post-L4.</p