Chromosome Painting Reveals Asynaptic Full Alignment of Homologs and HIM-8–Dependent Remodeling of X Chromosome Territories during Caenorhabditis elegans Meiosis

During early meiotic prophase, a nucleus-wide reorganization leads to sorting of chromosomes into homologous pairs and to establishing associations between homologous chromosomes along their entire lengths. Here, we investigate global features of chromosome organization during this process, using a chromosome painting method in whole-mount Caenorhabditis elegans gonads that enables visualization of whole chromosomes along their entire lengths in the context of preserved 3D nuclear architecture. First, we show that neither spatial proximity of premeiotic chromosome territories nor chromosome-specific timing is a major factor driving homolog pairing. Second, we show that synaptonemal complex-independent associations can support full lengthwise juxtaposition of homologous chromosomes. Third, we reveal a prominent elongation of chromosome territories during meiotic prophase that initiates prior to homolog association and alignment. Mutant analysis indicates that chromosome movement mediated by association of chromosome pairing centers (PCs) with mobile patches of the nuclear envelope (NE)–spanning SUN-1/ZYG-12 protein complexes is not the primary driver of territory elongation. Moreover, we identify new roles for the X chromosome PC (X-PC) and X-PC binding protein HIM-8 in promoting elongation of X chromosome territories, separable from their role(s) in mediating local stabilization of pairing and association of X chromosomes with mobile SUN-1/ZYG-12 patches. Further, we present evidence that HIM-8 functions both at and outside of PCs to mediate chromosome territory elongation. These and other data support a model in which synapsis-independent elongation of chromosome territories, driven by PC binding proteins, enables lengthwise juxtaposition of chromosomes, thereby facilitating assessment of their suitability as potential pairing partners

HAL-2 promotes homologous pairing during Caenorhabditis elegans meiosis by antagonizing inhibitory effects of synaptonemal complex precursors.

During meiosis, chromosomes align with their homologous pairing partners and stabilize this alignment through assembly of the synaptonemal complex (SC). Since the SC assembles cooperatively yet is indifferent to homology, pairing and SC assembly must be tightly coordinated. We identify HAL-2 as a key mediator in this coordination, showing that HAL-2 promotes pairing largely by preventing detrimental effects of SC precursors (SYP proteins). hal-2 mutants fail to establish pairing and lack multiple markers of chromosome movement mediated by pairing centers (PCs), chromosome sites that link chromosomes to cytoplasmic microtubules through nuclear envelope-spanning complexes. Moreover, SYP proteins load inappropriately along individual unpaired chromosomes in hal-2 mutants, and markers of PC-dependent movement and function are restored in hal-2; syp double mutants. These and other data indicate that SYP proteins can impede pairing and that HAL-2 promotes pairing predominantly but not exclusively by counteracting this inhibition, thereby enabling activation and regulation of PC function. HAL-2 concentrates in the germ cell nucleoplasm and colocalizes with SYP proteins in nuclear aggregates when SC assembly is prevented. We propose that HAL-2 functions to shepherd SYP proteins prior to licensing of SC assembly, preventing untimely interactions between SC precursors and chromosomes and allowing sufficient accumulation of precursors for rapid cooperative assembly upon homology verification

HAL-2 colocalizes with SYP proteins in aggregates that form when SC assembly is prevented.

<p>Co-staining of SYP-1 and HAL-2 in pachytene nuclei of indicated genotypes. In the wild-type nuclei, HAL-2 is localized to the nucleoplasm and exhibits very little colocalization with SYP-1, which is localized at the interface between paired homologs. In contrast, in mutants with severely disrupted SC assembly (cohesin mutant <i>scc-3(ku263)</i> and mutants lacking LE components HTP-3 or HIM-3), SYP-1 does not localize along chromosomes but instead becomes concentrated in nuclear aggregates (presumably polycomplexes); in these mutants, HAL-2 is detected in the nucleoplasm but also colocalizes with SYP-1 in the nuclear aggregates. Further, localization of HAL-2 in nuclear aggregates in <i>scc-3</i> mutants is SYP-dependent. Bar, 4 µm.</p

Defective nuclear reorganization and homologous pairing in <i>hal-2</i> mutants.

<p>(A) Chromosome organization in germline nuclei in regions of wild-type (wt) and <i>hal-2</i> mutant gonads extending from the premeiotic zone (left) through early pachytene (right). In the wild-type gonad, DAPI-stained chromatin appears widely dispersed in premeiotic nuclei and exhibits a highly clustered organization in nuclei within the transition zone (TZ), reflecting clustering of chromosomes during a period of active chromosome mobilization that begins soon after meiotic entry; DAPI signals appear more dispersed in nuclei that have exited the TZ and progressed into pachytene. The <i>hal-2</i> mutant gonad lacks nuclei with the clustered chromosome configuration characteristic of the TZ, indicating impairment of nuclear reorganization upon meiotic entry. Bar, 10 µm. (B) Left: Wild-type and <i>hal-2</i> mutant nuclei at mid-pachytene. The wild-type nuclei contain thick parallel tracks of DAPI-stained chromatin corresponding to aligned homolog pairs, whereas the <i>hal-2</i> mutant nuclei contain disorganized thin DAPI-stained chromatin tracks representing unaligned chromosomes. Right: Each panel shows DAPI-stained chromosomes in a single oocyte at diakinesis, the last stage of meiotic prophase. Whereas 6 bivalents are present in the wild-type oocyte, each representing a homolog pair held together by a chiasma, 12 smaller DAPI-stained bodies (univalents) are observed in the <i>hal-2</i> mutant oocyte, indicating an absence of chiasmata. Bar, 10 µm. (C) 5S rDNA FISH (left) and immunofluorescence (IF) of X chromosome PC-binding protein HIM-8 (right) in pachtyene nuclei from wild-type and <i>hal-2</i> germ lines. A single focus or two closely spaced foci is detected in wild-type nuclei, indicating paired homologs. Two widely spaced foci are seen in the <i>hal-2</i> mutant nuclei, indicating that both the X-PC and 5S rDNA locus were unpaired. Bar, 5 µm.</p

HAL-2 has additional roles in meiosis beyond preventing inappropriate association of SYP proteins with chromosomes.

<p>(A) Immunolocalization of SUN-1 S8-Pi in DAPI-stained gonads of the indicated genotypes, with meiosis progressing from left to right. Panels on the right are zoomed-in images of early (i; blue box) and later (ii; green box) stages of meiotic prophase. In the wild-type gonad, nuclei in the TZ (i) exhibit a clustered chromosome configuration and have multiple SUN-1 S8-Pi NE patches, whereas after progression into pachytene (ii), the chromosomes exhibit a more dispersed distribution within the nuclei and most nuclei have only a single SUN-1 S8-Pi focus remaining on the NE. In the <i>hal-2</i> mutant gonad, nuclei from both regions (i and ii) have dispersed chromosomes and either lack SUN-1 S8-Pi (i) or have low levels of diffuse SUN-1 S8-Pi NE signal (ii; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002880#pgen.1002880.s010" target="_blank">Figure S10</a>), while in the <i>syp-2</i> mutant, nuclei from both regions exhibit clustered chromosomes and multiple SUN-1 S8-Pi NE patches, reflecting prolonged persistence of chromosome mobilization. The <i>hal-2; syp-2</i> double mutant gonad does not exhibit persistent chromosome clustering and multiple persistent SUN-1 S8-Pi NE patches; chromosome clustering is restricted to a short TZ (i), and only a single SUN-1 S8-Pi NE focus persists beyond the TZ, in nuclei with dispersed chromosomes (ii), similar to wild-type gonads. Yellow lines depict the extent of the TZs, with the yellow arrow indicating extension of the TZ in the <i>syp-2</i> mutant beyond the region shown in the image. The <i>zyg-12::gfp</i> transgene was present in the gonads shown here. Bars, 10 µm. (B) Immunostaining of DNA strand exchange protein RAD-51 in early/mid-pachytene nuclei. While abundant RAD-51 foci are observed in both wild-type nuclei and <i>syp-3</i> mutant nuclei, very few foci were detected in the <i>hal-2</i> and <i>syp-3; hal-2</i> mutants, with most nuclei lacking RAD-51 foci. Bar, 2 µm. (C) Immunostaining of RAD-51 in mid-pachytene nuclei of <i>hal-2</i> mutants and wild-type controls. Abundant RAD-51 foci are observed in nuclei of <i>hal-2</i> germ lines exposed to 1 krad of γ irradiation, while few foci are detected in the nuclei of unirradiated <i>hal-2</i> controls. Bar, 2 µm.</p

<i>hal-2</i> mutants lack multiple markers of PC-mediated chromosomal movement.

<p>(A) IF images of early prophase nuclei stained for PLK-2 and SUN-1 phosphorylated on Ser12 (SUN-1 S12-Pi) in wild-type and <i>hal-2</i> animals. In the wild-type nuclei, SUN-1 S12-Pi and PLK-2 are concentrated together in bright patches at the nuclear envelope (NE). In the <i>hal-2</i> nuclei, PLK-2 and SUN-1 S12-Pi are not detected. Bar, 2 µm. (B) IF images of early prophase nuclei stained for ZYG-12::GFP and SUN-1 phosphorylated on Ser8 (SUN-1 S8-Pi) in wild-type and <i>hal-2</i> animals carrying the <i>zyg-12::gfp</i> transgene. In the wild-type nuclei, SUN-1 S8-Pi and ZYG-12::GFP are concentrated together in bright patches at the NE and are also detected at lower levels throughout the entire NE. In the <i>hal-2</i> nuclei shown, SUN-1 S8-Pi is not detected and ZYG-12::GFP is dispersed throughout the NE. Bar, 2 µm. (C) IF images of ZIM-2 (chromosome V PC-binding protein) in early prophase nuclei. A single bright focus near the NE is observed in the wild-type nuclei, whereas none is detected in the <i>hal-2</i> mutant nuclei. Bar, 2 µm.</p

Removal of SYP proteins in <i>hal-2</i> mutants partially restores chromosome clustering, markers of PC-mediated chromosome movement, and homologous pairing.

<p>(A) IF images of early prophase nuclei stained for PLK-2 and SUN-1 S12-Pi in <i>hal-2</i> and <i>hal-2; syp-2(ok307)</i> germ lines. Chromosome clustering, SUN-1 Ser12 phosphorylation and NE patches containing PLK-2 and SUN-1 S12-Pi (features that are missing in the <i>hal-2</i> nuclei) are restored in the <i>hal-2; syp-2</i> nuclei. Bar, 2 µm. (B) <i>hal-2</i> and <i>hal-2; syp-2</i> early prophase nuclei stained with antibodies against ZIM-2. Whereas ZIM-2 foci are not observed in the <i>hal-2</i> mutant nuclei, bright ZIM-2 foci near the NE are restored in the <i>hal-2; syp-2</i> double mutants. Bar, 2 µm. (C) Diagram of a hermaphrodite gonad depicting the 5 zones of equal lengths used for quantitation of pairing in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002880#pgen-1002880-g004" target="_blank">Figures 4D–4F</a>. In a wild-type gonad, Zone 1 contains premeiotic nuclei, Zone 2 includes some premeiotic nuclei, the TZ and some early pachytene nuclei, Zone 3 contains early and mid-pachytene nuclei, Zone 4 contains mid and late pachytene nuclei and Zone 5 consists of late pachytene nuclei. (D) Bar graph showing quantitation of pairing at the X-PC, measured as pairing of HIM-8 foci, for Zones 2–5 (the regions with most robust HIM-8 staining). While pairing of HIM-8 foci was abolished in the <i>hal-2</i> germ lines, high HIM-8 pairing levels were restored in the <i>hal-2; syp-2</i> double mutant, with pairing levels peaking in Zones 3 and 4 (94%; 87%) at levels approaching those of wild-type and the <i>syp-2</i> mutant (99%; 98%). Nonetheless pairing levels in <i>hal-2; syp-2</i> double mutants were significantly lower than those in <i>syp-2</i> gonads for all zones (Zone 2: p<0.0001, Zone 3: p = 0.012, Zone 4: p = 0.0002, Zone 5: p = 0.0194). Worms used in this analysis carried the <i>zyg-12::gfp</i> transgene. (E) Quantitation of ZIM-2 foci in nuclei from Zone 2, the region in which ZIM-2 staining is most robust in wild-type germ lines. Stacked bar graphs depict the percentage of Zone 2 nuclei with a single ZIM-2 focus (indicating paired V-PCs), two unpaired ZIM-2 foci (indicating unpaired V-PCs) or without any clear ZIM-2 foci (indicative of inactive PCs not engaged in chromosome mobilization). Worms used in this analysis carried the <i>zyg-12::gfp</i> transgene. (F) Bar graph depicting quantitation of pairing levels at the 5S rDNA locus assessed by FISH. A modest partial restoration of pairing at this non-PC locus was observed in the <i>syp-3(ok758); hal-2</i> double mutant: 5S rDNA pairing levels were effectively abolished in the <i>hal-2</i> mutant, whereas pairing in <i>syp-3; hal-2</i> double mutants showed a highly significant improvement over <i>hal-2</i> only in Zone 2 (p<0.0001). Further, <i>syp-3; hal-2</i> double mutants displayed significantly lower pairing levels than those in <i>syp-3</i> gonads for Zones 2–5 (p = 0.0023, p<0.0001, p<0.0001, p<0.0004).</p

<i>hal-2</i> mutants exhibit reduced chromosomal localization of HTP-1/2 that is rescued by removal of SYP proteins.

<p>(A) Co-immunolocalization of LE components HTP-3 and HTP-1/2 in pachytene nuclei of indicated genotypes. HTP-1/2 and HTP-3 are detected colocalized along the full lengths of paired and synapsed homologs in wild-type nuclei and along the axes of unpaired chromosomes in nuclei from the late pachytene region in a <i>syp-2</i> single mutant; HTP-1/2 and HTP-3 IF signals are present in similar ratios in these two genotypes. Images from the <i>hal-2</i> mutant show that the level of HTP-1/2 relative to HTP-3 was greatly reduced compared to wild-type and <i>syp-2</i> controls; however, HTP-1/2 localization was not abolished in the <i>hal-2</i> mutant, as shown in the inset, in which the HTP-1/2 IF signal has been adjusted to highlight the faint HTP-1/2 staining along the axes of the unpaired chromosomes. HTP-1/2 localization in <i>hal-2; syp-2</i> double mutant nuclei appears similar to the <i>syp-2</i> single mutant. Bar, 2 µm. (B) Each panel depicts the chromosomes of a single diakinesis oocyte of the indicated genotype, co-stained for HTP-1/2 and HTP-3. HTP-1/2 is detected on the chromosomes in the wild-type and <i>syp-2</i> control oocytes, but is not detected on the chromosomes in the <i>hal-2</i> mutant oocyte; HTP-1/2 staining is detected on the chromosomes in the <i>hal-2; syp-2</i> oocyte, reflecting rescue of the HTP-1/2 localization defect in <i>hal-2</i> mutants by the removal of SYP proteins. Bar, 2 µm.</p