Time-Course Analysis of Early Meiotic Prophase Events Informs Mechanisms of Homolog Pairing and Synapsis in Caenorhabditis elegans.

Segregation of homologous chromosomes during meiosis depends on their ability to reorganize within the nucleus, discriminate among potential partners, and stabilize pairwise associations through assembly of the synaptonemal complex (SC). Here we report a high-resolution time-course analysis of these key early events during Caenorhabditis elegans meiosis. Labeled nucleotides are incorporated specifically into the X chromosomes during the last 2 hr of S phase, a property we exploit to identify a highly synchronous cohort of nuclei. By tracking X-labeled nuclei through early meiotic prophase, we define the sequence and duration of chromosome movement, nuclear reorganization, pairing at pairing centers (PCs), and SC assembly. Appearance of ZYG-12 foci (marking attachment of PCs to the nuclear envelope) and onset of active mobilization occur within an hour after S-phase completion. Movement occurs for nearly 2 hr before stable pairing is observed at PCs, and autosome movement continues for ∼4 hr thereafter. Chromosomes are tightly clustered during a 2-3 hr postpairing window, during which the bulk of SC assembly occurs; however, initiation of SC assembly can precede evident chromosome clustering. SC assembly on autosomes begins immediately after PC pairing is detected and is completed within ∼3.5 hr. For the X chromosomes, PC pairing is contemporaneous with autosomal pairing, but autosomes complete synapsis earlier (on average) than X chromosomes, implying that X chromosomes have a delay in onset and/or a slower rate of SC assembly. Additional evidence suggests that transient association among chromosomes sharing the same PC protein may contribute to partner discrimination.

Evidence that masking of synapsis imperfections counterbalances quality control to promote efficient meiosis.

Reduction in ploidy to generate haploid gametes during sexual reproduction is accomplished by the specialized cell division program of meiosis. Pairing between homologous chromosomes and assembly of the synaptonemal complex at their interface (synapsis) represent intermediate steps in the meiotic program that are essential to form crossover recombination-based linkages between homologs, which in turn enable segregation of the homologs to opposite poles at the meiosis I division. Here, we challenge the mechanisms of pairing and synapsis during C. elegans meiosis by disrupting the normal 1:1 correspondence between homologs through karyotype manipulation. Using a combination of cytological tools, including S-phase labeling to specifically identify X chromosome territories in highly synchronous cohorts of nuclei and 3D rendering to visualize meiotic chromosome structures and organization, our analysis of trisomic (triplo-X) and polyploid meiosis provides insight into the principles governing pairing and synapsis and how the meiotic program is "wired" to maximize successful sexual reproduction. We show that chromosomes sort into homologous groups regardless of chromosome number, then preferentially achieve pairwise synapsis during a period of active chromosome mobilization. Further, comparisons of synapsis configurations in triplo-X germ cells that are proficient or defective for initiating recombination suggest a role for recombination in restricting chromosomal interactions to a pairwise state. Increased numbers of homologs prolong markers of the chromosome mobilization phase and/or boost germline apoptosis, consistent with triggering quality control mechanisms that promote resolution of synapsis problems and/or cull meiocytes containing synapsis defects. However, we also uncover evidence for the existence of mechanisms that "mask" defects, thus allowing resumption of prophase progression and survival of germ cells despite some asynapsis. We propose that coupling of saturable masking mechanisms with stringent quality controls maximizes meiotic success by making progression and survival dependent on achieving a level of synapsis sufficient for crossover formation without requiring perfect synapsis.

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.

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.

The synaptonemal complex shapes the crossover landscape through cooperative assembly, crossover promotion and crossover inhibition during Caenorhabditis elegans meiosis.

The synaptonemal complex (SC) is a highly ordered proteinaceous structure that assembles at the interface between aligned homologous chromosomes during meiotic prophase. The SC has been demonstrated to function both in stabilization of homolog pairing and in promoting the formation of interhomolog crossovers (COs). How the SC provides these functions and whether it also plays a role in inhibiting CO formation has been a matter of debate. Here we provide new insight into assembly and function of the SC by investigating the consequences of reducing (but not eliminating) SYP-1, a major structural component of the SC central region, during meiosis in Caenorhabditis elegans. First, we find an increased incidence of double CO (DCO) meiotic products following partial depletion of SYP-1 by RNAi, indicating a role for SYP-1 in mechanisms that normally limit crossovers to one per homolog pair per meiosis. Second, syp-1 RNAi worms exhibit both a strong preference for COs to occur on the left half of the X chromosome and a significant bias for SYP-1 protein to be associated with the left half of the chromosome, implying that the SC functions locally in promoting COs. Distribution of SYP-1 on chromosomes in syp-1 RNAi germ cells provides strong corroboration for cooperative assembly of the SC central region and indicates that SYP-1 preferentially associates with X chromosomes when it is present in limiting quantities. Further, the observed biases in the distribution of both COs and SYP-1 protein support models in which synapsis initiates predominantly in the vicinity of pairing centers (PCs). However, discontinuities in SC structure and clear gaps between localized foci of PC-binding protein HIM-8 and X chromosome-associated SYP-1 stretches allow refinement of models for the role of PCs in promoting synapsis. Our data suggest that the CO landscape is shaped by a combination of three attributes of the SC central region: a CO-promoting activity that functions locally at CO sites, a cooperative assembly process that enables CO formation in regions distant from prominent sites of synapsis initiation, and CO-inhibitory role(s) that limit CO number.

Differences between homologous alleles of olfactory receptor genes require the Polycomb Group protein Eed.

A number of mammalian genes are expressed from only one of the two homologous chromosomes, selected at random in each cell. These include genes subject to X-inactivation, olfactory receptor (OR) genes, and several classes of immune system genes. The means by which monoallelic expression is established are only beginning to be understood. Using a cytological assay, we show that the two homologous alleles of autosomal random monoallelic loci differ from each other in embryonic stem (ES) cells, before establishment of monoallelic expression. The Polycomb Group gene Eed is required to establish this distinctive behavior. In addition, we found that when Eed mutant ES cells are differentiated, they fail to establish asynchronous replication timing at OR loci. These results suggest a common mechanism for random monoallelic expression on autosomes and the X chromosome, and implicate Eed in establishing differences between homologous OR loci before and after differentiation.

X chromosomes alternate between two states prior to random X-inactivation.

Early in the development of female mammals, one of the two X chromosomes is silenced in half of cells and the other X chromosome is silenced in the remaining half. The basis of this apparent randomness is not understood. We show that before X-inactivation, the two X chromosomes appear to exist in distinct states that correspond to their fates as the active and inactive X chromosomes. Xist and Tsix, noncoding RNAs that control X chromosome fates upon X-inactivation, also determine the states of the X chromosomes prior to X-inactivation. In wild-type ES cells, X chromosomes switch between states; among the progeny of a single cell, a given X chromosome exhibits each state with equal frequency. We propose a model in which the concerted switching of homologous X chromosomes between mutually exclusive future active and future inactive states provides the basis for the apparently random silencing of one X chromosome in female cells.

Role of histone H3 lysine 27 methylation in X inactivation.

The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. Here we demonstrate that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation. Recruitment of the complex and methylation on the Xi depend on Xist RNA but are independent of its silencing function. Together, our results suggest a role for Eed-Ezh2-mediated H3-K27 methylation during initiation of both imprinted and random X inactivation and demonstrate that H3-K27 methylation is not sufficient for silencing of the Xi.

Xist RNA and the mechanism of X chromosome inactivation.

Dosage compensation in mammals is achieved by the transcriptional inactivation of one X chromosome in female cells. From the time X chromosome inactivation was initially described, it was clear that several mechanisms must be precisely integrated to achieve correct regulation of this complex process. X-inactivation appears to be triggered upon differentiation, suggesting its regulation by developmental cues. Whereas any number of X chromosomes greater than one is silenced, only one X chromosome remains active. Silencing on the inactive X chromosome coincides with the acquisition of a multitude of chromatin modifications, resulting in the formation of extraordinarily stable facultative heterochromatin that is faithfully propagated through subsequent cell divisions. The integration of all these processes requires a region of the X chromosome known as the X-inactivation center, which contains the Xist gene and its cis-regulatory elements. Xist encodes an RNA molecule that plays critical roles in the choice of which X chromosome remains active, and in the initial spread and establishment of silencing on the inactive X chromosome. We are now on the threshold of discovering the factors that regulate and interact with Xist to control X-inactivation, and closer to an understanding of the molecular mechanisms that underlie this complex process.