Caenorhabditis elegans DSB-3 reveals conservation and divergence among protein complexes promoting meiotic double-strand breaks

Meiotic recombination plays dual roles in the evolution and stable inheritance of genomes: Recombination promotes genetic diversity by reassorting variants, and it establishes temporary connections between pairs of homologous chromosomes that ensure their future segregation. Meiotic recombination is initiated by generation of double-strand DNA breaks (DSBs) by the conserved topoisomerase-like protein Spo11. Despite strong conservation of Spo11 across eukaryotic kingdoms, auxiliary complexes that interact with Spo11 complexes to promote DSB formation are poorly conserved. Here, we identify DSB-3 as a DSB-promoting protein in the nematode Caenorhabditis elegans. Mutants lacking DSB-3 are proficient for homolog pairing and synapsis but fail to form crossovers. Lack of crossovers in dsb-3 mutants reflects a requirement for DSB-3 in meiotic DSB formation. DSB-3 concentrates in meiotic nuclei with timing similar to DSB-1 and DSB-2 (predicted homologs of yeast/mammalian Rec114/REC114), and DSB-1, DSB-2, and DSB-3 are interdependent for this localization. Bioinformatics analysis and interactions among the DSB proteins support the identity of DSB-3 as a homolog of MEI4 in conserved DSB-promoting complexes. This identification is reinforced by colocalization of pairwise combinations of DSB-1, DSB-2, and DSB-3 foci in structured illumination microscopy images of spread nuclei. However, unlike yeast Rec114, DSB-1 can interact directly with SPO-11, and in contrast to mouse REC114 and MEI4, DSB-1, DSB-2, and DSB-3 are not concentrated predominantly at meiotic chromosome axes. We speculate that variations in the meiotic program that have coevolved with distinct reproductive strategies in diverse organisms may contribute to and/or enable diversification of essential components of the meiotic machinery.

Meiotic recombination is important for two reasons. It promotes genetic diversity by reassorting traits, and it creates temporary attachments between pairs of homologous chromosomes that are necessary for their future segregation at the meiosis I division. Recombination is initiated by the programmed introduction of DNA double-strand breaks (DSBs) (1). Some DSBs are repaired by a mechanism that leads to the formation of crossovers (COs) between homolog pairs, and the remaining DSBs are repaired as non-CO products, thereby restoring genome integrity. Although DSBs are required for CO formation, they may lead to genomic instability if they are not repaired or are repaired erroneously. Thus, DSB formation in meiotic cells is governed by regulatory and surveillance mechanisms that function to ensure that enough DSBs are created to guarantee a CO on each homolog pair while limiting excess DSBs that may endanger the genome (2). Without appropriate DSB formation and repair, COs may fail to form between homologs during meiotic prophase, resulting in unattached homologs (univalents) that missegregate during the meiotic divisions, leading to aneuploidy in the resulting progeny.Meiotic DSB formation is catalyzed by Spo11, a topoisomerase-like protein homologous to the catalytic A subunit of archaeal class VI topoisomerases that is well conserved across eukaryotic kingdoms (3–6). The mechanism of DNA breakage involves formation of a covalent linkage between the Spo11 protein and DNA, analogous to a key intermediate in the topisomerase reaction (1). Despite identification of structural and mechanistic conservation between Spo11 and TopVIA more than 20 y ago, however, counterparts of the archaeal TopVIB subunit that partner with Spo11 in “Spo11 core complexes” were not recognized until much later, reflecting substantial divergence both from TopVIB and among their eukaryotic orthologs (7–9).DSB formation also depends on multiple additional factors that play critical roles in determining the location, timing, levels, and regulation of DSB formation (2). Several of these auxiliary DSB-promoting factors, including Rec114, Mei4, and Mer2, were originally discovered through genetic screens in Saccharomyces cerevisiae designed to identify genes required for initiation of recombination (10–14) and similar screens in Schizosaccharomyces pombe (15, 16). In contrast to the high level of conservation observed for Spo11, but similar to the other subunits of the Spo11 core complex, many auxiliary DSB protein such Rec114, Mei4, and Mer2 are poorly conserved at the primary sequence level (1). Indeed, apart from limited homology detected between S. cerevisiae Rec114 and S. pombe Rec7 (17–19), high levels of sequence divergence had prevented identification of Rec114, Mei4, and Mer2 homologs outside of budding yeast until nonstandard bioinformatics approaches were applied (20, 21). Homologs of Rec114 and Mei4 that are required for meiotic recombination have now been identified in several species, including Mus musculus (21–23), S. pombe (17, 18, 24), and Arabidopsis thaliana (25, 26). Proteins discovered independently based on roles in meiotic recombination in the ascomycete Sordaria macrospora (Asy1) and the nematode Caenorhabditis elegans (DSB-1 and DSB-2) were also subsequently identified as putative Rec114 homologs (20, 27, 28), but Mei4 homologs were not yet identified in these organisms.Several studies have established that DSB auxiliary factors Rec114 and Mei4 work closely together with each other and with Mer2 to promote meiotic DSB formation. Physical interactions among these proteins and their orthologs have been demonstrated for several organisms (19, 21, 29–32), and coimmunoprecipitation experiments in M. musculus have further confirmed that these proteins interact with one another in vivo in a meiotic context (23). Recent biochemical analyses have shown that Rec114 and Mei4 together form individual complexes with a stoichiometry of two Rec114 molecules for every one Mei4 molecule and have further suggested that these complexes may self-assemble into large molecular condensates on chromatin during meiotic progression (33). In both S. cerevisiae and M. musculus, all three proteins have been reported to localize together in foci on meiotic prophase chromosomes (19, 23, 29, 32). Further, mouse REC114 and MEI4 and the Mer2 homolog IHO1 all localize predominantly at the meiotic chromosome axis (23, 32), contributing to the idea that they act as an intermediary between chromosome organization and DSB formation. Consistent with this view, chromatin immunoprecipitation experiments in both S. cerevisiae and S. pombe have shown that these proteins interact with both axis-enriched DNA sequences and with DSB sites (31, 34–36). Additionally, S. cerevisiae Rec114 and Mei4 interact with the Rec102 and Rec104 subunits that together comprise the TopVIB-like component of the Spo11 core complex (9, 19). Together these findings implicate Rec114–Mei4 in recruiting Spo11 to the meiotic chromosome axis.C. elegans DSB-1 and DSB-2, while clearly implicated in meiotic DSB formation, were difficult to recognize as Rec114 homologs owing to high sequence divergence (20, 27, 28). Further, C. elegans differs from yeast and mice regarding the relationships between DSB formation and meiotic chromosome organization. Whereas DSB-dependent recombination intermediates are required to trigger assembly of the synaptonemal complex (SC) between homologous chromosomes in yeast and mice, C. elegans can achieve full synapsis between aligned homologs even in the absence of DSB formation (6). Thus, there are substantial differences in the cellular environments in which DSB-promoting complexes have evolved and function in different organisms.In our current work, we identify DSB-3 as a protein that partners with DSB-1 and DSB-2 to promote SPO-11–dependent meiotic DSB formation in C. elegans. We demonstrate a requirement for DSB-3 in promoting the DSBs needed for CO formation, and we show that DSB-3 becomes concentrated in germ cell nuclei during the time when DSBs are formed, in a manner that is interdependent with DSB-1 and DSB-2. Through a combination of bioinformatics, interaction data, and colocalization analyses, we identify DSB-3 as a likely Mei4 homolog and establish DSB-1–DSB-2–DSB-3 as functional counterpart of the Rec114-Mei4 complex. Despite homology and a shared role in promoting DSB formation, we find that C. elegans DSB-1, DSB-2, and DSB-3 are distributed broadly on chromatin rather than becoming concentrated preferentially on chromosome axes as observed for mouse REC114–MEI4 complexes. This work highlights the evolutionary malleability of protein complexes that serve essential, yet auxiliary, roles in meiotic recombination. Rapid diversification of such proteins may reflect a relaxation of constraints enabled by changes in another aspect of the reproductive program, or alternatively, they may reflect a capacity of alterations in such proteins to have an immediate impact on reproductive success.     

Uptake and tissue-specific distribution of selected polychlorinated biphenyls in developing chicken embryos

Fertilized chicken eggs were injected with high doses of individual polychlorinated biphenyl (PCB) congeners (0.5 microg of PCB 77, 9.8 microg of PCB 153, or 10.9 microg of PCB 180) before incubation to investigate the structure-specific uptake of these compounds by the embryo and their accumulation in brain and liver tissue. In accordance with earlier publications, a gradual uptake and accumulation of these compounds was observed during the last week of embryonic development. The PCB uptake and distribution to the specific tissues did not appear to be structure dependent. Wet-weight liver PCB concentrations (18, 266, and 278 ng/g at hatching for PCB 77, PCB 153, and PCB 180, respectively) were consistently two- to fourfold higher than carcass levels (7 ng/g of PCB 77, 117 ng/g of PCB 153, and 81 ng/g of PCB 180 at hatching). Whereas liver and carcass concentrations increased exponentially between day 13 of incubation and hatching, PCB levels in brain tissue remained unaltered (range, 0.6-1.0 ng/g of PCB 77 and 8-12 ng/g of PCB 153 and PCB 180 throughout the last week of incubation). Lipid analysis of the organs suggested that the lipid composition of brain may be an important factor explaining the low PCB accumulation in this tissue.     

5-HTR<Subscript>2A</Subscript> and 5-HTR<Subscript>3A</Subscript> but not 5-HTR<Subscript>1A</Subscript> antagonism impairs the cross-modal reactivation of deprived visual cortex in adulthood

Visual cortical areas show enhanced tactile responses in blind individuals, resulting in improved behavioral performance. Induction of unilateral vision loss in adult mice, by monocular enucleation (ME), is a validated model for such cross-modal brain plasticity. A delayed whisker-driven take-over of the medial monocular zone of the visual cortex is preceded by so-called unimodal plasticity, involving the potentiation of the spared-eye inputs in the binocular cortical territory. Full reactivation of the sensory-deprived contralateral visual cortex is accomplished by 7 weeks post-injury. Serotonin (5-HT) is known to modulate sensory information processing and integration, but its impact on cortical reorganization after sensory loss, remains largely unexplored. To address this issue, we assessed the involvement of 5-HT in ME-induced cross-modal plasticity and the 5-HT receptor (5-HTR) subtype used. We first focused on establishing the impact of ME on the total 5-HT concentration measured in the visual cortex and in the somatosensory barrel field. Next, the changes in expression as a function of post-ME recovery time of the monoamine transporter 2 (vMAT2), which loads 5-HT into presynaptic vesicles, and of the 5-HTR_1A and 5-HTR_3A were assessed, in order to link these temporal expression profiles to the different types of cortical plasticity induced by ME. In order to accurately pinpoint which 5-HTR exactly mediates ME-induced cross-modal plasticity, we pharmacologically antagonized the 5-HTR_1A, 5-HTR_2A and 5-HTR_3A subtypes. This study reveals brain region-specific alterations in total 5-HT concentration, time-dependent modulations in vMAT2, 5-HTR_1A and 5-HTR_3A protein expression and 5-HTR antagonist-specific effects on the post-ME plasticity phenomena. Together, our results confirm a role for 5-HTR_1A in the early phase of binocular visual cortex plasticity and suggest an involvement of 5-HTR_2A and 5-HTR_3A but not 5-HTR_1A during the late cross-modal recruitment of the medial monocular visual cortex. These insights contribute to the general understanding of 5-HT function in cortical plasticity and may encourage the search for improved rehabilitation strategies to compensate for sensory loss.