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TITLE Recombination-Independent Recognition of DNA Homology for Meiotic Silencing in Neurospora Crassa

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TITLE Recombination-Independent Recognition of DNA Homology for Meiotic Silencing in Neurospora Crassa bioRxiv preprint doi: https://doi.org/10.1101/573907; this version posted March 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. TITLE Recombination-independent recognition of DNA homology for meiotic silencing in Neurospora crassa AUTHORS Nicholas Rhoades1, Germano Cecere2, Thomas Hammond1 and Eugene Gladyshev3 * AFFILIATIONS 1. School of Biological Sciences, Illinois State University, Normal IL 61790, USA 2. Group Mechanisms of Epigenetic Inheritance, Department of Developmental and Stem Cell Biology, Institut Pasteur, Paris 75015, France 3. Group Fungal Epigenomics, Department of Mycology, Institut Pasteur, Paris 75015, France * corresponding author ([email protected]) ABSTRACT Pairing of homologous chromosomes represents a key aspect of meiosis in nearly all sexually reproducing species. While meiotic pairing requires the formation of double-strand DNA breaks in some organisms, in many others it can proceed in the apparent absence of DNA breakage and recombination. The mechanistic nature of such recombination-independent pairing represents a fundamental question in molecular biology. Using “meiotic silencing by unpaired DNA” (MSUD) in the fungus Neurospora crassa as a model system, we demonstrate the existence of a cardinally new solution to the problem of inter-chromosomal homology recognition during meiosis. Here we take advantage of the unique ability of MSUD to efficiently detect and silence (by RNA interference) any relatively short DNA fragment lacking a homologous allelic partner. We show that MSUD does not require the function of eukaryotic RecA proteins and the type II topoisomerase- like protein Spo11. We further show that MSUD can recognize patterns of weak interspersed homology in which short units of sequence identity are arrayed with a periodicity of 11 base-pairs (bp). Taken together, these results reveal the function of a recombination-independent homology-directed process in guiding the expression of small interfering RNAs and suggest that meiotic chromosomes can be evaluated for sequence homology at base-pair resolution by a mechanism that operates on intact DNA molecules. 1 bioRxiv preprint doi: https://doi.org/10.1101/573907; this version posted March 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. MAIN TEXT Proper pairing of homologous chromosomes in meiosis is essential for ensuring fertility and preventing birth defects. While the general role of recombination during meiosis has been firmly established (1), many organisms (including the fruit fly D. melanogaster and the roundworm C. elegans) feature “non-canonical” programs in which homologous chromosomes can identify one another in the apparent absence of DNA breakage and recombination (1). Such recombination-independent pairing was previously suggested to rely on various forms of “bar-coding” by transcription, proteins, RNAs, or landmark genomic features such as centromeres and telomeres (2-6). Alternatively, there exists a possibility that recombination-independent pairing may be guided directly at the level of DNA homology (7, 8), but the experimental evidence for this type of homologous inter-chromosomal communication in meiosis remains sparse (9). In the model fungus Neurospora crassa, a process known as meiotic silencing by unpaired DNA (MSUD) can identify non-homologous DNA sequences present at allelic chromosomal positions (10). Once detected, such sequences undergo transient post-transcriptional silencing by RNA interference (10). The ability of MSUD to detect non-homologous sequences as short as ~1 kbp, irrespective of nucleotide composition and transcriptional capacity, strongly suggests that a general homology search is involved (11). Furthermore, because MSUD occurs within a relatively short time interval (a few hours at the beginning of prophase I), the underlying search mechanism must be very efficient. Previous studies have identified a substantial number of factors required for MSUD (11, 12), but they have not explored the possible connection between MSUD and meiotic recombination. The genome of N. crassa encodes only one RecA-like protein, MEI-3RAD51, which is largely dispensable during vegetative growth but becomes essential in meiosis (13). This lethal phenotype can be fully suppressed by inactivating Spo11, a conserved type II topoisomerase-like protein that generates recombination-initiating double-strand DNA breaks (14). Thus, N. crassa can complete meiosis and produce viable (but frequently aneuploid) progeny in the absence of both MEI-3RAD51 and Spo11 (8). We took advantage of this experimental system to test if MEI-3RAD51 and Spo11 were required for recognition of non-homologous DNA during MSUD. Because Spo11-deficient crosses tend to produce aberrant spores, we had to avoid using standard spore features, such as shape or color, in our analysis. Instead, we chose to assay the expression of a fluorescent fusion protein, hH1-GFP (15), directly in the meiotic cell. To create appropriate reporter strains, the hH1- gfp construct was integrated by homologous recombination as the replacement of the mei-3 gene (Fig. 1A). When the construct is provided by the female parent, many additional nuclei in the fruiting body are expected to express hH1-GFP. Alternatively, when the construct is provided by the male parent, hH1-GFP expression should be restricted to the generative lineage comprising premeiotic, meiotic and postmeiotic 2 bioRxiv preprint doi: https://doi.org/10.1101/573907; this version posted March 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. nuclei. To preclude the hH1-gfp construct from being inactivated in premeiotic nuclei by repeat-induced point mutation (RIP), crosses were performed in appropriate RIP-deficient genetic backgrounds (16, 17). We first analyzed a cross in which meiotic recombination and MSUD remained functional, while only one copy of mei-3::hH1-gfp was present in the diploid nucleus (Fig. 1B and 1C: mei-3+/Δ, spo11+/+). As expected, no meiotic expression of hH1-GFP was observed. This result was obtained regardless of whether the reporter construct was provided by the female or the male parent. The expression of hH1-GFP in this heterozygous condition could be readily restored by the partial attenuation of MSUD (Fig. 1C: mei-3+/Δ, spo11+/Δ, sad-2+/Δ). This suppression effect was achieved by using a dominant deletion allele of sad-2 (11). Taken together, these results indicate that the mei-3::hH1-gfp reporter provides a reliable readout of MSUD activity. We next asked if mei-3::hH1-gfp, when present in only one copy, was detected by MSUD in the complete absence of MEI-3RAD51 and Spo11. Here we analyzed a spo11Δ/Δ cross in which the reporter construct was matched against a standard mei-3Δ deletion allele produced by the Neurospora Genome Project (18). We found that the mei-3::hH1-gfp allele was fully silenced in this situation (Fig. 1C: mei-3Δ/Δ, spo11Δ/Δ, “unpaired” hH1-gfp). The expression of hH1-GFP was restored when two identical mei-3::hH1-gfp alleles were provided (Fig. 1C: mei-3Δ/Δ, spo11Δ/Δ, “paired” hH1-gfp). Taken together, these results demonstrate that MSUD remains functional in the absence of MEI-3RAD51 and Spo11. While Spo11 is required for normal levels of meiotic pairing in N. crassa (19), residual Spo11-independent recombination has also been reported (20). To investigate if such non-canonical recombination could have occurred in our spo11Δ/Δ crosses, we examined the level of linkage between two widely-separated genetic markers, mat a and csr-1Δ, on Chromosome I (Fig. 1D). Notably, this interval includes the his-3 locus, which was previously shown to exhibit substantial levels of Spo11-independent recombination in crosses between certain Neurospora strains (19, 20). We used the csr-1Δ marker for the following reasons: (i) only loss-of-function csr-1 alleles can confer resistance to cyclosporin A, (ii) this resistance is recessive, and (iii) csr-1+ remains stable during the sexual phase (17). Thus, following a csr-1+/Δ cross, partial diploids with two different Chromosomes I (produced by nondisjunction in meiosis I and corresponding to the genotype csr-1+/Δ) can be avoided by germinating spores on cyclosporin-containing medium. Through this approach, we selectively isolated csr-1Δ progeny and performed additional genotyping at the mat locus with a PCR- based assay, which was specifically designed to detect a potential mixture of mat A and mat a DNA (Methods). 3 bioRxiv preprint doi: https://doi.org/10.1101/573907; this version posted March 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. We started by examining a mei-3Δ/Δ, spo11Δ/Δ cross that had already been assayed for MSUD (Fig. 1D; Supplementary Table 2: cross “X5”). Analysis of 513 csr-1Δ progeny of this cross failed to identify a single instance of the recombinant genotype mat A, csr-1Δ (Fig. 1D). As a control, we also examined the degree of linkage between mat a and csr-1Δ in a mei-3+/Δ, spo11+/Δ cross, in which meiotic recombination was at least partially active (Fig. 1D, Supplementary Table 2: cross “X9”). A substantial proportion of recombinant progeny was recovered (26%, Fig. 1D: mei-3+/Δ, spo11+/Δ), demonstrating that our assay was sensitive enough to detect recombination even in this sub-optimal situation (i.e., when both MEI-3RAD51 and Spo11 were expressed from single “unpaired” mei-3+ and spo11+ alleles, respectively).
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