James D. Higgins

Arabidopsis meiotic crossover hot spots overlap with H2A.Z nucleosomes at gene promoters

PRDM9 directs human meiotic crossover hot spots to intergenic sequence motifs, whereas budding yeast hot spots overlap regions of low nucleosome density (LND) in gene promoters. To investigate hot spots in plants, which lack PRDM9, we used coalescent analysis of genetic variation in Arabidopsis thaliana. Crossovers increased toward gene promoters and terminators, and hot spots were associated with active chromatin modifications, including H2A.Z, histone H3 Lys4 trimethylation (H3K4me3), LND and low DNA methylation. Hot spot–enriched A-rich and CTT-repeat DNA motifs occurred upstream and downstream, respectively, of transcriptional start sites. Crossovers were asymmetric around promoters and were most frequent over CTT-repeat motifs and H2A.Z nucleosomes. Pollen typing, segregation and cytogenetic analysis showed decreased numbers of crossovers in the arp6 H2A.Z deposition mutant at multiple scales. During meiosis, H2A.Z forms overlapping chromosomal foci with the DMC1 and RAD51 recombinases. As arp6 reduced the number of DMC1 or RAD51 foci, H2A.Z may promote the formation or processing of meiotic DNA double-strand breaks. We propose that gene chromatin ancestrally designates hot spots within eukaryotes and PRDM9 is a derived state within vertebrates.

Pathways to meiotic recombination in Arabidopsis thaliana

Meiosis is a central feature of sexual reproduction. Studies in plants have made and continue to make an important contribution to fundamental research aimed at the understanding of this complex process. Moreover, homologous recombination during meiosis provides the basis for plant breeders to create new varieties of crops. The increasing global demand for food, combined with the challenges from climate change, will require sustained efforts in crop improvement. An understanding of the factors that control meiotic recombination has the potential to make an important contribution to this challenge by providing the breeder with the means to make fuller use of the genetic variability that is available within crop species. Cytogenetic studies in plants have provided considerable insights into chromosome organization and behaviour during meiosis. More recently, studies, predominantly in Arabidopsis thaliana, are providing important insights into the genes and proteins that are required for crossover formation during plant meiosis. As a result, substantial progress in the understanding of the molecular mechanisms that underpin meiosis in plants has begun to emerge. This article summarizes current progress in the understanding of meiotic recombination and its control in Arabidopsis. We also assess the relationship between meiotic recombination in Arabidopsis and other eukaryotes, highlighting areas of close similarity and apparent differences.

Expression and functional analysis of AtMUS81 in Arabidopsis meiosis reveals a role in the second pathway of crossing-over.

Meiotic crossovers/chiasmata, that are required to ensure chromosome disjunction, arise via the class I interference-dependent pathway or via the class II interference-free pathway. The proportions of these two classes vary considerably between different organisms. In Arabidopsis, about 85% of chiasmata are eliminated in Atmsh4 mutants, denoting that these are class I events. In budding and fission yeasts Msh4-independent crossovers arise largely or entirely via a Mus81-dependent pathway. To investigate the origins of the 15% residual (AtMSH4-independent) chiasmata in Arabidopsis we conducted a cytological and molecular analysis of AtMUS81 meiotic expression and function. Although AtMUS81 functions in somatic DNA repair and recombination, it is more highly expressed in reproductive tissues. The protein is abundantly present in early prophase I meiocytes, where it co-localizes, in a double-strand break-dependent manner, with the recombination protein AtRAD51. Despite this, an Atmus81 mutant shows normal growth and has no obvious defects in reproductive development that would indicate meiotic impairment. A cytological analysis confirmed that meiosis was apparently normal in this mutant and its mean chiasma frequency was similar to that of wild-type plants. However, an Atmsh4/Atmus81 double mutant revealed a significantly reduced mean chiasma frequency (0.85 per cell), compared with an Atmsh4 single mutant (1.25 per cell), from which we conclude that AtMUS81 accounts for some, but not all, of the 15% AtMSH4-independent residual crossovers. It is possible that other genes are responsible for these residual chiasmata. Alternatively the AtMUS81 pathway coexists with an alternative parallel pathway that can perform the same functions.

Meiotic Adaptation to Genome Duplication in Arabidopsis arenosa

Whole genome duplication (WGD) is a major factor in the evolution of multicellular eukaryotes, yet by doubling the number of homologs, WGD severely challenges reliable chromosome segregation, a process conserved across kingdoms. Despite this, numerous genome-duplicated (polyploid) species persist in nature, indicating early problems can be overcome. Little is known about which genes are involved--only one has been molecularly characterized. To gain new insights into the molecular basis of adaptation to polyploidy, we investigated genome-wide patterns of differentiation between natural diploids and tetraploids of Arabidopsis arenosa, an outcrossing relative of A. thaliana. We first show that diploids are not preadapted to polyploid meiosis. We then use a genome scanning approach to show that although polymorphism is extensively shared across ploidy levels, there is strong ploidy-specific differentiation in 39 regions spanning 44 genes. These are discrete, mostly single-gene peaks of sharply elevated differentiation. Among these peaks are eight meiosis genes whose encoded proteins coordinate a specific subset of early meiotic functions, suggesting these genes comprise a polygenic solution to WGD-associated chromosome segregation challenges. Our findings indicate that even conserved meiotic processes can be capable of nimble evolutionary shifts when required.

AtMSH5 partners AtMSH4 in the class I meiotic crossover pathway in Arabidopsis thaliana, but is not required for synapsis

MSH5, a meiosis-specific member of the MutS-homologue family of genes, is required for normal levels of recombination in budding yeast, mouse and Caenorhabditis elegans. In this paper we report the identification and characterization of the Arabidopsis homologue of MSH5 (AtMSH5). Transcripts of AtMSH5 are specific to reproductive tissues, and immunofluorescence studies indicate that expression of the protein is abundant during prophase I of meiosis. In a T-DNA tagged insertional mutant (Atmsh5-1), recombination is reduced to about 13% of wild-type levels. The residual chiasmata are randomly distributed between cells and chromosomes. These data provide further evidence for at least two pathways of meiotic recombination in Arabidopsis and indicate that AtMSH5 protein is required for the formation of class I interference-sensitive crossovers. Localization of AtMSH5 to meiotic chromosomes occurs at leptotene and is dependent on DNA double-strand break formation and strand exchange. Localization of AtMSH5 to the chromatin at mid-prophase I is dependent on expression of AtMSH4. At late zygotene/early pachytene a proportion of AtMSH5 foci co-localize with AtMLH1 which marks crossover-designated sites. Chromosome synapsis appears to proceed normally, without significant delay, in Atmsh5-1 but the pachytene stage is extended by several hours, indicative of the operation of a surveillance system that monitors the progression of prophase I.

Spatiotemporal Asymmetry of the Meiotic Program Underlies the Predominantly Distal Distribution of Meiotic Crossovers in Barley

This work characterizes factors involved in the predominantly distal location of meiotic crossovers in barley. Recombination initiates first in the distal regions and later in the interstitial regions; manipulating meiotic progression with higher temperatures produced more interstitial crossovers that could improve mapping of agronomical traits and reduce linkage drag. Meiosis involves reciprocal exchange of genetic information between homologous chromosomes to generate new allelic combinations. In cereals, the distribution of genetic crossovers, cytologically visible as chiasmata, is skewed toward the distal regions of the chromosomes. However, many genes are known to lie within interstitial/proximal regions of low recombination, creating a limitation for breeders. We investigated the factors underlying the pattern of chiasma formation in barley (Hordeum vulgare) and show that chiasma distribution reflects polarization in the spatiotemporal initiation of recombination, chromosome pairing, and synapsis. Consequently, meiotic progression in distal chromosomal regions occurs in coordination with the chromatin cycles that are a conserved feature of the meiotic program. Recombination initiation in interstitial and proximal regions occurs later than distal events, is not coordinated with the cycles, and rarely progresses to form chiasmata. Early recombination initiation is spatially associated with early replicating, euchromatic DNA, which is predominately found in distal regions. We demonstrate that a modest temperature shift is sufficient to alter meiotic progression in relation to the chromosome cycles. The polarization of the meiotic processes is reduced and is accompanied by a shift in chiasma distribution with an increase in interstitial and proximal chiasmata, suggesting a potential route to modify recombination in cereals.

Inter-Homolog Crossing-Over and Synapsis in Arabidopsis Meiosis Are Dependent on the Chromosome Axis Protein AtASY3

In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR), we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC) formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation.

The Fanconi Anemia Ortholog FANCM Ensures Ordered Homologous Recombination in Both Somatic and Meiotic Cells in Arabidopsis

Arabidopsis FANCM, a homolog of human FANCM, is involved in the suppression of somatic homologous recombination. Here, it is shown that At-FANCM also plays a key role in meiosis, as it ensures the controlled formation of genetic exchange by suppressing erroneous interactions between parental genomes and by controlling meiotic crossover formation between homologs. The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)–independent pathway. In addition, it is required for double-strand break–induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.

Chromosome synapsis in Arabidopsis: analysis of the transverse filament protein ZYP1 reveals novel functions for the synaptonemal complex

With respect to history, plants have provided an ideal system for cytogenetical analysis of the synaptonemal complex (SC). However, until recently, the identification of the genes that encode the SC in plants has proved elusive. In recent years, Arabidopsis thaliana was developed as a model system for plant meiosis research. As a result, there was substantial progress in the isolation of meiotic genes and this has recently led to the isolation of the first plant SC gene, ZYP1. The ZYP1 gene encodes a transverse filament (TF) protein that is predicted to have structural similarity to TF proteins found in other organisms. Analysis of plants deficient in ZYP1 expression has provided important insights into the function of the SC in plants. Loss of ZYP1 has only a limited effect on the overall level of recombination. However, it is associated with extensive nonhomologous recombination leading to multivalent formation at metaphase I. This phenomenon was not previously reported in other organisms. It is important to note that cytological analysis of the ZYP1 deficient lines indicates that SC formation is not required for the imposition of crossover interference.

A strategy to investigate the plant meiotic proteome

The analysis of meiosis in higher plants has benefited considerably in recent years from the completion of the genome sequence of the model plant Arabidopsis thaliana and the development of cytological techniques for this species. A combination of forward and reverse genetics has provided important routes toward the identification of meiotic genes in Arabidopsis. Nevertheless identification of certain meiotic genes remains a challenge due to problems such as limited sequence conservation between species, existence of closely related gene families and in some cases functional redundancy between gene family members. Hence there is a requirement to develop new experimental approaches that can be used in conjunction with existing methods to enable a greater range of plant meiotic genes to be identified. As one potential route towards this goal we have initiated a proteomics-based approach. Unfortunately, the small size of Arabidopsis anthers makes an analysis in this species technically very difficult. Therefore we have initially focussed on Brassica oleracea which is closely related to Arabidopsis, but has the advantage of possessing significantly larger anthers. The basic strategy has been to use peptide mass-finger printing and matrix-assisted laser desorption ionization time of flight mass spectrometry to analyse proteins expressed in meiocytes during prophase I of meiosis. Initial experiments based on the analysis of proteins from staged anther tissue proved disappointing due to the low level of detection of proteins associated with meiosis. However, by extruding meiocytes in early prophase I from individual anthers prior to analysis a significant enrichment of meiotic proteins has been achieved. Analysis suggests that at least 18% of the proteins identified by this route have a putative meiotic function and that this figure could be as high as one-third of the total. Approaches to increase the enrichment of proteins involved in meiotic recombination and chromosome synapsis are also described.