15 research outputs found
ATM/ATR kinases link the synaptonemal complex and DNA double-strand break repair pathway choice
DNA double-strand breaks (DSBs) are deleterious lesions, which must be repaired precisely to maintain genomic stability. During meiosis, programmed DSBs are repaired via homologous recombination (HR) while repair using the nonhomologous end joining (NHEJ) pathway is inhibited, thereby ensuring crossover formation and accurate chromosome segregation.1,2 How DSB repair pathway choice is implemented during meiosis is unknown. In C. elegans, meiotic DSB repair takes place in the context of the fully formed, highly dynamic zipper-like structure present between homologous chromosomes called the synaptonemal complex (SC).3,4,5,6,7,8,9 The SC consists of a pair of lateral elements bridged by a central region composed of the SYP proteins in C. elegans. How the structural components of the SC are regulated to maintain the architectural integrity of the assembled SC around DSB repair sites remained unclear. Here, we show that SYP-4, a central region component of the SC, is phosphorylated at Serine 447 in a manner dependent on DSBs and the ATM/ATR DNA damage response kinases. We show that this SYP-4 phosphorylation is critical for preserving the SC structure following exogenous (Îł-IR-induced) DSB formation and for promoting normal DSB repair progression and crossover patterning following SPO-11-dependent and exogenous DSBs. We propose a model in which ATM/ATR-dependent phosphorylation of SYP-4 at the S447 site plays important roles both in maintaining the architectural integrity of the SC following DSB formation and in warding off repair via the NHEJ repair pathway, thereby preventing aneuploidy.This work was supported by CIHR grant 119468 to M.Z., a MRC core-funded grant to E.M.-P., and National Institutes of Health grant R01GM072551 to M.P.C.Peer reviewe
Tissue-Specific Split sfGFP System for Streamlined Expression of GFP Tagged Proteins in the Caenorhabditis elegans Germline
Identifying protein localization is a useful tool in analyzing protein function. Using GFP-fusion tags, researchers can study the function of endogenous proteins in living tissue. However, these tags are considerably large, making them difficult to insert, and they can potentially affect the normal function of these proteins. To improve on these drawbacks, we have adopted the split sfGFP system for studying the localization of proteins in the Caenorhabditis elegans germline. This system divides the “super folder” GFP into 2 fragments, allowing researchers to use CRISPR/Cas9 to tag proteins more easily with the smaller subunit, while constitutively expressing the larger subunit from another locus. These two parts are able to stably interact, producing a functional GFP when both fragments are in the same cellular compartment. Our data demonstrate that the split sfGFP system can be adapted for use in C. elegans to tag endogenous proteins with relative ease. Strains containing the tags are homozygous viable and fertile. These small subunit tags produce fluorescent signals that matched the localization patterns of the wild-type protein in the gonad. Thus, our study shows that this approach could be used for tissue-specific GFP expression from an endogenous locus
CRL4 regulates recombination and synaptonemal complex aggregation in the Caenorhabditis elegans germline.
To maintain the integrity of the genome, meiotic DNA double strand breaks (DSBs) need to form by the meiosis-specific nuclease Spo11 and be repaired by homologous recombination. One class of products formed by recombination are crossovers, which are required for proper chromosome segregation in the first meiotic division. The synaptonemal complex (SC) is a protein structure that connects homologous chromosomes during meiotic prophase I. The proper assembly of the SC is important for recombination, crossover formation, and the subsequent chromosome segregation. Here we identify the components of Cullin RING E3 ubiquitin ligase 4 (CRL4) that play a role in SC assembly in Caenorhabditis elegans. Mutants of the CRL4 complex (cul-4, ddb-1, and gad-1) show defects in SC assembly manifested in the formation of polycomplexes (PCs), impaired progression of meiotic recombination, and reduction in crossover numbers. PCs that are formed in cul-4 mutants lack the mobile properties of wild type SC, but are likely not a direct target of ubiquitination. In C. elegans, SC assembly does not require recombination and there is no evidence that PC formation is regulated by recombination as well. However, in one cul-4 mutant PC formation is dependent upon early meiotic recombination, indicating that proper assembly of the SC can be diminished by recombination in some scenarios. Lastly, our studies suggest that CUL-4 deregulation leads to transposition of the Tc3 transposable element, and defects in formation of SPO-11-mediated DSBs. Our studies highlight previously unknown functions of CRL4 in C. elegans meiosis and show that CUL-4 likely plays multiple roles in meiosis that are essential for maintaining genome integrity
A Novel Role for α-Importins and Akirin in Establishment of Meiotic Sister Chromatid Cohesion in Caenorhabditis elegans
The CSN/COP9 Signalosome Regulates Synaptonemal Complex Assembly during Meiotic Prophase I of <i>Caenorhabditis elegans</i>
<div><p>The synaptonemal complex (SC) is a conserved protein structure that holds homologous chromosome pairs together throughout much of meiotic prophase I. It is essential for the formation of crossovers, which are required for the proper segregation of chromosomes into gametes. The assembly of the SC is likely to be regulated by post-translational modifications. The CSN/COP9 signalosome has been shown to act in many pathways, mainly via the ubiquitin degradation/proteasome pathway. Here we examine the role of the CSN/COP9 signalosome in SC assembly in the model organism <i>C. elegans</i>. Our work shows that mutants in three subunits of the CSN/COP9 signalosome fail to properly assemble the SC. In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes. The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers. The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes. We also find a marked increase in apoptosis in <i>csn</i> mutants that specifically eliminates nuclei with aggregated SC proteins. <i>csn</i> mutants exhibit defects in germline proliferation, and an almost complete pachytene arrest due to an inability to activate the MAPK pathway. The work described here supports a previously unknown role for the CSN/COP9 signalosome in chromosome behavior during meiotic prophase I.</p></div
SC central element assembly defects in <i>csn</i> mutants.
<p>A) CSN alleles used in this study: black rectangles represent exons, black lines introns, gray areas represent UTR regions, red lines region deleted and purple, green and blue rectangles the protein domains. PAM and PINT are subdomains of the PCI domain, B) Micrographs of SYP-1 (red or grey scaled) and DAPI (blue) stained wild-type and <i>csn-5</i> mutants nuclei representing the various stages of the <i>C. elegans</i> gonad. Images are projections through half of a three-dimensional data stacks. Scale bar is 2 µm. PMT = pre-meiotic tip, TZ = transition zone, EP = early pachytene, MP = mid pachytene, LP = late pachytene. SYP-1 aggregates appear in the TZ-like stage of the gonad and persist through the LP-like stage. C) Whole gonad from wild-type and <i>csn</i> mutants SYP-1 and DAPI stained. Images show smaller gonads in <i>csn</i> mutants and lack of oocytes progressing through diakinesis. Scale Bar 16 µm. SYP-1 (grayscale) staining only of gonads showing aggregation throughout the gonad, starting at transition zone.</p
Quantification of the lack of oocytes and fecundity test.
<p>A) Relative sizes of the pre-meiotic tips for wild-type and the <i>csn</i> mutants. The size of the mitotic zone is reduced in <i>csn</i> mutants. n = 10 for each strain p<0.0005 for wild-type vs. <i>csn-2</i>; p = 0.005 for wild-type vs. <i>csn-5</i> and p<0.05 for <i>csn-2</i> vs. <i>csn-5</i>; Mann Whitney Test B) Quantification of the number of gonads that contained oocytes in diakinesis for the <i>csn</i> mutants, *<i>p</i><sub>MW</sub><0.0005 and **<i>p</i><sub>MW</sub><0.005, Mann Whitney Test C) Top: the average number of oocytes in diakinesis for the <i>csn</i> mutants and the <i>csn</i> mutant, apoptosis checkpoint double mutants. Bottom: the average number of eggs laid for <i>csn</i> mutants and apoptosis checkpoint double mutants. <i>csn</i> mutants have a severe reduction in the number of oocytes and lay no eggs.</p
Quantification of the SYP-1 aggregates.
<p>A) Schematic representation of the zones of the <i>C. elegans</i> gonad. PMT = pre-meiotic tip, TZ = transition zone, EP = early pachytene, MP = mid pachytene, LP = late pachytene. B–E) Quantification of SYP-1 aggregates in zones of the gonad. Percent of nuclei with: no SYP-1 (black), linear SYP-1 (blue), aggregated SYP-1 (purple pink and red) and other (yellow), zones as in A, n nuclei scored wild type: 1123, <i>csn-2</i>: 868, <i>csn-5</i>: 1020, <i>csn-6</i>: 85, p<0.0005 for pairwise comparisons; Fisher's Exact Test F) Representative images of nuclei scored in C–D all taken from the same gonad in late pachytene of <i>csn-2</i> mutants, G) Western Blot confirming the reduction of expression of SYP-1 in <i>csn</i> mutants. Normalization values (α-SYP-1/α-TUB) shown are the average of 2 different experiments. Normalized intensities: wild-type 0.72±0.26, <i>csn-2</i> 0.24±0.20 and <i>csn-5</i> 0.58±0.11.</p
<i>csn</i> mutant genetically interact with the ubiquitination-neddylation pathway in the regulation of SC assembly and recombination.
<p>A–D) Quantification of SYP-1 aggregates. A and C are data from all gonad, while B and D is from late pachytene nuclei. Percent of nuclei with: no SYP-1 (black), linear SYP-1 (blue), aggregated SYP-1 (purple pink and red) and other (yellow), zones as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004757#pgen-1004757-g002" target="_blank">Figure 2A</a>, n nuclei scored for whole gonad <i>csn-2</i>: with pL4440 = 2023 with <i>ned-8(RNAi)</i> = 430, with <i>uba-1(RNAi)</i> = 1121, <i>csn-5</i>: with pL4440 = 2096, with <i>ned-8(RNAi)</i> = 441, with <i>uba-1(RNAi)</i> = 1014. E) Quantitative analysis of COSA-1 foci in late pachytene wild-type: Percent of nuclei with: zero (black) one (orange), two (red), three (pink) four (magenta), five (purple), six (blue) and seven (gray), n nuclei scored for pL4440 = 57, with <i>ned-8(RNAi)</i> = 47, with <i>uba-1(RNAi)</i> = 25, <i>csn-2</i>: with pL4440 = 168, with <i>ned-8(RNAi)</i> = 66, with <i>uba-1(RNAi)</i> = 126, <i>csn-5</i>: with pL4440 = 152, with <i>ned-8(RNAi)</i> = 47 with, <i>uba-1(RNAi)</i> = 83, pL4440 = empty vector control vs. <i>ned-8(RNAi)</i> on <i>csn-2</i> or <i>csn-5</i> p = 0.002, p<0.001, Fisher's Exact Test, pL4440 vs. <i>uba-1(RNAi)</i> on <i>csn-2</i> or <i>csn-5</i> p<0.001 Mann Whitney Test). F–H) Quantification of SYP-1 aggregates in zones of the gonad for the indicated genotypes, as performed in figure, number of total nuclei scored: wild-type 25 n = 641, <i>rfl-1</i> at 25 n = 1674, <i>cul-4</i> n = 542 2, I) Schematic representation of the pathway examined in the experiment.</p
Pairing stabilization is affected in <i>csn</i> mutants.
<p>A) Analysis of pairing stabilization between wild-type, <i>syp-1(me17)</i>, <i>csn</i> mutants. A schematic representation of the timing of meiotic stages relative to the zones in the <i>C elegans</i> gonad. zone 1 = pre-meiotic tip, zone 2 and 3 = transition from mitosis to meiosis, zone 4–6 = pachytene. The black arrow represents the movement of nuclei through the stages (zones) of meiosis. <i>csn</i> mutants show defects in pairing stabilization number of nuclei counted and <i>p</i>-values can be found in Sup.Tables 1 and 2. B) High magnification micrographs of individual nuclei. Images are projections through three-dimensional data stacks. 5S FISH probe foci are in green and DAPI stained chromosomes are in blue. zone 4 = early pachytene, zone 5 = mid pachytene, zone 6 = late pachytene. Scale bar is 2 µm.</p