The Ecm11-Gmc2 complex promotes synaptonemal complex formation through assembly of transverse filaments in budding yeast

During meiosis, homologous chromosomes pair at close proximity to form the synaptonemal complex (SC). This association is mediated by transverse filament proteins that hold the axes of homologous chromosomes together along their entire length. Transverse filament proteins are highly aggregative and can form an aberrant aggregate called the polycomplex that is unassociated with chromosomes. Here, we show that the Ecm11-Gmc2 complex is a novel SC component, functioning to facilitate assembly of the yeast transverse filament protein, Zip1. Ecm11 and Gmc2 initially localize to the synapsis initiation sites, then throughout the synapsed regions of paired homologous chromosomes. The absence of either Ecm11 or Gmc2 substantially compromises the chromosomal assembly of Zip1 as well as polycomplex formation, indicating that the complex is required for extensive Zip1 polymerization. We also show that Ecm11 is SUMOylated in a Gmc2-dependent manner. Remarkably, in the unSUMOylatable ecm11 mutant, assembly of chromosomal Zip1 remained compromised while polycomplex formation became frequent. We propose that the Ecm11-Gmc2 complex facilitates the assembly of Zip1 and that SUMOylation of Ecm11 is critical for ensuring chromosomal assembly of Zip1, thus suppressing polycomplex formation

Telomere maintenance in liquid crystalline chromosomes of dinoflagellates

The organisation of dinoflagellate chromosomes is exceptional among eukaryotes. Their genomes are the largest in the Eukarya domain, chromosomes lack histones and may exist in liquid crystalline state. Therefore, the study of the structural and functional properties of dinoflagellate chromosomes is of high interest. In this work, we have analysed the telomeres and telomerase in two Dinoflagellata species, Karenia papilionacea and Crypthecodinium cohnii. Active telomerase, synthesising exclusively Arabidopsis-type telomere sequences, was detected in cell extracts. The terminal position of TTTAGGG repeats was determined by in situ hybridisation and BAL31 digestion methods and provides evidence for the linear characteristic of dinoflagellate chromosomes. The length of telomeric tracts, 25-80 kb, is the largest among unicellular eukaryotic organisms to date. Both the presence of long arrays of perfect telomeric repeats at the ends of dinoflagellate chromosomes and the existence of active telomerase as the primary tool for their high-fidelity maintenance demonstrate the general importance of these structures throughout eukaryotes. We conclude that whilst chromosomes of dinoflagellates are unique in many aspects of their structure and composition, their telomere maintenance follows the most common scenario

Tidying-up the plant nuclear space: domains, functions, and dynamics

Understanding how the packaging of chromatin in the nucleus is regulated and organized to guide complex cellular and developmental programmes, as well as responses to environmental cues is a major question in biology. Technological advances have allowed remarkable progress within this field over the last years. However, we still know very little about how the 3D genome organization within the cell nucleus contributes to the regulation of gene expression. The nuclear space is compartmentalized in several domains such as the nucleolus, chromocentres, telomeres, protein bodies, and the nuclear periphery without the presence of a membrane around these domains. The role of these domains and their possible impact on nuclear activities is currently under intense investigation. In this review, we discuss new data from research in plants that clarify functional links between the organization of different nuclear domains and plant genome function with an emphasis on the potential of this organization for gene regulation

G2/M-checkpoint activation in fasciata1 rescues an aberrant S-phase checkpoint but causes genome instability

Abstract The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1KO and ATRKO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of-function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that are described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.</jats:p

Proposed model for the function of the Ecm11-Gmc2 complex.

<p>The association of Ecm11 with Gmc2, forming the E-G complex (Ecm11-Gmc2), facilitates its loading at the synapsis initiation complex (SIC). In parallel, Zip1 is recruited to the SIC. The presence of the SIC and Zip1 with the E-G complex facilitates the SUMOylation of Ecm11. The SUMOylated E-G complex selectively promotes the chromosomal assembly of Zip1 while polycomplex formation is discouraged. See discussion for details.</p

Ecm11 and Gmc2 are necessary for the efficient assembly of Zip1 on chromosomes and in the polycomplex.

<p>(A) Zip1 localization is discontinuous on meiotic prophase chromosomes in the <i>ecm11</i> and <i>gmc2</i> mutants. The localization of Zip1 along with Red1, a component of meiotic chromosome axes, was examined on spread chromosomes. Bar, 5 µm. (B, C) Quantitative analysis of the Zip1 localization. Zip1 stretch area represents the size of one continuous Zip1 staining. See Results and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003194#s4" target="_blank">Materials and Methods</a> for more details. (D) Polycomplex formation was abolished in <i>ecm11</i> and <i>gmc2</i> mutants. Zip1 localization was examined in the <i>spo11</i> mutant background. The white arrowhead indicates the location of the polycomplex. Small speckle-like Zip1 staining likely represents the centromeric localization of Zip1. Chromosome spreads were prepared using cells at 20 hours after introduction into meiosis when, in wild type, cells at the pachytene stage are enriched. (E) Quantification of spread nuclei exhibiting a polycomplex.</p

SUMOylation of Ecm11 at Lysine 5 is essential for facilitating the chromosomal assembly of Zip1.

<p>(A) The predominant role of Lysine 5 in facilitating the chromosomal assembly of Zip1. Meiotic chromosomes of wild type cells or <i>ecm11</i> mutants in which SUMOylation was compromised were examined for their Zip1 localization and also for Red1. Bar, 5 µm. (B, C) Quantitative analyses of Zip1 localization. See Figure legend of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003194#pgen-1003194-g002" target="_blank">Figure 2B, 2C</a> and also Results and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003194#s4" target="_blank">Materials and Methods</a> for details. (D) Polycomplex formation was elevated when Lysine 5 was mutated. Meiotic chromosomes carrying the mutations indicated were stained for the Zip1 protein and the fractions of chromosome spreads carrying a polycomplex were calculated. At least 100 chromosome spreads were counted for each strain.</p

Ecm11 and Gmc2 are required for efficient meiotic crossing over.

<p>(A) Sporulation is delayed in the <i>ecm11</i> and <i>gmc2</i> mutants. Diploid cells were introduced into meiosis and spore formation was examined at indicated time points. (B) Crossing over is reduced in the <i>ecm11</i> and <i>gmc2</i> mutants. Diploid cells carrying one circular and one linear chromosome III were introduced into meiosis and crossing over between these chromosomes was measured by Southern blotting. The amount of linear dimer and trimer chromosomes represents the efficiency of crossing over. (C) Quantification of crossover products shown in (B). The amount of recombinants was expressed as the ratio (percentage) of the combined signal of linear dimer and trimer bands per the sum of linear monomer, dimer and trimer bands. Error bars represent SEM.</p

Ecm11 and Gmc2 associate with each other and are localized to the synapsis initiation sites at early prophase I and later to the SC central region.

<p>(A) Ecm11 and Gmc2 colocalize throughout meiotic prophase I. A diploid strain carrying <i>ECM11-FLAG</i> and <i>myc-GMC</i>2 was introduced into meiosis, chromosomes were surface spread and Ecm11 and Gmc2 were immunostained with anti-FLAG and anti-myc antibodies. The boxed areas were magnified and shown below. (B) Both Ecm11 and Gmc2 colocalize with Zip1, and are localized to the area between paired homologs. A diploid strain carrying either <i>ECM11-myc</i> or <i>myc-GMC2</i> was introduced into meiosis and spread chromosomes were immunostained for Ecm11 (top), Gmc2 (bottom) and Zip1. The boxed areas were magnified and shown below, with either the Ecm11 or Gmc2 localization along with the DAPI or Zip1 staining. (C) Ecm11 colocalizes with Zip3 at early prophase I. A diploid strain carrying <i>ECM11-FLAG</i> and <i>ZIP3-myc</i> was introduced into meiosis and spread chromosomes were stained for Ecm11 and Zip3 using anti-FLAG and anti-myc antibodies. Bar, 5 µm in (A–C). (D) Ecm11 and Gmc2 show interaction using the yeast two hybrid system. Medium lacking tryptophan and leucine was used to maintain the DBD fusion plasmid (marked with <i>TRP1</i>) and the AD fusion plasmid (marked with <i>LEU2</i>). Growth on medium lacking adenine (shown on the left) reflects the expression level of the <i>GAL4-ADE2</i> reporter gene and is thus a measure of the interaction between two fusion proteins. Shown on the right is growth on medium lacking tryptophan and leucine as a reference. (E) Ecm11 and Gmc2 show interaction in the co-immunoprecipitation assay. Extracts from meiotic cells with both Ecm11 and Gmc2 tagged, only Ecm11 tagged with untagged Gmc2, or only Gmc2 tagged with untagged Ecm11, were subjected to immunoprecipitation experiments with antibodies shown. Immunoprecipitates were analysed by Western blotting with antibodies as indicated.</p