Increased genome size is caused by heterochromatin addition in two non-related bat species, Hesperoptenus doriae and Philetor brachypterus (Vespertilionidae, Chiroptera, Mammalia).

The average genome size (GS) of bats, which are the only mammals capable of powered flight, is approximately 18% smaller than that of closely related mammalian orders. The low nuclear DNA content of Chiroptera is comparable to that of birds, which are also characterized by a high metabolic rate. Only a few chiropteran taxa possess notable amounts of constitutive heterochromatin. Here, we studied the karyotypes of two non-related vesper bat species with unusually high amounts of constitutive heterochromatin: Hesperoptenus doriae and Philetor brachypterus. Conventional staining methods and whole-chromosome painting with probes derived from Myotis myotis (2n = 44), showing a karyotype close to that of the presumed ancestor of Vespertilionidae, revealed Robertsonian fusions as the main type of rearrangement leading to the exceptionally reduced diploid chromosome number of 2n = 26 in both species. Moreover, both karyotypes are characterized by large blocks of pericentromeric heterochromatin composed of CMA-positive and DA-DAPI-positive segments. In H. doriae, the heterochromatin accumulation has resulted in a genome size of 3.22 pg (1C), which is 40% greater than the mean genome size for the family. For P. brachypterus, a genome size of 2.94 pg was determined, representing an increase of about 28%. Most notably, in H. doriae, the presence of additional constitutive heterochromatin correlates with an extended mitotic cell cycle duration in vitro. A reduction in diploid chromosome number to 30 or lower is discussed as a possible cause of the accumulation of pericentromeric heterochromatin in Vespertilionidae.

Conservation and Variability of Meiosis Across the Eukaryotes.

Comparisons among a variety of eukaryotes have revealed considerable variability in the structures and processes involved in their meiosis. Nevertheless, conventional forms of meiosis occur in all major groups of eukaryotes, including early-branching protists. This finding confirms that meiosis originated in the common ancestor of all eukaryotes and suggests that primordial meiosis may have had many characteristics in common with conventional extant meiosis. However, it is possible that the synaptonemal complex and the delicate crossover control related to its presence were later acquisitions. Later still, modifications to meiotic processes occurred within different groups of eukaryotes. Better knowledge on the spectrum of derived and uncommon forms of meiosis will improve our understanding of many still mysterious aspects of the meiotic process and help to explain the evolutionary basis of functional adaptations to the meiotic program.

Tetrahymena meiosis: Simple yet ingenious.

The presence of meiosis, which is a conserved component of sexual reproduction, across organisms from all eukaryotic kingdoms, strongly argues that sex is a primordial feature of eukaryotes. However, extant meiotic structures and processes can vary considerably between organisms. The ciliated protist Tetrahymena thermophila, which diverged from animals, plants, and fungi early in evolution, provides one example of a rather unconventional meiosis. Tetrahymena has a simpler meiosis compared with most other organisms: It lacks both a synaptonemal complex (SC) and specialized meiotic machinery for chromosome cohesion and has a reduced capacity to regulate meiotic recombination. Despite this, it also features several unique mechanisms, including elongation of the nucleus to twice the cell length to promote homologous pairing and prevent recombination between sister chromatids. Comparison of the meiotic programs of Tetrahymena and higher multicellular organisms may reveal how extant meiosis evolved from proto-meiosis.

Spatial constraints on chromosomes are instrumental to meiotic pairing.

In most eukaryotes, the meiotic chromosomal bouquet (comprising clustered chromosome ends) provides an ordered chromosome arrangement that facilitates pairing and recombination between homologous chromosomes. In the protist Tetrahymena thermophila, the meiotic prophase nucleus stretches enormously, and chromosomes assume a bouquet-like arrangement in which telomeres and centromeres are attached to opposite poles of the nucleus. We have identified and characterized three meiosis-specific genes [meiotic nuclear elongation 1-3 (MELG1-3)] that control nuclear elongation, and centromere and telomere clustering. The Melg proteins interact with cytoskeletal and telomere-associated proteins, and probably repurpose them for reorganizing the meiotic prophase nucleus. A lack of sequence similarity between the Tetrahymena proteins responsible for telomere clustering and bouquet proteins of other organisms suggests that the Tetrahymena bouquet is analogous, rather than homologous, to the conserved eukaryotic bouquet. We also report that centromere clustering is more important than telomere clustering for homologous pairing. Therefore, we speculate that centromere clustering may have been the primordial mechanism for chromosome pairing in early eukaryotes.

An MCM family protein promotes interhomolog recombination by preventing precocious intersister repair of meiotic DSBs.

Recombinational repair of meiotic DNA double-strand breaks (DSBs) uses the homologous chromosome as a template, although the sister chromatid offers itself as a spatially more convenient substrate. In many organisms, this choice is reinforced by the recombination protein Dmc1. In Tetrahymena, the repair of DSBs, which are formed early in prophase, is postponed to late prophase when homologous chromosomes and sister chromatids become juxtaposed owing to tight parallel packing in the thread-shaped nucleus, and thus become equally suitable for use as repair templates. The delay in DSB repair is achieved by rejection of the invading strand by the Sgs1 helicase in early meiotic prophase. In the absence of Mcmd1, a meiosis-specific minichromosome maintenance (MCM)-like protein (and its partner Pamd1), Dmc1 is prematurely lost from chromatin and DNA synthesis (as monitored by BrdU incorporation) takes place in early prophase. In mcmd1Δ and pamd1Δ mutants, only a few crossovers are formed. In a mcmd1Δ hop2Δ double mutant, normal timing of Dmc1 loss and DNA synthesis is restored. Because Tetrahymena Hop2 is believed to enable homologous strand invasion, we conclude that Dmc1 loss in the absence of Mcmd1 affects only post-invasion recombination intermediates. Therefore, we propose that the Dmc1 nucleofilament becomes dismantled immediately after forming a heteroduplex with a template strand. As a consequence, repair synthesis and D-loop extension starts in early prophase intermediates and prevents strand rejection before the completion of homologous pairing. In this case, DSB repair may primarily use the sister chromatid. We conclude that Mcmd1‒Pamd1 protects the Dmc1 nucleofilament from premature dismantling, thereby suppressing precocious repair synthesis and excessive intersister strand exchange at the cost of homologous recombination.

A chromatin-associated protein required for inducing and limiting meiotic DNA double-strand break formation.

Programmed DNA double-strand breaks (DSBs) are required for meiotic recombination, but the number is strictly controlled because they are potentially harmful. Here we report a novel protein, Pars11, which is required for Spo11-dependent DSB formation in the protist Tetrahymena. Pars11 localizes to chromatin early in meiotic prophase in a Spo11-independent manner and is removed before the end of prophase. Pars11 removal depends on DSB formation and ATR-dependent phosphorylation. In the absence of the DNA damage sensor kinase ATR, Pars11 is retained on chromatin and excess DSBs are generated. Similar levels of Pars11 persistence and DSB overproduction occur in a non-phosphorylatable pars11 mutant. We conclude that Pars11 supports DSB formation by Spo11 until enough DSBs are formed; thereafter, DSB production stops in response to ATR-dependent degradation of Pars11 or its removal from chromatin. A similar DSB control mechanism involving a Rec114-Tel1/ATM-dependent negative feedback loop regulates DSB formation in budding yeast. However, there is no detectable sequence homology between Pars11 and Rec114, and DSB numbers are more tightly controlled by Pars11 than by Rec114. The discovery of this mechanism for DSB regulation in the evolutionarily distant protist and fungal lineages suggests that it is conserved across eukaryotes.

A streamlined cohesin apparatus is sufficient for mitosis and meiosis in the protist Tetrahymena.

In order to understand its diverse functions, we have studied cohesin in the evolutionarily distant ciliate model organism Tetrahymena thermophila. In this binucleate cell, the heritable germline genome is maintained separately from the transcriptionally active somatic genome. In a previous study, we showed that a minimal cohesin complex in Tetrahymena consisted of homologs of Smc1, Smc3, and Rec8, which are present only in the germline nucleus, where they are needed for normal chromosome segregation as well as meiotic DNA repair. In this study, we confirm that a putative homolog of Scc3 is a member of this complex. In the absence of Scc3, Smc1 and Rec8 fail to localize to germline nuclei, Rec8 is hypo-phosphorylated, and cells show phenotypes similar to depletion of Smc1 and Rec8. We also identify a homolog of Scc2, which in other organisms is part of a heterodimeric complex (Scc2/Scc4) that helps load cohesin onto chromatin. In Tetrahymena, Scc2 interacts with Rec8 and Scc3, and its absence causes defects in mitotic and meiotic divisions. Scc2 is not required for chromosomal association of cohesin, but Rec8 is hypo-phosphorylated in its absence. Moreover, we did not identify a homolog of the cohesin loader Scc4, and no evidence was found of auxiliary factors, such as Eco1, Pds5, or WAPL. We propose that in Tetrahymena, a single, minimal cohesin complex performs all necessary functions for germline mitosis and meiosis, but is dispensable for transcription regulation and chromatin organization of the somatic genome.

DNA double-strand break formation and repair in Tetrahymena meiosis.

The molecular details of meiotic recombination have been determined for a small number of model organisms. From these studies, a general picture has emerged that shows that most, if not all, recombination is initiated by a DNA double-strand break (DSB) that is repaired in a recombinogenic process using a homologous DNA strand as a template. However, the details of recombination vary between organisms, and it is unknown which variant is representative of evolutionarily primordial meiosis or most prevalent among eukaryotes. To answer these questions and to obtain a better understanding of the range of recombination processes among eukaryotes, it is important to study a variety of different organisms. Here, the ciliate Tetrahymena thermophila is introduced as a versatile meiotic model system, which has the additional bonus of having the largest phylogenetic distance to all of the eukaryotes studied to date. Studying this organism can contribute to our understanding of the conservation and diversification of meiotic recombination processes.

BIME2, a novel gene required for interhomolog meiotic recombination in the protist model organism Tetrahymena.

Meiotic recombination is initiated by DNA double-strand breaks (DSBs). Most DSBs are converted into nonreciprocal exchanges (gene conversions) or crossovers (COs) between sister chromatids. Only a minority of DSBs are processed toward interhomolog COs, the precursors of the chiasmata that connect homologous chromosomes. Dmc1, the meiosis-specific paralog of the universal recombination protein Rad51, is required for interhomolog COs; in its absence, univalents are primarily formed. Here, we report a ciliate-specific novel meiotic gene, BIME2, which also promotes interhomolog crossing over. In the bime2Δ mutant, DSBs are formed and repaired normally, but bivalent formation is strongly reduced. Bime2 protein forms foci on chromatin during meiotic prophase, and chromatin localization of Bime2 and Dmc1 is largely interdependent. Bime2 distantly resembles budding yeast Rdh54/Tid1 and the vertebrate Rad54B helicases and may have similar functions in promoting or stabilizing Dmc1 nucleoprotein filaments.

A single cohesin complex performs mitotic and meiotic functions in the protist tetrahymena.

The cohesion of sister chromatids in the interval between chromosome replication and anaphase is important for preventing the precocious separation, and hence nondisjunction, of chromatids. Cohesion is accomplished by a ring-shaped protein complex, cohesin; and its release at anaphase occurs when separase cleaves the complex's α-kleisin subunit. Cohesin has additional roles in facilitating DNA damage repair from the sister chromatid and in regulating gene expression. We tested the universality of the present model of cohesion by studying cohesin in the evolutionarily distant protist Tetrahymena thermophila. Localization of tagged cohesin components Smc1p and Rec8p (the α-kleisin) showed that cohesin is abundant in mitotic and meiotic nuclei. RNAi knockdown experiments demonstrated that cohesin is crucial for normal chromosome segregation and meiotic DSB repair. Unexpectedly, cohesin does not detach from chromosome arms in anaphase, yet chromosome segregation depends on the activity of separase (Esp1p). When Esp1p is depleted by RNAi, chromosomes become polytenic as they undergo multiple rounds of replication, but fail to separate. The cohesion of such bundles of numerous chromatids suggests that chromatids may be connected by factors in addition to topological linkage by cohesin rings. Although cohesin is not detected in transcriptionally active somatic nuclei, its loss causes a slight defect in their amitotic division. Notably, Tetrahymena uses a single version of α-kleisin for both mitosis and meiosis. Therefore, we propose that the differentiation of mitotic and meiotic cohesins found in most other model systems is not due to the need of a specialized meiotic cohesin, but due to additional roles of mitotic cohesin.

Meiosis gene inventory of four ciliates reveals the prevalence of a synaptonemal complex-independent crossover pathway.

To establish which meiosis genes are present in ciliates, and to look for clues as to which recombination pathways may be treaded by them, four genomes were inventoried for 11 meiosis-specific and 40 meiosis-related genes. We found that the set of meiosis genes shared by Tetrahymena thermophila, Paramecium tetraurelia, Ichthyophthirius multifiliis, and Oxytricha trifallax is consistent with the prevalence of a Mus81-dependent class II crossover pathway that is considered secondary in most model eukaryotes. There is little evidence for a canonical class I crossover pathway that requires the formation of a synaptonemal complex (SC). This gene inventory suggests that meiotic processes in ciliates largely depend on mitotic repair proteins for executing meiotic recombination. We propose that class I crossovers and SCs were reduced sometime during the evolution of ciliates. Consistent with this reduction, we provide microscopic evidence for the presence only of degenerate SCs in Stylonychia mytilus. In addition, lower nonsynonymous to synonymous mutation rates of some of the meiosis genes suggest that, in contrast to most other nuclear genes analyzed so far, meiosis genes in ciliates are largely evolving at a slower rate than those genes in fungi and animals.

Mus81 nuclease and Sgs1 helicase are essential for meiotic recombination in a protist lacking a synaptonemal complex.

Mus81 resolvase and Sgs1 helicase have well-established roles in mitotic DNA repair. Moreover, Mus81 is part of a minor crossover (CO) pathway in the meiosis of budding yeast, plants and vertebrates. The major pathway depends on meiosis-specific synaptonemal complex (SC) formation, ZMM proteins and the MutLγ complex for CO-directed resolution of joint molecule (JM)-recombination intermediates. Sgs1 has also been implicated in this pathway, although it may mainly promote the non-CO outcome of meiotic repair. We show in Tetrahymena, that homologous chromosomes fail to separate and JMs accumulate in the absence of Mus81 or Sgs1, whereas deletion of the MutLγ-component Mlh1 does not affect meiotic divisions. Thus, our results are consistent with Mus81 being part of an essential, if not the predominant, CO pathway in Tetrahymena. Sgs1 may exert functions similar to those in other eukaryotes. However, we propose an additional role in supporting homologous CO formation by promoting homologous over intersister interactions. Tetrahymena shares the predominance of the Mus81 CO pathway with the fission yeast. We propose that in these two organisms, which independently lost the SC during evolution, the basal set of mitotic repair proteins is sufficient for executing meiotic recombination.

The Tetrahymena meiotic chromosome bouquet is organized by centromeres and promotes interhomolog recombination.

In order to form crossovers and to undergo reductional segregation during meiosis, homologous chromosomes must pair. In Tetrahymena, meiotic prophase nuclei elongate immensely, and, within the elongated nucleus, chromosomes are arranged with telomeres assembled at one pole and centromeres at the opposite pole. This organisation is an exaggerated form of the bouquet, a meiotic chromosome arrangement that is widely conserved among eukaryotes. We show that centromere function is crucial for the formation of Tetrahymena's stretched bouquet and, thereby, for homologue pairing. This finding adds to previous reports of the importance of centromeres in chromosome pairing in budding yeast and in Drosophila. Tetrahymena's bouquet is an ataxia telangiectasia- and RAD3-related (ATR)-dependent meiotic DNA damage response that is triggered by meiotic DNA double-strand breaks (DSBs), suggesting that the bouquet is needed for DSB repair. However, in the present study we show that although homologous pairing is impeded in the absence of the bouquet, DSB repair takes place nevertheless. Moreover, recombinational DSB repair, as monitored by bromodeoxyuridine incorporation, takes place only after exit from the bouquet stage. Therefore, we conclude that the bouquet is not required for DSB repair per se, but may be necessary for the alignment of homologous loci in order to promote homologous crossovers over alternative repair pathways.

The recombinases Rad51 and Dmc1 play distinct roles in DNA break repair and recombination partner choice in the meiosis of Tetrahymena.

Repair of programmed DNA double-strand breaks (DSBs) by meiotic recombination relies on the generation of flanking 3' single-stranded DNA overhangs and their interaction with a homologous double-stranded DNA template. In various common model organisms, the ubiquitous strand exchange protein Rad51 and its meiosis-specific homologue Dmc1 have been implicated in the joint promotion of DNA-strand exchange at meiotic recombination sites. However, the division of labor between these two recombinases is still a puzzle. Using RNAi and gene-disruption experiments, we have studied their roles in meiotic recombination and chromosome pairing in the ciliated protist Tetrahymena as an evolutionarily distant meiotic model. Cytological and electrophoresis-based assays for DSBs revealed that, without Rad51p, DSBs were not repaired. However, in the absence of Dmc1p, efficient Rad51p-dependent repair took place, but crossing over was suppressed. Immunostaining and protein tagging demonstrated that only Dmc1p formed strong DSB-dependent foci on meiotic chromatin, whereas the distribution of Rad51p was diffuse within nuclei. This suggests that meiotic nucleoprotein filaments consist primarily of Dmc1p. Moreover, a proximity ligation assay confirmed that little if any Rad51p forms mixed nucleoprotein filaments with Dmc1p. Dmc1p focus formation was independent of the presence of Rad51p. The absence of Dmc1p did not result in compensatory assembly of Rad51p repair foci, and even artificial DNA damage by UV failed to induce Rad51p foci in meiotic nuclei, while it did so in somatic nuclei within one and the same cell. The observed interhomologue repair deficit in dmc1Δ meiosis is consistent with a requirement for Dmc1p in promoting the homologue as the preferred recombination partner. We propose that relatively short and/or transient Rad51p nucleoprotein filaments are sufficient for intrachromosomal recombination, whereas long nucleoprotein filaments consisting primarily of Dmc1p are required for interhomolog recombination.

Novel requirements for HAP2/GCS1-mediated gamete fusion in Tetrahymena.

The ancestral gamete fusion protein, HAP2/GCS1, plays an essential role in fertilization in a broad range of taxa. To identify factors that may regulate HAP2/GCS1 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic Δhap2/gcs1 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2, whose products are necessary for membrane pore formation following mating type recognition and adherence. GFU2 is predicted to be a single-pass transmembrane protein, while GFU1, though lacking obvious transmembrane domains, has the potential to interact directly with membrane phospholipids in the cytoplasm. Like Tetrahymena HAP2/GCS1, expression of GFU1 is required in both cells of a mating pair for efficient fusion to occur. To explain these bilateral requirements, we propose a model that invokes cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in Tetrahymena's multiple mating type system.

The nuclear envelope protein Matefin/SUN-1 is required for homologous pairing in C. elegans meiosis.

We identify a highly specific mutation (jf18) in the Caenorhabditis elegans nuclear envelope protein matefin MTF-1/SUN-1 that provides direct evidence for active involvement of the nuclear envelope in homologous chromosome pairing in C. elegans meiosis. The reorganization of chromatin in early meiosis is disrupted in mtf-1/sun-1(jf18) gonads, concomitant with the absence of presynaptic homolog alignment. Synapsis is established precociously and nonhomologously. Wild-type leptotene/zygotene nuclei show patch-like aggregations of the ZYG-12 protein, which fail to develop in mtf-1/sun-1(jf18) mutants. These patches remarkably colocalize with a component of the cis-acting chromosomal pairing center (HIM-8) rather than the centrosome. Our data on this mtf-1/sun-1 allele challenge the previously postulated role of the centrosome/spindle organizing center in chromosome pairing, and clearly support a role for MTF-1/SUN-1 in meiotic chromosome reorganization and in homolog recognition, possibly by mediating local aggregation of the ZYG-12 protein in meiotic nuclei.

Mug20, a novel protein associated with linear elements in fission yeast meiosis.

In the fission yeast, Schizosaccharomyces pombe, homologous chromosomes efficiently pair and recombine during meiotic prophase without forming a canonical synaptonemal complex (SC). Instead, it features simpler filamentous structures, the so-called linear elements (LinEs), which bear some resemblance to the axial/lateral element subunits of the SC. LinEs are required for wild-type recombination frequency. Here, we recognized Mug20, the product of a meiotically upregulated gene, as a LinE-associated protein. GFP-tagged Mug20 and anti-Mug20 antibody co-localized completely with Rec10, one of the major constituents of LinEs. In the absence of Mug20, LinEs failed to elongate beyond their initial state of nuclear dots. Foci of recombination protein Rad51 and genetic recombination were reduced. Since meiotic DNA double-strand breaks (DSBs), which initiate recombination, are induced at sites of preformed LinEs, we suggest that reduced recombination is a consequence of incomplete LinE extension. Therefore, we propose that Mug20 is required to extend LinEs from their sites of origin and thereby to increase DSB proficient regions on chromosomes.

Arrested crossover precursor structures form stable homologous bonds in a Tetrahymena meiotic mutant.

Meiotic DNA double-strand breaks produce reciprocally exchanged DNA strands, which mature into chiasmata that hold homologous chromosomes together as bivalents. These bivalents are subsequently separated in the first meiotic division. In a mutant lacking the newly identified Tetrahymena gene APRO1 (Anaphase promoting 1), meiosis is arrested by the end of prophase. Mature chiasmata are not formed but bivalents are connected via a molecular precursor structure. In-depth analysis of this arrested intermediate structure may help to elucidate the noncanonical molecular recombination pathway in Tetrahymena.

MRE11 and COM1/SAE2 are required for double-strand break repair and efficient chromosome pairing during meiosis of the protist Tetrahymena.

Programmed DNA double-strand breaks (DSBs) are generated during meiosis to initiate homologous recombination. Various aspects of DSB formation, signaling, and repair are accomplished or governed by Mre11, a component of the MRN/MRX complex, partially in cooperation with Com1/Sae2/CtIP. We used Tetrahymena to study evolutionarily conserved and changed functions of Mre11 and Com1. There is a difference between organisms with respect to the dependency of meiotic DSB formation on Mre11. By cytology and an electrophoresis-based assay for DSBs, we found that in Tetrahymena Mre11p is not required for the formation and ATR-dependent signaling of DSBs. Its dispensability is also reflected by wild-type-like DSB-dependent reorganization of the meiotic nucleus and by the phosphorylation of H2A.X in mre11∆ mutant. However, mre11∆ and com1∆ mutants are unable to repair DSBs, and chromosome pairing is reduced. It is concluded that, while MRE11 has no universal role in DNA damage signaling, its requirement for DSB repair is conserved between evolutionarily distant organisms. Moreover, reduced chromosome pairing in repair-deficient mutants reveals the existence of two complementing pairing processes, one by the rough parallel arrangement of chromosomes imposed by the tubular shape of the meiotic nucleus and the other by repair-dependent precise sequence matching.

SUMOylation is required for normal development of linear elements and wild-type meiotic recombination in Schizosaccharomyces pombe.

In the fission yeast, Schizosaccharomyces pombe, synaptonemal complexes (SCs) are not formed during meiotic prophase. However, structures resembling the axial elements of SCs, the so-called linear elements (LinEs) appear. By in situ immunostaining, we found Pmt3 (S. pombe's SUMO protein) transiently along LinEs, suggesting that SUMOylation of some component(s) of LinEs occurs during meiosis. Mutation of the SUMO ligase Pli1 caused aberrant LinE formation and reduced genetic recombination indicating a role for SUMOylation of LinEs for the regulation of meiotic recombination. Western blot analysis of TAP-tagged Rec10 demonstrated that there is a Pli1-dependent posttranslational modification of this protein, which is a major LinE component and a distant homolog of the SC protein Red1. Mass spectrometry (MS) analysis revealed that Rec10 is both phosphorylated and ubiquitylated, but no evidence for SUMOylation of Rec10 was found. These findings indicate that the regulation of LinE and Rec10 function is modulated by Pli1-dependent SUMOylation of LinE protein(s) which directly or indirectly regulates Rec10 modification. On the side, MS analysis confirmed the interaction of Rec10 with the known LinE components Rec25, Rec27, and Hop1 and identified the meiotically upregulated protein Mug20 as a novel putative LinE-associated protein.