A Caenorhabditis elegans cohesion protein with functions in meiotic chromosome pairing and disjunction - PubMed (original) (raw)

A Caenorhabditis elegans cohesion protein with functions in meiotic chromosome pairing and disjunction

P Pasierbek et al. Genes Dev. 2001.

Abstract

We have studied four Caenorhabditis elegans homologs of the Rad21/Scc1/Rec8 sister-chromatid cohesion protein family. Based on the RNAi phenotype and protein localization, it is concluded that one of them, W02A2.6p, is the likely worm ortholog of yeast Rec8p. The depletion of C. elegans W02A2.6p (called REC-8) by RNAi, induced univalent formation and splitting of chromosomes into sister chromatids at diakinesis. Chromosome synapsis at pachytene was defective, but primary homology recognition seemed unaffected, as a closer-than-random association of homologous fluorescence in situ hybridization (FISH) signals at leptotene/zygotene was observed. Depletion of REC-8 also induced chromosome fragmentation at diakinesis. We interpret these fragments as products of unrepaired meiotic double-stranded DNA breaks (DSBs), because fragmentation was suppressed in a spo-11 background. Thus, REC-8 seems to be required for successful repair of DSBs. The occurrence of DSBs in REC-8-depleted meiocytes suggests that DSB formation does not depend on homologous synapsis. Anti-REC-8 immunostaining decorated synaptonemal complexes (SCs) at pachytene and chromosomal axes in bivalents and univalents at diakinesis. Between metaphase I and metaphase II, REC-8 is partially lost from the chromosomes. The partial loss of REC-8 from chromosomes between metaphase I and metaphase II suggests that worm REC-8 might function similarly to yeast Rec8p. The loss of yeast Rec8p from chromosome arms at meiosis I and centromeres at meiosis II coordinates the disjunction of homologs and sister chromatids at the two meiotic divisions.

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Figures

Figure 1

Figure 1

Phylogenetic tree of Rad21/Scc1/Rec8 family members, calculated by the Phylo_Win program using the Neighbor Joining method (Galtier et al. 1996). The tree is based on a Clustal W-alignment with minor modifications (Thompson et al. 1994) of the amino terminus of Scc1/Rec8 proteins. S.c. = Saccharomyces cerevisiae, S.p. = Schizosaccharomyces pombe, D.m. = Drosophila melanogaster, H.s. = Homo sapiens, C.e. = Caenorhabditis elegans, A.t. = Arabidopsis thaliana. For protein sequences used see Parisi et al. (1999), Bhatt et al. (1999), and Klein et al. (1999). The next noncohesion protein hit (EMBL/GenBank accession no. AF279455) in a PSI-BLAST search with H. sapiens Scc1p as the query, served as the outgroup. The scale bar denotes the number of substitutions per site.

Figure 2

Figure 2

Expression of K08A8.3p (COH-1), F10G7.4p (COH-2), and W02A2.6p (REC-8) in Caenorhabditis elegans N2. Antisera against the three proteins detected single prominent bands. The minor bands most likely correspond to breakdown products. COH-1 and COH-2 have predicted molecular weights of 75 kD, whereas the bands detected by the respective antisera indicate a slightly aberrant migration. The band detected by the REC-8 antiserum corresponds exactly to the predicted 89 kD of the protein. All antisera could be blocked efficiently by recombinant protein, indicating that the observed signals are specific (not shown).

Figure 3

Figure 3

Immunolocalization of Rec8p homologs. COH-1 is present in the nuclei of embryos (A) and of the distal tip cell (D). It also localizes to chromosomes in pachytene (G) to diakinesis (H). COH-2 is localized in embryonic nuclei (B) and forms foci from the mitotic up to the pachytene zone of the gonad (E, I). In pachytene nuclei the foci are exclusively peripheral (I). REC-8 can not be detected in embryos (C). It is homogeneously distributed in early nuclei of the gonad (F) and is condensed into threads in pachytene nuclei (J). Bars: 5 μm in A for A–C, 10 μm in E for D–F, 5 μm in H for G–J.

Figure 4

Figure 4

RNAi-induced reduction in viability and induction of Him phenotype for rec-8. Worms were soaked in dsRNA for 12 h and then transferred to fresh plates every 24 h. The percentage of viability and sex of the laid offspring was determined for each 24 h interval. Viability was strongly reduced for the first two intervals. At later times, when a sufficiently high number of viable embryos appeared because of fading of the RNAi-effect, increased incidence of male offspring was observed. No data could be collected for time points later than 96 h as worms ran out of sperm and produced unfertilized eggs. In total, viability was determined for ∼3000 embryos.

Figure 5

Figure 5

REC-8 immunostaining pattern throughout gonadal mitosis and meiosis. (A) Grains in nuclei of the mitotic zone. (B) Formation of short threads (arrows) in the transition zone (leptotene/zygotene). (C) REC-8 delineates synaptonemal complexes (SCs) in pachytene. In D and E immunostaining is shown on top and DAPI at the bottom, in F immunostaining is on the left and DAPI on the right. Diakinesis: Labeling of chromosomal axes in bivalents (D) and spo-11 univalents (E). Insert in D shows an enlarged DAPI-stained bivalent (arrows) with the inverted image of the REC-8 axes superimposed. (F) From top to bottom: Metaphase I (side view, two bivalents in focus, arrows denote equator), early anaphase I (top view of half-nucleus; the other half is in a different focal plane underneath), late anaphase I (separating nuclei out of alignment due to preparation), metaphase II (side view, three chromosomes in the focal plane; arrows denote equator, polar body outside the area shown). Bar in C represents 10 μm in A–C; bar in D, 5 μm in D–F.

Figure 6

Figure 6

Immunostaining of HIM-3 (A,B,E left: green) and REC-8 (C,D,E right: red) of spread nuclei at leptotene/zygotene (A,C) and pachytene (B,D,E). Note the parallel association of forming axial elements (presynaptic alignment) in A. Both proteins delineate the six synaptonemal complexes (SCs) at pachytene (B,D) and show nearly perfect colocalization by double immunostaining (E, center). Counterstaining of chromatin by DAPI (blue). Bar: 10 μm.

Figure 7

Figure 7

Cytological effects of REC-8 depletion. (A,B) Separated sister chromatids and chromosomal fragments (arrows; some fragments slightly out of focus) at diakinesis (DAPI staining). (C) Sister chromatids are separated, but no fragments occur after REC-8 depletion in spo-11 deletion animals. (D) Wild-type (untreated) diakinesis showing six bivalents (DAPI). (E) Dispersed HIM-3 immunostaining in pachytene zone after RNAi. (F) Immunolocalization of HIM-3 in wild-type pachytene. Bar: 10 μm.

Figure 8

Figure 8

Detection of homologous pairing at pachytene in wild-type (A) and REC-8-depleted (B) gonads, as determined by two color FISH (green: chromosome I locus; red: chromosome V locus; blue: DNA staining by DAPI). Pairing is deficient in nuclei lacking the protein. Arrows indicate split sister chromatids. Bar: 10 μm.

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