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

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

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

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

Inability to initiate recombination during triplo-X meiosis impairs establishment of exclusive, pairwise interactions.

<p><b>A.</b> Major classes of X chromosome synapsis configurations in triplo-X nuclei illustrated by 3-D surface renderings of individual nuclei stained for HIM-8 (white), HTP-3 (red), SYP-1 (green), and DAPI (blue). Scored nuclei were from the latter half of the region corresponding to mid-pachytene in diploids, which corresponds to the location of S-phase labeled nuclei at the 24 h time point (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen-1003963-g003" target="_blank">Figure 3B</a>). Red dotted traces indicate regions of HTP-3 staining where SYP-1 was not detected, and yellow dotted traces indicate regions of HTP-3 and SYP-1 overlap associated with the X chromosomes. Nuclei i and ii both show synapsis between a presumed pair of Xs and exclusion of the third X to a distinct domain of DAPI staining. In i, the third X lacks SYP-1 (red arrow). In ii, the third X shows SYP-1 over part of its axis length (yellow arrow), and absence of SYP-1 over the remainder (red arrow); for clarity, top two-thirds of this nucleus is rendered. Frequency of classes is indicated in the schematics. The remaining three nuclei showed all three X chromosome axes together for part of their length. Nuclei from 3 germ lines were analyzed. <b>B.</b> The major class of X chromosome synapsis configuration in triplo-X <i>spo-11</i> nuclei, illustrated as in A. The X chromosomes occupy a single, unitary DAPI domain, indicating that the third X is not excluded; the majority of HTP-3 staining within the X chromosome domain appears to localize to a single SC (yellow triangle). The remaining three nuclei showed partial exclusion of the third X chromosome, which showed some SYP-1 loading. Nuclei from 4 germ lines were analyzed.</p

Male germ cells containing supernumerary chromosomes show a greater capacity to apply H3K9me2.

<p><b>A.</b> DAPI staining (white) and IF for SUN-1 S8-Pi (green) and H3K9me2 (red) in whole mount male diploid (1X:2A) and triploid (2X:3A) germ lines. Green and red lines are drawn as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen-1003963-g006" target="_blank">Figure 6</a>; gray lines indicate meiotic zone used for scoring in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen.1003963.s008" target="_blank">Figure S8</a>, from onset of meiotic SUN-1 S8-Pi staining through the end of the pachytene stage. Bar = 10 µm. Insets (right) show H3K9me2 (red) staining patterns in DAPI-stained mid- to late pachytene nuclei (gray). Bars = 4 µm. <b>B.</b> Quantitation of the number of H3K9me2 domains in late pachytene nuclei of hermaphrodite vs. male germ lines with a single odd X chromosome (compare 3X:2A vs. 1X:2A), or a triploid karyotype (compare 3X:3A vs. 2X:3A). 3-D-rendered images were used for scoring. Each stacked bar represents the late pachytene region of a single germ line of the indicated genotype, comprising 18–47 nuclei in hermaphrodites and 14–26 nuclei in males; the “>3” category includes nuclei in which contiguous H3K9me2 domains occupied half or more of the nuclear volume. <b>C.</b> Coincidence of H3K9me2 (blue) staining with synapsis defects in triploid hermaphrodites (3X:3A) and males (3X:2A) illustrated by 3-D surface renderings of half-nuclei from the late pachytene region. HTP-3 (red), SYP-1 (green), and DAPI (gray) are shown. Unsynapsed regions (arrows) are marked in both sexes, whereas aberrantly synapsed regions are not marked in the hermaphrodite (open triangle), but robustly marked in the male (filled triangle).</p

X chromosome territories usually occupy a unitary domain early in the synapsis window.

<p><b>A.</b> Use of S-phase labeling to identify a highly synchronous cohort of meiotic prophase nuclei. Image of the TZ region of a diploid germ line (DAPI in gray) labeled with EdU (red) for 5 h prior to fixation; circles indicate synchronous nuclei displaying a single labeled chromosome pair, <i>i.e.</i>, the late-replicating X chromosomes. Bar = 4 µm. <b>B.</b> 3-D renderings of nuclei early in the synapsis window, illustrating the major class in which PCs are paired and X chromosome territories are “extended” (length greater than twice the width). In all karyotypes the territory comprises a unitary spatial domain suggesting rough alignment. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003963#pgen.1003963.s003" target="_blank">Figure S3</a> for all classes. Germ lines were injected with fluorescently labeled dNTPs 10 h prior to fixation and HIM-8 staining (green); nuclei displaying labeled X chromosomes are found in the late TZ in these samples. <b>C.</b> Quantitation of the spatial organization of X chromosome territories in nuclei fixed at 10 h post labeling. Categorization is based on X-PC pairing status and labeled X chromosome territory shape as indicated in the schematics. Informative classes are highlighted in green and orange. Nuclei from 4–6 germ lines were analyzed.</p