Elsevier

Experimental Cell Research

Volume 289, Issue 2, 1 October 2003, Pages 245-255
Experimental Cell Research

Regular article
The Caenorhabditis elegans SCC-3 homologue is required for meiotic synapsis and for proper chromosome disjunction in mitosis and meiosis

https://doi.org/10.1016/S0014-4827(03)00266-0Get rights and content

Abstract

The product of the Caenorhabditis elegans ORF F18E2.3 is homologous to the cohesin component Scc3p. By antibody staining the product of F18E2.3 is found in interphase and early meiotic nuclei. At pachytene it localizes to the axes of meiotic chromosomes but is no longer detectable on chromatin later in meiosis or in mitoses. Depletion of the gene product by RNAi results in aberrant mitoses and meioses. In meiosis, homologous pairing is defective during early meiotic prophase and at diakinesis there occur univalents consisting of loosely connected sister chromatids or completely separated sisters. The recombination protein RAD-51 accumulates in nuclear foci at higher numbers during meiotic prophase and disappears later than in wild-type worms, suggesting a defect in the repair of meiotic double-stranded DNA breaks. Embryos showing nuclei of variable size and anaphase bridges, indicative of mitotic segregation defects, are frequently observed. In the most severely affected gonads, nuclear morphology cannot be related to any specific stage. The cytological localization and the consequences of the lack of the protein indicate that C. elegans SCC-3 is essential for sister chromatid cohesion both in mitosis and in meiosis.

Introduction

Chromosomes replicate during S-phase of the cell cycle and from that time on consist of two identical DNA molecules, the sister chromatids. During mitosis, the sister chromatids of each chromosome are segregated equally to daughter nuclei, which thereby obtain identical copies of the genetic material. Sister chromatids have to remain connected from S-phase through the onset of mitosis until their centromeres have oriented toward and attached to the opposite poles of the mitotic spindle. If they separated precociously, nondisjunction could occur, leading to genetically unbalanced daughter cells. Moreover, the association of chromatids after replication allows the use of the sister molecule as template for the replacement of deleted or damaged DNA tracts by mechanisms of recombinational DNA repair. Therefore, the association of sister chromatids is actively maintained by a process called sister chromatid cohesion [for review see 1].

A group of proteins, central for sister chromatid cohesion, form a complex, the cohesin, which is highly conserved from yeast to humans. Cohesin localizes to replicated chromosomes in mitosis and meiosis and is required for the establishment and maintenance of cohesion between sister chromatids. Two of the four core cohesin subunits, Smc1 and Smc3, belong to the structural maintenance of chromosomes (SMC) family, members of which are also found in condensin, a protein complex with a role in chromosome condensation [for review see 2]. The other two are the sister chromatid cohesion proteins Scc1/Mcd1/Rad21 and Scc3(Irr1) [for review see [3], [4]].

In the budding yeast, cohesion occurs along the entire chromosome arm until the onset of anaphase whereas in vertebrates most of the cohesin is lost along the arms before prophase, and only a small amount (probably in the centromeric region) is maintained until the onset of anaphase [5]. The release of cohesion at anaphase occurs by the APC-dependent cleavage of Scc1/Mcd1 [6], [7], for reviews see [1], [8]. In yeast, the cleavage by the protease Esp1, the so-called separase, is facilitated by the phosphorylation of Scc1 by the Polo kinase Cdc5 [9].

In meiosis, sister chromatid cohesion serves additional functions. First, it supports interhomolog and intersister interactions during the initiation of recombination [10], and second, it stabilizes chiasmata. The physical link between homologs by chiasmata is necessary for their faithful disjunction at the first meiotic division. It was postulated by Darlington [11] and Maguire [12], [13], [14] that cohesion between sister chromatids either at or distal to chiasmata is necessary for their stabilization. Loss of cohesion distally to chiasmata allows homologs to separate at meiosis I, but sister chromatids of each homolog migrate together as they remain connected by cohesion at and/or around the centromere [15], [16], [17]. Prior to the second meiotic division, cohesion is released in the centromeric regions, and this allows the separation of sister chromatids. It is not yet known by which mechanism the differential loss of cohesion in meiosis is governed, but it seems to involve the protection of the centromeric cohesin from cleavage at meiosis I [18], [19].

The specific demands of stepwise resolution of cohesion and perhaps also additional functions in meiotic recombination have apparently led to the evolution of a meiosis-specific version of cohesin. The most notable difference is the substitution of the Scc1/Mcd1/Rad21 subunit by Rec8. In mammals and in Schizosaccharomyces pombe an exclusively or predominantly meiotic version of the Scc3 homolog, called SA3/STAG3 and Rec11, seems to exist [20], [21], [22], for reviews see [2], [4].

The product of the Caenorhabditis elegans gene F18E2.3 was identified in database searches as the only worm member of the Scc3 family of cohesion proteins comprising the mitotic Scc3/SA1/STAG1, SA2/STAG2 of vertebrates and their homologs in Arabidopsis and Drosophila and the meiotic SA3/STAG3 and Rec11 [2], [23]. F18E2.3 is not exclusively germline-expressed in the worm [24], [25] and the corresponding protein is therefore likely to play a role both in mitosis and meiosis.

Here we studied the mitotic and meiotic functions and the cellular localization of C. elegans SCC-3 by depletion of the protein by RNA interference and by immunocytology.

Section snippets

Worm strains and culture conditions

The wild-type (N2 Bristol) strain and the spo-11(ok79) mutant were obtained from the Caenorhabditis Genetics Center (University of Minnesota, St. Paul, MN). Worms were grown on NGM plates with Escherichia coli OP50 [26].

Protein depletion

The HIM-3 and REC-8 proteins were depleted by double-stranded RNA interference (RNAi). RNAi was performed with a fragment of the corresponding target sequences (him-3 ZK381.1 cDNA spanning nucleotides 3 to 864 and rec-8 W02A2.6 cDNA spanning nucleotides 630 to 2339). DNA

SCC-3 depletion causes severe defects in gonads and embryos

To inhibit the production of wild-type levels of the scc-3 gene product by RNAi [28], scc-3 double-stranded RNA was administered to worms by feeding them on dsRNA-expressing bacteria. dsRNA applied from early larval stages (L1–L2) onward led to a Sterile–Uncoordinated (Unc) phenotype and a protruding vulva in the adults, which are characteristic of cell division defects [31], [32], [33]. The viability of F1 laid by these worms was 1.2% (1/83), whereas in the wild-type control group it was 94%

The phenotypes created by the loss of F18E2.3 protein and its cytological localization confirm its homology to the Scc3/STAG family of cohesion proteins

On the basis of its similarity, the protein encoded by the C. elegans gene F18E2.3 was considered to be a homolog of the Scc3 cohesin subunit [23]. The Scc3 family includes known orthologs from a variety of fungi, plants, and animals [2], [4]. In S. pombe and in mammals, meiotic variants of Scc3 that probably function in the context of a meiosis-specific cohesin complex were found [22], [41] Here we show that the product of F18E2.3 is the likely Scc3 homolog of C. elegans with functions both in

Acknowledgements

We are grateful to Anton Gartner (M.P.I. Biochemistry, Munich, GER), Anne Villeneuve (Stanford University, CA), and Monique Zetka (McGill University, Montreal, Canada) for the RAD-51, SYP-1, and HIM-3 antibodies, respectively. We thank Bonnie Wohlrab for technical assistance. This work was supported by grants from the Austrian Science Fund to D.S. (S8211) and J.L. (P14642).

References (53)

  • D.G. Albertson et al.

    Cell cycling and DNA replication in a mutant blocked in cell division in the nematode Caenorhabditis elegans

    Dev. Biol.

    (1978)
  • J.E. Sulston et al.

    Abnormal cell lineages in mutants of the nematode Caenorhabditis elegans

    Dev. Biol.

    (1981)
  • P. Goldstein

    Multiple synaptonemal complexes (polycomplexes)origin, structure and function

    Cell Biol. Int. Rep.

    (1987)
  • S. Kaitna et al.

    The aurora B kinase AIR-2 regulates kinetochores during mitosis and is required for separation of homologous chromosomes during meiosis

    Curr. Biol.

    (2002)
  • E. Sonoda et al.

    Sec1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells

    Dev. Cell

    (2001)
  • T.U. Tanaka

    Bi-orienting chromosomes on the mitotic spindle

    Curr. Opin. Cell Biol.

    (2002)
  • C.H. Haering et al.

    Molecular architecture of SMC proteins and the yeast cohesin complex

    Mol. Cell

    (2002)
  • K. Nasmyth

    Disseminating the genomejoining, resolving, and separating sister chromatids during mitosis and meiosis

    Annu. Rev. Genet.

    (2001)
  • T. Hirano

    The ABCs of SMC proteinstwo-armed ATPases for chromosome condensation, cohesion, and repair

    Genes Dev.

    (2002)
  • A.R. Ball et al.

    The structural maintenance of chromosomes (SMC) family of proteins in mammals

    Chromosome Res.

    (2001)
  • F. Uhlmann et al.

    Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1

    Nature

    (1999)
  • S. Hauf et al.

    Cohesin cleavage by separase required for anaphase and cytokinesis in human cells

    Science

    (2001)
  • K. Nasmyth et al.

    Splitting the chromosomecutting the ties that bind sister chromatids

    Science

    (2000)
  • C.D. Darlington

    Recent Advances in Cytology

    (1932)
  • M.P. Maguire

    The mechanism of chiasma maintenancea study based upon behaviour of acentric fragments produced by crossovers in heterozygous paracentricinversions

    Cytologia

    (1982)
  • B.H. Lee et al.

    Spo13 regulates cohesin cleavage

    Genes Dev.

    (2002)
  • Cited by (0)

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