(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Differentiated function and localisation of SPO11-1 and PRD3 on the chromosome axis during meiotic DSB formation in Arabidopsis thaliana [1]

['Christophe Lambing', 'Department Of Plant Sciences', 'University Of Cambridge', 'Cambridge', 'United Kingdom', 'Rothamsted Research', 'Harpenden', 'School Of Biosciences', 'University Of Birmingham', 'Edgbaston']

Date: 2022-10

During meiosis, DNA double-strand breaks (DSBs) occur throughout the genome, a subset of which are repaired to form reciprocal crossovers between chromosomes. Crossovers are essential to ensure balanced chromosome segregation and to create new combinations of genetic variation. Meiotic DSBs are formed by a topoisomerase-VI-like complex, containing catalytic (e.g. SPO11) proteins and auxiliary (e.g. PRD3) proteins. Meiotic DSBs are formed in chromatin loops tethered to a linear chromosome axis, but the interrelationship between DSB-promoting factors and the axis is not fully understood. Here, we study the localisation of SPO11-1 and PRD3 during meiosis, and investigate their respective functions in relation to the chromosome axis. Using immunocytogenetics, we observed that the localisation of SPO11-1 overlaps relatively weakly with the chromosome axis and RAD51, a marker of meiotic DSBs, and that SPO11-1 recruitment to chromatin is genetically independent of the axis. In contrast, PRD3 localisation correlates more strongly with RAD51 and the chromosome axis. This indicates that PRD3 likely forms a functional link between SPO11-1 and the chromosome axis to promote meiotic DSB formation. We also uncovered a new function of SPO11-1 in the nucleation of the synaptonemal complex protein ZYP1. We demonstrate that chromosome co-alignment associated with ZYP1 deposition can occur in the absence of DSBs, and is dependent on SPO11-1, but not PRD3. Lastly, we show that the progression of meiosis is influenced by the presence of aberrant chromosomal connections, but not by the absence of DSBs or synapsis. Altogether, our study provides mechanistic insights into the control of meiotic DSB formation and reveals diverse functional interactions between SPO11-1, PRD3 and the chromosome axis.

Most eukaryotes rely on the formation of gametes with half the number of chromosomes for sexual reproduction. Meiosis is a specialised type of cell division essential for the transition between a diploid and a haploid stage during gametogenesis. In early meiosis, programmed-DNA double strand breaks (DSBs) occur across the genome. These DSBs are processed by a set of proteins and the broken ends are repaired using the genetic information from the homologous chromosomes. These reciprocal exchanges of information between two chromosomes are called crossovers. Crossovers physical link chromosomes in pairs which is essential to ensure their correct segregation during the two rounds of meiotic division. Crossovers are also essential for the creation of genetic diversity as they break genetic linkages to form novel allelic blocks. The formation of DSBs is not completely understood in plants. Here we studied the function of SPO11-1 and PRD3, two proteins involved in the formation of DSBs in Arabidopsis. We discovered functional differences in their respective mode of recruitment to the chromosomes, their interactions with proteins forming the chromosome core and their roles in chromosome co-alignment. These indicate that, although SPO11-1 and PRD3 share a role in the formation of DSBs, the two proteins have additional and distinct roles beside DSB formation.

Funding: Funding was provided by European Research Council grant ERC-2015-CoG-681987 ‘SynthHotSpot’ to IRH, a BBSRC grant-aided support as part of the Institute Strategic Program Designing Future Wheat Grant (BB/P016855/1) and an Institutional Sponsorship Fund as part of the UKRI grant (BB/W510543/1) to CL, a Basic Science Research Program through the National Research Foundation of Korea (NRF-2020R1A2C2007763) to KC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

To gain insights into meiotic DSB control in Arabidopsis, we used immunocytogenetic assays to explore the interrelationship of key components of the recombination machinery and chromosome axis. A recent ASY1 immunoprecipitation pull-down assay combined with mass spectrometry identified interacting proteins in Brassica [ 32 ]. Among the proteins co-immunoprecipitated, PRD3 was the only DSB-promoting factor identified [ 32 ]. This prompted us to focus our study on SPO11-1 and PRD3, and to investigate their respective localisation and function in relation to the chromosome axis. This revealed distinct localisation of PRD3 and SPO11-1 on meiotic chromosomes. Specifically, we found that PRD3 is relatively enriched on the chromosome axis, while SPO11-1 is enriched on the chromatin loops. We demonstrate that the association of PRD3 with the axis correlates with the timing of DSB formation during prophase I. We also show that the linear meiotic chromosome axis is required for the formation of DSBs, but not for the recruitment of SPO11-1, and that PRD3 physically interacts with several components of the chromosome axis. We identified a new function of SPO11-1 in the nucleation of the SC transverse filament protein ZYP1 [ 50 ], which is independent of DSB formation and not shared with PRD3. Lastly, we revealed that a delay in meiotic progression is linked with aberrant chromosomal connections, rather than the absence of recombination or synapsis. Our data provide mechanistic insights into the function of the DSB complex and the role of the chromosome axis in the initiation of meiotic recombination in plants.

Many aspects of the spatial and temporal control of meiotic DSB formation remain elusive. For instance, mechanistic links between DSB formation and the chromosome axis are well documented in budding yeast [ 39 , 43 – 44 ], but less so in plants. In budding yeast and mice, numerical control of DSB formation is established by inter-homolog engagement, whereby connections between homologous chromosomes are sufficient to suppress formation of DSBs [ 45 – 47 ]. A second pathway limiting DSB numbers is dependent on Tel1 and Mec1 kinases in budding yeast [ 48 ]. In Arabidopsis, the homolog of Tel1 (ATM) is a negative regulator of meiotic DSB formation [ 49 ], but whether other pathways are present remains unclear.

Meiotic DSB formation occurs in the context of a linear chromosome axis, onto which the chromatin is organized in loop-axis arrays [ 21 , 22 ]. The Arabidopsis chromosome axis is composed of cohesin complexes containing REC8, the coiled-coil proteins ASY3 (a functional ortholog of Red1, SYCP2 and SYCP3) and ASY4, and the HORMA-containing protein ASY1 (a functional ortholog of Hop1, HORMAD1 and HORMAD2) [ 23 – 32 ]. REC8 cohesin organizes the chromatin around an axial structure, while ASY3 and ASY1 promote inter-homolog recombination [ 22 , 28 , 29 , 33 – 35 ]. The co-alignment of the two homologous chromosome axes leads to the formation of the synaptonemal complex, which consists of transverse filaments connecting the two axes and forming a tripartite structure that influences crossover formation [ 36 – 38 ]. In Arabidopsis and budding yeast, chromatin immunoprecipitation and sequencing (ChIP-seq) of REC8 revealed that cohesin is enriched in regions depleted of SPO11-oligonucleotides, consistent with DSBs forming in the chromatin loops away from the axis sites [ 33 , 34 , 39 , 40 ]. Depletion of meiotic cohesin causes a reduction in DSBs and early recombination markers in mice and plants [ 33 , 41 , 42 ]. A link between the axis and DSB formation was further demonstrated in budding yeast, where the PHD finger protein Spp1 simultaneously interacts with Mer2 on the chromosome axis, and H3K4me3 on the chromatin loops [ 43 , 44 ]. Through these interactions, Spp1 supports the tethering of recombination hotspots to the axis during DSB formation.

Meiotic DSBs are formed by a conserved topoisomerase-VI-like complex containing SPO11 and MTOPVIB [ 2 , 3 ]. In Arabidopsis, two SPO11 proteins are required for meiotic DSB formation; SPO11-1 and SPO11-2, in addition to the auxiliary proteins PRD1, PRD2, PRD3 and DFO [ 4 – 8 ]. During DSB formation, SPO11 remains covalently attached to the DNA 5′ ends at the break sites, allowing immunoprecipitation of SPO11-oligonucleotide complexes followed by sequencing, and thus identification of DSB locations [ 9 – 11 ]. Mapping SPO11-oligonucleotides genome-wide in plants, animals and fungi has revealed that meiotic DSBs are heterogeneously distributed throughout genomes [ 9 – 13 ]. For example, SPO11 DSB hotspots are associated with gene promoter regions that are nucleosome depleted in budding yeast [ 14 ]. Similarly, Arabidopsis SPO11-1 DSB hotspots are located in nucleosome-depleted regions at the 5′ and 3′ ends of genes and in specific transposons families [ 10 ]. In contrast, mice and human SPO11 DSB hotspots are spatially controlled by PRDM9 and are associated with intergenic regions and specific DNA motifs [ 15 – 17 ]. Epigenetic information on the chromatin and DNA also influences DSB formation [ 10 , 18 ]. For example, H3K9me2 and DNA methylation were found to repress DSB frequency in Arabidopsis, including within transposable elements [ 18 – 20 ].

Meiosis is a specialized cell division that is required for sexual reproduction and leads to the generation of genetic diversity [ 1 ]. Meiosis halves the chromosome number of the genome to form haploid gametes (e.g. sperm and egg cells), which upon fusion, restore the diploid state [ 1 ]. Furthermore, programmed DNA double-strand breaks (DSBs) are formed during meiosis and are repaired via homologous recombination between each pair of chromosomes to generate crossovers and non-crossovers [ 1 ]. Crossovers consist of reciprocal exchanges of genetic information between homologous chromosomes, which break pre-existing genetic linkages to form new combinations of alleles [ 1 ]. Meiotic DSB formation and repair are tightly regulated such that the integrity of the genome is preserved, while allowing creation of genetic diversity.

Results

SPO11-1 localisation is partially dependent on PRD2, SPO11-2 and MTOPVIB, but not PRD1 or PRD3 To test if the recruitment of SPO11-1 to meiotic chromatin requires other components of the DSB machinery, we immunostained for ASY1 and SPO11-1-MYC in mutants defective in DSB formation, namely prd1, prd2, prd3, mtopVIB and spo11-2 [2,6–8]. SPO11-1-MYC foci associated with chromatin were detected in all mutants (Fig 1B), suggesting that each component of the DSB machinery is not strictly essential to recruit SPO11-1-MYC to meiotic chromatin. However, we observed significant differences in SPO11-1-MYC foci numbers between the mutants (Fig 1B). The localisation of SPO11-1-MYC was not affected in prd1 (Mann-Whitney-Wilcoxon test (MWW) P = 0.192) and prd3 (MWW, P = 0.464), with foci numbers not significantly different to those in wild type (Fig 1B and 1D and S2 Table). In contrast, the number of SPO11-1-MYC foci was reduced to 77.5% in prd2 (MWW, P = 7.5x10-4), while they were reduced to 55.2% and 36.1% in spo11-2 (MWW P = 1.3x10-5) and mtopVIB (MWW P = 1.1x10-7), respectively (Fig 1B and 1D and S2 Table). A recent study reported that the reduction of SPO11-1-MYC foci was slightly more severe in spo11-2 than in mtopVIb, which we did not observe in our study [53]. However, as we used a different SPO11-1-MYC transgenic line this may have contributed to the difference in the phenotype observed. Nevertheless, both studies are concordant in showing that SPO11-1 foci localisation is severely disrupted in spo11-2 and mtopvib. Therefore, PRD1 and PRD3 are not required for SPO11-1-MYC foci formation, while PRD2, SPO11-2 and MTOPVIB support the localisation of SPO11-1-MYC during meiosis. As SPO11-1 forms a catalytic complex with SPO11-2 and MTOPVIB [2], this likely explains the stronger defect in SPO11-1 localisation observed in these mutants.

PRD3 physically interacts with chromosome axis proteins We hypothesised that the reduced number of RAD51 foci between early and late prophase may be linked to the remodeling of the chromosome axis, and the progressive synapsis taking place between homologous chromosomes in late prophase I [51]. To identify components of the DSB machinery that may interact with the chromosome axis, we revisited data from an ASY1-affinity immunoprecipitation pull-down assay from isolated Brassica meiocytes, which is closely related to Arabidopsis. We found that PRD3 was the only known component of the DSB machinery to be immunoprecipitated with ASY1 (S6 Table) [32]. Moreover, peptides for PRD3 were found in the two-independent ASY1 pull-down experiments that have the highest recovery for ASY3 peptides, which is another component of the chromosome axis. [32]. The proteins identified from co-IP-MS were searched against the Arabidopsis TAIR10 proteome to identify orthologues using BLAST. This resulted in 482 proteins and the proteins were used as seeds to query the STRING database version 11.5 [56], which returned a total of 7,124 interactions. Among this network, PRD3 forms an interacting sub-network with ASY1, ASY3 and PCH2 (Fig 3A). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. PRD3 interacts with components of the chromosome axis. (A) String protein association network showing the linkage between ASY1, ASY3, PCH2 and PRD3. The width of the string represents the strength of the association between the two proteins. (B) Immunostaining of ASY1, ASY3, SMC3 and REC8 in wild type and prd3. Scale bars = 10 μM. (C) Serial drop dilutions (undiluted, 1/10 dilution, 1/100 dilution) of yeast-two hybrid colonies grown on selective media to test the interaction between PRD3, ASY1, ASY3 and PCH2. “+” indicates presence of the Arabidopsis coding sequence in the AD- or BD-fused vector used for transforming yeast whereas “-”indicates absence of the Arabidopsis coding sequence in the AD- or BD-fused vector. SD-LT means synthetic defined medium lacking leucine and tryptophan and this medium selects for co-transformants yeast cells. SD-LTH means synthetic defined medium lacking leucine, tryptophan and histidine. SD-LTHA means synthetic defined medium lacking leucine, tryptophan, histidine and adenine. SD-LTH and SD-LTHA test for protein interaction under low- and high-stringency conditions, respectively. (D) Co-localisation assay of the fusion proteins PRD3, ASY1, ASY3 and PCH2 in Arabidopsis protoplasts. “+” indicates presence of the coding sequence in the CFP or YFP-fusion vector during protoplast transfection whereas “-”indicates absence of the coding sequence in the CFP or YFP-fusion vector. Scale bars = 20μM. https://doi.org/10.1371/journal.pgen.1010298.g003 To confirm a physical interaction between Arabidopsis PRD3, ASY1 and ASY3, we used the GAL4 yeast two-hybrid system in budding yeast. Consistent with the Brassica affinity proteomic data, we observed that PRD3 interacts with ASY1 on the less stringent medium SD -LTH, but not on the most stringent medium SD -LTHA, which may suggest weak or transient interaction between the two proteins in this heterologous system (Fig 3C). In contrast, PRD3 strongly interacts with ASY3 on both SD -LTH and SD -LTHA media (Fig 3C). We also tested the interaction between PRD3 and PCH2, since PCH2 is recruited to the axis when chromosomes are synapsing and remodels the axis through protein-protein interactions [57]. We observed that yeast co-transformed with PRD3 and PCH2 were able to grow on both SD -LTH and SD -LTHA selective media. Yeast transformed with BD-PCH2 also grew on these media, but at a lower rate, which suggests that PRD3 can interact with PCH2 in a yeast two-hybrid assay (Fig 3C). Lastly, we observed that PRD3 and PCH2 can self-dimerise as yeast co-transformed with AD-PRD3 and BD-PRD3 or AD-PCH2 and BD-PCH2 grew on the restrictive medium (Fig 3C). Additionally, we noted that yeast transformed with AD-PCH2 were slow to grow on SD-LT (Fig 3C), suggesting that the expression of this heterologous protein causes some toxicity to the yeast cells. To further test the interaction between PRD3 and ASY1, ASY3 or PCH2 in Arabidopsis, the proteins were fused to YFP or CFP fluorescent proteins and transiently expressed in protoplasts. Expression of PRD3-YFP showed that the protein mainly localises in the nucleus (Fig 3D). Co-expression of PRD3-YFP and ASY1-CFP or PRD3-YFP and ASY3-CFP revealed extensive overlap of the two fluorescent signals in the nuclei, indicating a propensity of the two proteins to co-localise in the nucleus. In contrast, it appeared that PCH2-CFP localised in both the cytoplasm and nucleus, which is in accord with its localisation in meiotic cells [58]. The expression of PRD3-YFP in the presence of PCH2-CFP shows that PRD3-YFP is found localising in these two cellular compartments and the fluorescent signal overlaps with the signal of PCH2-CFP (Fig 3D). Overall, our data suggest that PRD3 can interact with components of the chromosome axis consistent with the co-localisation of PRD3 with ASY1 on the Arabidopsis meiotic chromosome axes.

[END]
---
[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010298

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.