Msh4 and Msh5 function in SC-independent chiasma formation during the streamlined meiosis of Tetrahymena

Abstract

ZMM proteins have been defined in budding yeast as factors that are collectively involved in the formation of interfering crossovers (COs) and synaptonemal complexes (SCs), and they are a hallmark of the predominant meiotic recombination pathway of most organisms. In addition to this so-called class I CO pathway, a minority of crossovers are formed by a class II pathway, which involves the Mus81-Mms4 endonuclease complex. This is the only CO pathway in the SC-less meiosis of the fission yeast. ZMM proteins (including SC components) were always found to be co-occurring and hence have been regarded as functionally linked. Like the fission yeast, the protist Tetrahymena thermophila does not possess a SC, and its COs are dependent on Mus81-Mms4. Here we show that the ZMM proteins Msh4 and Msh5 are required for normal chiasma formation, and we propose that they have a pro-CO function outside a canonical class I pathway in Tetrahymena. Thus, the two-pathway model is not tenable as a general rule.

Keywords: DSB repair; crossover pathways; meiosis; recombination; synaptonemal complex.

Figure 1

Meiosis in Tetrahymena. (A) Meiosis is initiated by the mating of two starving cells (I). Each contains a diploid generative MIC and a somatic MAC. The MICs undergo synchronous meiosis in the two partners whereas the MACs do not participate. The MIC elongates during meiotic prophase (II–III) in response to DSB formation. During the stage of maximal elongation (IV), homologous chromosomes pair. When DSBs are repaired by homologous recombination, the MIC shortens again (V–VI), and distinct chromatin threads become visible. Five condensed bivalents appear from diakinesis to metaphase I. Chromosomes and chromatids separate during first and second meiotic division, respectively. Classification of early meiotic stages (I–VI) according to Sugai and Hiwatashi (1974). (B) Microscopical images (Giemsa staining) of corresponding stages. Bar, 10 µm.

Figure 2

Tetrahymena Msh4 and Msh5 homologs and msh4 and msh5 knockout strains. (A) Unrooted phylogenetic tree of MutS1 homologs from T. thermophila, human, and budding and fission yeast reconstructed from a protein sequence alignment of the MutS_II-MutS_V domain regions. Tetrahymena MutS homologs were collected by searching the Tetrahymena proteome derived from the TetraFGD RNAseq transcriptome with the Pfam MutS_V domain model. Sequences for all other species are based on Lin et al. (2007). The MrBayes consensus tree is shown with Bayesian posterior probabilities in percent illustrating the degree of support for each node on the tree (Ronquist et al. 2012). The scale bar indicates the branch length representing the expected number of substitutions per site. (B) Full replacement of wild-type MSH4 copies by KO cassettes is shown by Southern hybridization with a sequence flanking the 3′ end of MSH4 as a probe to BglII fragments of wild type (WT) and msh4 knockout (mating type B and C clones) genomic DNA. The msh5 knockout strains (mating type B and C) show extensive but not complete replacement of wild-type MSH5 copies by knockout cassettes. Also for MSH5 KO, the 3′ flanking sequence was used as a Southern probe to BglII fragments of genomic DNA.

Figure 3

Reduced chiasma formation in the msh4Δ mutant. (A) Five bivalents in the wild type. In diplonema, homologous chromosomes twist around each other. In diakinesis, homologous arms are tightly associated, and only the centromeric regions (arrowheads) are separated. In metaphase I, tension at the kinetochores separates the proximal regions of homologous arms. Bar, 5 µm. (B) In the mutant, diakinesis-metaphase I nuclei show only univalents, mixtures of univalents and bivalents, or only bivalents (from the left). Arrowheads denote rod bivalents; the arrow indicates a ring bivalent. (C) Univalents in a spo11Δ control. Chromosomes are stained with Giemsa in A–C. (D, Top) Since bound arms accomodate an unknown number of chiasmata, chiasma reduction was indirectly estimated by scoring diakinesis-metaphase I chromosomes as having two arms bound (ring bivalents), one arm bound (rod bivalents), and no arms bound (pairs of univalents). (Bottom) Numbers of bound arms/nucleus scored for the wild type, the msh4Δ mutant, and the spo11Δ control (n = 100 nuclei for each).

Figure 4

DSB and JM formation appear normal in the msh4Δ mutant. (A) Dmc1 foci, cytological markers of DSBs, appear similar in meiotic nuclei (arrows) of the wild type and the mutant. Foci appear from stage II onward, reach a maximum at stage IV, and disappear by stage V. Cells were subjected to a spreading technique, which removes free protein from nuclei. The antibody used detects chromatin-bound Dmc1 in the MICs but also Rad51 in the MACs (Howard-Till et al. 2011). (B) FISH to elongated nuclei was used to assess pairing of homologous loci (see text). Examples of unpaired and paired loci are shown. (C) BrdU administered to cells early in meiosis is not incorporated during stage IV, but only from stage V onward. Stage numbers correspond to the categories shown in Figure 1A. Bar, 10 µm.

Figure 5

Lack of Sgs1 restores bivalent formation in the msh4Δ mutant. (A) The msh4Δ mutant shows a mixture of bivalents and univalents, whereas bivalents in the msh4Δ sgs1 RNAi double mutant resemble those of the wild type except for a slight condensation defect. Giemsa staining. Bar, 5 µm. (B) Comparison of distances between homologous loci in diakinesis-metaphase I nuclei of the three genotypes and of spo11Δ as a nonbivalent forming control. Fifty nuclei of each genotype are plotted in the order of increasing distances. (Inset) Examples of a wild-type diakinesis-metaphase I nucleus with a single FISH signal where the corresponding chromosome arms are connected (left), and of a msh4Δ nucleus with two signals, indicating separated arms (right). Chromatin was stained with DAPI.

Figure 6

Meiosis in msh5Δ. (A) Dmc1 localization to elongated prophase nuclei (left) is normal. (B) Incorporation of BrdU (left) indicates normal recombination-related DNA synthesis and JM formation. (C) Three examples of abnormal diakinesis-metaphase I stages with univalents and bivalents. Giemsa staining. Bar, 10 µm.