Identification and Characterization of
Suppressors of Nonhomologous Synapsis during
C. elegans Meiosis
Ka-Lun Law
Department of Biology, McGill University
Montreal, Quebec, Canada
February, 2011
A thesis submitted to McGill University in partial fulfillment
of the requirement of the degree of Doctor of Philosophy
©
Ka-Lun Law, 2011
Table of Contents
Abstract/ Résumé …………………………………………………............. 5
Acknowledgements ……………………………………………………… 11
Chapter I: Literature Review …………………………………………….. 13
Overview of meiosis …………………………………………………………………... 14
Meiotic prophase events ………………………………………………………………. 15
Meiotic chromosome pairing ………………………………………………………….. 20
Chromosome movement during meiotic pairing stages ………………………………. 22
HIM-3 family functions during meiosis ………………………………………………. 25
The biological significance of chaperonin complex and its relevance to meiosis ……. 28
C. elegans as a model to study meiosis ……………………………………………….. 31
Rationale for study ……………………………………………………………………. 33
References for chapter I ……………………………………………………………….. 34
Appendix for chapter I ………………………………………………………………… 45
Table 1.1
Figure 1.2-1.6
Chapter II: Isolation of him-3(vv6) Suppressors ………………………… 46
Summary of chapter II ………………………………………………………………… 47
Materials and methods ………………………………………………………………… 48
Results ……………………………………………………………………………….... 52
Isolating the suppressors from screen ……………………………………………………………………. 52
Cytological studies of the suppressors …………………………………………………………………… 54
Mapping and analysis of suppressors ……………………………………………………………………. 54
References for chapter II ……………………………………………………………… 62
2
Appendix for chapter II ……………………………………………………………….. 63
Chart 2.1-2.2
Figure 2.3
Table 2.4
Figure 2.5-2.6
Chapter III: Characterization of the Suppressor cct-4(vv39) ……………. 64
Summary of chapter III ……………………………………………………………….. 65
Materials and methods ………………………………………………………………… 66
Results ………………………………………………………………………………… 71
vv39 corresponds to a mutation in the CCT chaperonin subunit CCT-4 ………………………………. 71
CCT-4 colocalizes with chromatin throughout the germline …………………………………………. 73
cct-4(RNAi) results in germline defects ………………………………………………………………... 74
CCT-4 is required to assemble axes competent for PC protein recruitment and SC assembly ………... 74
Loss of CCT-1 and CCT-3 recapitulates cct-4(RNAi) axes morphogenesis defects …………………… 75
cct-4(vv39) mutants exhibit meiotic cell cycle delay ………………………………………………….. 75
him-3(vv6) mutants have defects in axes morphogenesis and synapsis progression that are suppressed by
CCT-4vv39 ………………………………………………………………………………………………. 76
cct-4(vv39) restores autosomal PC protein recruitment in him-3(vv6) mutants ……………………….. 77
Defective ZYG-12/SUN-1 patch formation in him-3(vv6) mutants is restored by CCT-4vv39 ………… 78
him-3(vv6) mutants have extensive nonhomologous synapsis ………………………………………… 80
Defective initial pairing of him-3(vv6) mutants is partially rescued by CCT-4vv39 …………………….. 81
CCT-4vv39 partially suppresses nonhomologous synapsis in him-3(vv6) mutants ……………………… 82
CCT-4vv39 restores RAD-51 kinetics to wild-type levels in him-3(vv6) mutant germlines …………...... 83
CCT-4 partially rescues the homolog pairing defects of htp-1(gk149) mutants ……………………….. 84
References for chapter II ……………………………………………………………… 88
Appendix for chapter III ………………………………………………………………. 91
Figure 3.1-3.23
Chapter IV: Discussion and Conclusion ………………………………… 92
Isolation of genetic suppressors of meiotic defects …………………………………… 93
A role for CCT function in the meiotic prophase ……………………………………... 95
HIM-3vv6 results in delayed axes morphogenesis ……………………………………... 97
CCT-4vv39 suppresses the axes morphogenesis defects of him-3(vv6) ………………... 98
Mechanisms of suppression of nonhomologous synapsis during meiotic prophase ….. 99
Conclusion and future directions …………………………………………………….. 102
References for chapter IV ……………………………………………………………. 103
3
Appendix for chapter IV ………………………………………………….. ………… 105
Figure 4.1……………………………………………………………………………………………... 106
Glossary ……………………………………………………………………………… 107
4
Abstract
Meiosis is a specialized process that allows the generation of haploid gametes
from diploid cells. The proper segregation of homologous chromosomes during meiosis
I depends on the initial alignment of chromosomes, the stabilization of this alignment
through synapsis and the formation of chiasmata between homologs. Previous studies
have demonstrated that HIM-3, a structural component of the meiotic chromosome axes,
is required for these processes through the recruitment of the autosomal pairing center
proteins (ZIMs) and of the synaptonemal complex component SYP-1. The him-3(vv6)
mutation results in the substitution of a highly conserved amino acid of the HORMA
domain, believed to mediate protein-protein interactions. HIM-3 levels in vv6 mutant
germlines appear to be normal and the protein is loaded to the chromosome axes, but
nevertheless him-3(vv6) mutants exhibit defects in autosomal homolog alignment, in
meiotic cell cycle progression as indicated by extension of leptotene/zygotene stages, in
nonhomologous synapsis, in recombination progression and in chiasmata formation. In
him-3(vv6) mutants, the morphology of chromosome axes appears to be immature and
noncontiguous at early meiosis. In addition, the ZIMs fail to localize to the pairing
centers, however the X chromosome pairing center protein HIM-8 remains unaffected.
This suggests that the presence of a single pairing center protein is sufficient to license
synapsis globally, irrespective to homology, resulting in nonhomologous synapsis in
him-3(vv6) mutants.
The objective of this study is to further understand the mechanism that regulates
chromosome alignment and synapsis. An EMS-based suppressor screen was performed
with the him-3(vv6) allele to identify other factors that regulate alignment and synapsis
5
formation between homologous chromosomes. Four dominant suppressors (vv38, vv39,
vv41 and vv50) and one semi-dominant suppressor (vv52) that rescue the embryonic
lethality phenotype of him-3(vv6) were isolated from the screen. All of the suppressor
mutants showed a less extensive leptotene/zygotene region in comparison to him-3(vv6),
indicating they have better meiotic cell cycle progression.
Genetic mapping was
performed on the three strongest suppressors: vv38, vv39, and vv52. vv38 was identified
to be an intragenic suppressor, with the mutation also located inside the HORMA
domain.
vv52 was mapped to a finite interval on chromosome II, and vv39 was
identified as a novel allele of cct-4, which encodes the delta subunit of type II
chaperonin complex. In wild-type germlines, CCT-4 is localized to the cytoplasm and to
the nucleus, indicating that it has a nuclear role. In addition, a portion of CCT-4 is found
to colocalize with chromatin during leptotene/zygotene, suggesting that it has a function
at chromosome pairing stages.
In cct-4(RNAi) germlines, the morphology of
chromosome axes appears to be noncontiguous, ZIM-3 fails to be recruited to the pairing
centers and SYP-1 fails to localize to the axes, indicating that CCT-4 is required for axes
morphogenesis, loading of ZIMs and SC formation. Interestingly, RNAi against two
other chaperonin complex subunits CCT-1 and CCT-3 also showed similar axes
morphogenesis and synaptonemal complex formation defects, indicating that CCT-4
function may be mediated through the chaperonin complex. In cct-4(vv39) mutants, the
morphology of chromosome axes appeared immature at early leptotene, but the
recruitment of ZIMs and synapsis formation are still sustained at later meiotic stages. In
him-3(vv6); cct-4(vv39) mutants, the morphology of chromosome axes is restored and
localization of ZIM-3 also appears to be normal, resulting in the suppression of other
6
subsequent defects including homolog alignment and nonhomologous synapsis. It has
been previously shown that synapsis takes place precociously in htp-1(gk174) mutants,
due to SYP-1 loading onto immature chromosome axes; and interestingly, CCT-4vv39 is
also able to rescue the homolog alignment defects in this mutant. It is possible CCT4vv39 rescues the alignment defects by delaying axes morphogenesis and thus stops the
precocious loading of SYP-1. Homologous chromosomes would then be able to align
without the interference of premature synapsis. These results are consistent with the
hypothesis that CCT-4 mediates timely axes morphogenesis through folding of axes
component HIM-3.
This study is the first to provide insight on the function of molecular chaperonin in
mediating meiotic processes and opens a completely new area of research. It would be
interesting in the future to further study the nuclear CCT chaperonin complex and its
clients to learn more about their roles during meiotic prophase.
7
Résumé
La méiose est un processus spécialisé qui permet la production de gamètes
haploïdes à partir de cellules diploïdes. La ségrégation des chromosomes homologues
durant la méiose I dépend de l'alignement initial de chromosomes, la stabilisation de
cette alignement par la synapse et la formation de chiasmas entre les homologues. Des
études antérieures ont démontré que HIM-3, un élément des axes chromosome
méiotique, est requis pour ces processus par le recrutement des protéines autosomique
centre de liaison (ZIMS) et de la composante synaptonémal complexe (SYP-1). Le him3(vv6) mutation correspond à la substitution d'un acide aminé très conservé du domaine
HORMA, considérés comme des médiateurs des interactions protéine-protéine. HIM-3
dans vv6 germlines semble être normale et la protéine est chargée aux axes des
chromosomes, mais him-3(vv6) mutants présentent des défauts dans l'alignement
homologue autosomique, dans la progression du cycle cellulaire méiotique indiqué par
l'extension des stades leptotène/zygotène, en non homologue synapsis, dans la
progression de la recombinaison et la formation des chiasmas. En him-3(vv6) mutants,
la morphologie du chromosome axes semble être immature et non contigus au début
méiose. En outre, le ZIMS ne parviennent pas localiser à centres de liaison, mais la
protéine du centre de liaison du chromosome X HIM-8 reste inchangée. Ceci indique
que la présence d'une protéine du centre est suffisante pour initialiser synapsis partout,
quel que soit l'homologie.
L'objectif de cette étude est de mieux comprendre le mécanisme qui régle
l'alignement des chromosomes et synapse. Un crible d'EMS a été réalisé avec him-3(vv6)
allèle pour identifier d'autres facteurs qui réglent l'alignement et la formation de la
8
synapse entre chromosomes homologues. Quatre suppresseurs dominants (vv38, vv39,
vv41 et vv50) et un suppresseur de semi-dominant (vv52) ont été isolés. Tous les
mutants suppresseurs ont montré une region leptotène/zygotène moins étendue par
rapport à him-3(vv6), indiquant qu'ils ont une meilleure progression du cycle cellulaire
méiotique. vv38 a été identifié comme un suppresseur intragénique, avec la mutation
trouve aussi dans le domaine HORMA. vv52 a été localisé sur un intervalle fini sur le
chromosome II, et vv39 a été identifié comme un nouvel allèle de cct-4, qui code pour la
sous-unité delta de type II chaperonin complexe. En germlines de type sauvage, CCT-4
est localisée dans le cytoplasme et dans le noyau, ce qui indique qu'il a un rôle nucléaire.
En outre, une partie de la CCT-4 se trouve à colocalisées avec la chromatine au cours du
leptotène/zygotène, ce qui suggère qu'il a une fonction à des stades d’alignement des
chromosomes.
Dans cct-4(RNAi) germlines, la morphologie du chromosome axes
semble être non contigus, ZIM-3 ne parvient pas être recrutés à des centres de liaison et
SYP-1 ne parvient pas localiser à axes, ce qui indique que CCT-4 est requis pour les
axes morphogenèse, le chargement de ZIMS et la formation du complexe synaptonémal.
RNAi contre deux autres sous-unités complexes chaperonine CCT-1 et CCT-3 a
également montré des défauts de la morphogenèse axes et de la formation du complexe
synaptonémal, indiquant que CCT-4 fonction peut être médiée par le complexe
chaperonine. Dans cct-4(vv39) mutants, la morphologie des chromosomes axes est
immatures à début leptotène, mais le recrutement des ZIMs et la formation de la synapse
sont soutenue à des stades ultérieurs de la méiose.
Dans him-3(vv6); cct-4(vv39)
mutants, la morphologie des axes des chromosomes est rétabli et la localisation des
ZIM-3 semble également être normale, ce qui entraîne la suppression d'autres défauts
9
ultérieurs, comme l'alignement des chromosomes et non homologue synapsis. Il a été
montré précédemment que la synapse a lieu précocement dans htp-1(gk174) mutants, en
raison de SYP-1 chargement sur les axes chromosome immature et CCT-4vv39 est aussi
capable de sauver les défauts d'alignement homologue chez ce mutant. Il est possible
CCT-4vv39 sauve les défauts d'alignement en retardant la morphogenèse axes et s'arrête
donc le chargement précoce de SYP-1. Chromosomes homologues serait alors en mesure
d'aligner sans l'intervention de synapsis prématurée. Ces résultats sont cohérents avec
l'hypothèse que la morphogenèse du chromosome axes est réglée par CCT-4 en pliage de
HIM-3.
Cette étude est la première à donner un aperçu sur la fonction de chaperonine
moléculaire dans la médiation des processus méiotique et ouvre un domaine entièrement
nouveau de la recherche. Il serait intéressant à l'avenir d'approfondir l'étude du complexe
chaperonine CCT nucléaire et de ses clients pour d’apprendre davantage sur leur rôle au
cours de la prophase méiotique.
10
Acknowledgments
First of all I would like to thank my supervisor Dr. Monique Zetka for giving me an
opportunity to pursue my Ph.D. degree in her lab. I was only a new graduate with a
Bachelor Degree when I joined her team. She taught me a lot of new techniques in
genetics and in molecular biology. Despite my experiments did not work all the time,
she always remained positive and encouraged me to try different approaches. She was
very patient with me even though I did not always explain myself clearly. She always
had a lot of great ideas in her mind and was very good in explaining them. She also
contributed ideas to this thesis as well as the editing. Secondly I would like to thank my
mother, who unconditionally stayed by my side for the past few years of my study.
Things did not always go well in the past few years, either at work or at home. But no
matter what happened, she would stand by me to give me a lot of encouragement and
support. I am also very thankful to the past and present members of Zetka lab. Paul
Bent and Will Goodyer, who were undergraduate students when I first joined the lab,
taught me a lot of techniques about how to properly handle C. elegans and showed me
around the Stewart Biology Building. Brent McGranth, who was our technician at that
time, showed me how to make plates and solutions. He also helped me when I was
doing the EMS mutagenesis. Malek Jundi, Melanie Mah and Noemie Riendeau, who
joined the lab when I was doing the screen, helped me in various tasks including picking
worms and doing the initial linkage group analysis. Dr. Susi Kaitna, who was the postdoc in our lab, helped me solved a lot of scientific problems. Yvonne Quan, who was a
Master student, made the lab livelier and also gave me a pet turtle. Dr. Florence Couteau,
Dr. Buget Saribek, Sara Labella, Aleksandar Vujin, Jasmin Hanafi, Marvra Nasir, Sam
11
Shinn, who are the present lab members, helped me with lot of things and gave me
valuable comments and information regarding my project. I also need to thank them for
contributing ideas and suggestions for this thesis. I would like to thank my supervisory
committee members: Dr. Richard Roy and Dr. Jackie Vogel, who provided me guidance
and lot of wonderful ideas for my project. I would like to thank members of the Roy lab
for useful discussions and ideas during our weekly lab meeting. I would like to thank
the Hekimi lab, the Roy lab, the Fagotto lab, the Schöck lab, the Nilson lab and the Dent
lab for kindly sharing their equipments and reagents. I am very thankful to Dr. Steven
Jones and his group for doing the Illumina Sequencing and the bioinformatics. I would
like to thank Anne Villeneuve’s and Adrianna LaVolpe’s lab for sharing the antibodies. I
would also like to thank the Caenorhabditis Genetics Center for providing me with
certain C. elegans strains. Lastly I would like to thank NSERC and CIHR for supporting
my project.
12
Chapter I:
Literature Review
13
Overview of meiosis
Meiosis plays a critical role in all sexually reproducing organisms by accurately
reducing the ploidy of diploid cells in half to generate haploid gametes. In contrast to
mitosis where DNA replication is followed by segregation of the resulting sister
chromatids to opposite poles, during meiosis a single round of DNA replication is
followed by segregation of homologous chromosomes during two successive rounds of
cell division, meiosis I (MI) and meiosis II (MII).
The reduction of chromosome
number relies on the segregation of homologous chromosomes at meiosis I, which
depends on the earlier events during meiotic prophase I: pairing of homologous
chromosomes, synapsis (marked by the formation of the synaptonemal complex; SC),
and recombination that leads to the formation of crossovers, cytologically evident as
chiasmata (reviewed by Zickler and Kleckner, 1998; 1999). At metaphase I, homologs
align at the equatorial plate and sister chromatids coorient to face the same pole; correct
biorientation relies on chiasma formation between the homologs (reviewed by Page and
Hawley, 2003). At anaphase I, homologous chromosomes segregate from one another,
while sister chromatids remain connected to each other at their centromeres; anaphase II
resembles a mitotic division in that sister kinetochores face opposite poles and sister
chromatids are segregated. Completion of the meiotic divisions results in the production
of four haploid daughter cells and because of crossovers, the daughter cells are not
genetically identical to the parental cell, resulting in genetic diversity. Although meiosis
is a well-studied process, how the homologs initially recognize each other and align, and
how such alignment is coordinated with synapsis and recombination are still not fully
understood. Understanding the mechanisms that govern meiotic chromosome
14
segregation is clinically important because aneuploidy and polyploidy are major causes
of human miscarriage and developmental abnormalities (Robison et al., 2001), and
studies of the recombination process contribute to the understanding of DNA repair
mechanisms and the maintenance of genome integrity (reviewed by Thorslund and West,
2007).
Meiotic prophase events
The first meiotic division is distinguished by four interlinked processes:
chromosome pairing, synapsis, recombination, and segregation of homologous
chromosomes (reviewed by Walker and Hawley, 2000), and successful segregation
depends on the earlier events. Meiotic prophase is divided into five stages: leptotene,
zygotene, pachytene, diplotene and diakinesis, that have been defined by chromosome
morphogenesis and by chromosome behaviors (reviewed by Roeder, 1997; reviewed by
Zickler and Kleckner, 1998; reviewed by Cohen and Pollard, 2001) (Table 1.1). Correct
chromosome morphogenesis is required for meiotic chromosome pairing and
recombination and is initiated during premeiotic S phase. (reviewed by Zickler and
Kleckner, 1998). During pre-meiotic S phase, newly replicated sister chromatids are
linked together by the conserved cohesin protein complex (reviewed by Hagstrom and
Meyer, 2003; reviewed by Uhlmann, 2004). In budding yeast, this cohesin complex
consists of four core components that are highly conserved in eukaryotes: Smc1 and
Smc3, two members of the structural maintenance of chromosome (SMC) family; Scc3
(reviewed by Revenkova and Jessberger, 2005; reviewed by Nasmyth, 2005); and a
member of the kleisin family specific for meiosis called Rec8 that replaces the mitotic
15
counterpart Scc1 (Klein et al., 1999; Watanabe and Nurse, 1999). Rec8 is cleaved by
separase at anaphase I (Buonomo et al., 2000) and is required for proper reductional
chromosome segregation in budding yeast (Watanabe and Nurse, 1999). Rec8 homologs
have also been reported in other eukaryotes and carry out a similar function (Parisi et al.,
1999; Pasierbek et al., 2001). Establishment of meiotic sister chromatid cohesion is
required for the formation a proteinaceous structure known as the axial element
(reviewed by Zickler and Kleckner, 1998). Axial element formation is a cytological
characteristic of leptotene chromosomes, and was initially identified by electron
microscopy as a structure that forms along the length of sister chromatids before
synapsis takes place (Comings and Okada, 1970). Some of the protein components of
the axial element have been identified, including Red1 in S. cerevisiae and HIM-3 in C.
elegans (Smith and Roeder, 1997; Zetka et al., 1999). Similar to other organisms, in C.
elegans sister chromatid cohesion is required for axial element formation since in both
rec-8 and scc-3 mutants, HIM-3 fails to localize (Pasierbek et al., 2001; Pasierbek et al.,
2003).
During leptotene and zygotene stages, homologous chromosomes recognize each
other and align along their lengths at a distance; this arrangement is stabilized by
synapsis at pachtytene (reviewed by Zickler, 2006).
In both C. elegans and D.
melanogaster, homologue pairing and synapsis are independent of recombination and its
initiation (McKim et al., 1998; Dernburg et al., 1998). However in other organisms
such as S. cerevisiae, proper homologue alignment and synapsis are dependent on the
formation of meiotic double-strand DNA breaks (DSB) during recombination initiation
(Peoples et al., 2002).
Synapsis is defined by the formation of the synaptonemal
16
complex (SC), consisting of two lateral elements that are derived from axial elements;
chromatin loops are anchored to the two lateral elements, which are connected together
by transverse filaments in the central region of the SC to form a tripartite structure
(reviewed by Roeder, 1997; reviewed by Page and Hawley, 2003; Reviewed by Page and
Hawley, 2004) (Figure 1.2). Transverse filament/central region components have been
identified and characterized, including Zip1 in S. cerevisiae, SCP1 in rats, Syn1 in mice
and human, and SYP-1 in C. elegans (Meuwissen et al., 1992; Sym et al., 1993; Dobson
et al., 1994; Liu et al., 1996; MacQueen et al., 2002). Components of the lateral/axial
elements include Cor1 in hamsters and SCP3 in rats (Dobson et al., 1994; Lammers et
al., 1994). The SC is thought to function as a zipper that binds the homologous
chromosomes together (reviewed by Cohen and Pollard, 2001) and plays a role in
promoting interhomolog exchange.
Mutations in S. cerevisiae Zip1 reduce
recombination frequencies by 50-70% (Sym et al., 1993), and crossing over is abolished
in C. elegans syp-1 mutants (MacQueen et al., 2002). However, synapsis per se does
not distinguish homology and the SC can form between nonhomologous DNA sequences.
The first evidence came from light microscopy analysis of maize microsporocytes
heterozygous for different types of chromosomal rearrangements and translocations,
showing full synapsis of all chromosome pairs (McClintock, 1933).
Similar
observations were made in the analysis of reciprocal-translocation heterozygotes in C.
elegans (Goldstein, 1986). Nonhomologous synapsis also occurs in the yeast mutants of
mre11S and hop2 which also exhibit defects in meiotic recombination (Nairz and Klein,
1997; Leu et al., 1998; Tsubouchi and Roeder, 2003), and in C. elegans mutants of him3 and htp-1 that are defective in chromosome pairing (Couteau et al., 2004; Couteau and
17
Zetka, 2005).
In budding yeast, meiotic recombination is initiated by the formation of DSBs that
requires the products of at least 10 genes: SPO11, MRE11, RAD50, XRS2, MER2,
MRE2, MEK1, ME14, REC104, REC114, and REC102 (Alani et al., 1990; Cao et al.,
1990; Bharagava et al., 1992; Ivanov et al., 1992; Menees et al., 1992; Johzuka and
Ogawa, 1995; Ogawa et al., 1995; Rockmill et al., 1995; Bullard et al., 1996). Spo11, a
highly conserved type II topoisomerase, is required for meiotic DSB formation
(reviewed by Villeneuve and Hillers, 2001), but a conserved role for other genes is less
clear. Once DSBs are formed, the 5’ ends of the breaks are degraded to expose the 3’end single stranded DNA (ssDNA) tails; these tails then invade the intact template to
prime DNA synthesis and initiate repair of DSBs with homologous sequences during
homologous recombination (reviewed by Villeneuve and Hillers, 2001). The ssDNA
invasion is catalyzed by two proteins that are closely related to bacterial RecA, Rad51
and Dmc1 (reviewed by Masson and West, 2001). While Rad51 also functions in DSB
repair in somatic cells, Dmc1 is present exclusively in meiosis (reviewed by Masson and
West, 2001).
Interestingly, the Dmc1 gene is absent in both C. elegans and D.
melanogaster, raising the possibility that Rad51 in these two organisms may be modified
to retain some Dmc1-like characteristic (reviewed by Villeneuve and Hillers, 2001).
DNA synthesis and strand displacement lead to the formation of double Holliday
junctions, which are in turn resolved into both crossover and non-crossover
recombination products (reviewed by Kunz and Schär, 2004). In many organisms such
as yeast, mouse and C. elegans; the crossover outcome is promoted by two members of
MutS DNA mismatch-repair family, Msh4 and Msh5 (Ross-Macdonald and Roeder,
18
1994; Hollingsworth et al., 1995; Colaiácovo et al., 2003; Hoffmann and Borts, 2004;
Kolas and Cohen, 2004). These proteins function as a heterodimer and are thought to
recognize and stabilize a nascent recombination intermediate, allowing recruitment of
additional factors and resolution along the crossover pathway (reviewed by Kunz and
Schär, 2004). Meiotic crossovers do not occur close to each other and are almost always
widely spread apart, a phenomenon known as crossover interference that has been
proposed to require the SC (reviewed by Bishop and Zickler, 2004). Recent studies have
suggested that interference does not strictly require SC as it can occur in the absence of
Zip1 in budding yeast, a component required for SC formation (Fung et al., 2004).
Instead, the crossover designation event in this organism is thought to precede SC
assembly and initiate an interference signal that is transmitted along the chromosome
axes, blocking nearby crossover designations as the signal travels (reviewed by Bishop
and Zickler, 2004). C. elegans is proficient in inhibiting multiple crossovers as the vast
majority of homologs in this organisms experience only one crossover per meiosis
(reviewed by van Veen and Hawley, 2003).
The complex events occurring during meiotic prophase are monitored by
checkpoints. In budding yeast, mutants that are defective in meiotic recombination and
chromosome synapsis undergo checkpoint-induced arrest at pachytene (Roeder and
Bailis, 2000). In mammals, two checkpoints may exist; one responding to synapsis
failure and the other responding to unrepaired DSBs (Odorisio et al., 1998; Giacomo et
al., 2005). In C. elegans, evidence from a number of studies suggests the existence of
two distinct checkpoints: a synapsis checkpoint that monitors synapsis of chromosomes
(Bhalla et al., 2005), and a recombination checkpoint that is activated by damage DNA
19
or by unrepaired meiotic recombination intermediates (Gartner et al., 2000). While the
synapsis checkpoint monitors the presence of synapsis and induces apoptosis in late
pachytene nuclei, there is no known checkpoint that monitors synapsis between
nonhomologous chromosome in the yeast, in the mouse and in the nematode. For
example, in both S. cerevisiae and mouse, hop2 mutants show an extensive amount of
nonhomologous synapsis and undergo checkpoint-mediated arrest at pachytene, but this
checkpoint is triggered by the failure of repairing meiotic double-strand breaks and not
by the nonhomologous synapsis itself (Petukhova et al., 2003; Tsubouchi and Roeder,
2003). Studies in C. elegans have suggested the existence of a chromosome axis-based
surveillance system that prevents nonhomologous synapsis by inhibiting the loading of
SC components onto the chromosome axis before homologous pairing is properly
established (Couteau and Zetka, 2005). HTP-1 is a component of meiotic chromosome
axis and in htp-1 mutants, initial pairing of homologous chromosomes is severely
impaired, but nevertheless SC formation is not inhibited and synapsis occurs between
nonhomologous chromosomes (Couteau and Zetka, 2005; Martinez-Perez et al., 2005).
This result suggests that HTP-1 functions in coordinating pairing and synapsis by
transmitting a “wait synapsis” signal that may target SYP-1 or other SC components by
sequesting them or by preventing the premature loading of SC components before
pairing between homologous chromosomes is properly established (Martinez-Perez et
al., 2005).
Meiotic chromosome pairing
The process by which homologous chromosomes find each other and pair during
20
meiotic prophase remains largely mysterious in most organisms. In C. elegans and D.
melanogaster, pairing is promoted by specific sites or regions that are comprised of
repetitive sequences (McKim et al., 1993; Zetka and Rose, 1995; McKee et al., 2000;
reviewed by Page and Hawley, 2003). One of the best characterized pairing sites is a
240 base pair repeat sequence found in the heterochromatin of X and Y chromosomes of
D. melanogaster that facilitates pairing and segregation of the sex chromosomes in
males (McKee, 1996). In C. elegans, studies of chromosome rearrangements have
shown that a single site near the end of each chromosome is required to promote pairing,
synapsis and recombination along the length of the chromosome (McKim et al., 1988;
reviewed by Zetka and Rose, 1995); these sites are known as the homologue recognition
regions (HRR) or pairing centers (PCs). The PC of each of the six chromosomes in C.
elegans is bound by one of four zinc-finger proteins: ZIM-1 (Chromosomes II and III),
ZIM-2 (Chromosome V), ZIM-3 (Chromosome I and IV), and HIM-8 (Chromosome X)
(Phillips et al., 2005, Phillips and Dernburg, 2006). Mutants in the zim genes and him-8
result in the disruptions of pairing and synapsis specific for their corresponding
chromosomes, indicating that these proteins are required for PC function (Phillips et al.,
2005; Phillips and Dernburg, 2006). Recently a number of repetitive sequence motifs
have been identified bioinformatically in the genetically defined PC regions (Sanford
and Perry, 2001) and these motifs have been shown to be able to recruit PC proteins in
vivo (Phillips et al., 2009). Furthermore, insertion of these motifs into a chromosome
lacking the endogenous PC is sufficient to restore pairing, synapsis and recombination
(Phillips et al., 2009).
In addition to the PC and associated proteins, chromosome axes also play a role
21
in promoting pairing. In C. elegans, HIM-3, HTP-1 and HTP-3 are all components of
axes and their disruption results in defects in alignment, synapsis and recombination
(Zetka et al., 1999; Couteau and Zetka, 2005; Goodyer et al., 2008). In budding yeast,
Red1 and Hop1 are components of the axial element and are also required for
homologous chromosomes pairing (Hollingsworth and Byer, 1989; Hollingsworth et al.,
1990; Smith and Roeder, 1997). Studies in rice have shown that the axial element
component PAIR3 is required for bouquet formation, pairing, recombination and SC
formation (Wang et al., 2010).
Together these results suggest that axes provide a
structural platform that facilitates pairing between homologous chromosomes.
Chromosome movement during meiotic pairing stages
The homolog pairing process is accompanied by dramatic meiotic chromosome
movements that have been widely observed in different organisms including, R.
norvegicus (Parvinen et al., 1976), S. pombe (Chikashige et al., 2007), S. cerevisiae
(Trelles-Sticken et al., 2000), and C. elegans (Sato et al., 2009; Penkner et al., 2009;
Baudrimont et al., 2010). During early meiosis, telomeres are anchored within the inner
surface of the nuclear envelope and clustered together within a limited area, a
phenomenon known as chromosomal bouquet formation (reviewed by Zickler and
Kleckner, 1998). The mechanistic basis for bouquet formation is not known, and a few
organisms exhibit a variant situation; for example in C. elegans, only one end of each
chromosome is attached to the nuclear envelope (Goldstein, 1986).
The proposed
function of bouquet formation is to promote homologue alignment by bringing all
chromosomes together within a finite area, thus spatially restricting the area for the
22
homology search (reviewed by Zickler, 2006). Bouquet formation has been studied
extensively in S. pombe where the telomeres are linked to the spindle pole body (SPB)
and the cytoplasmic microtubule network through a protein bridge consisting Sad1p and
Kms1p (reviewed by Starr and Fisher, 2005). Sad1p is an inner nuclear envelope protein
that contains a SUN domain that recruits Kms1p to the outer nuclear envelope by
interacting with its C-terminal KASH domain, thereby enabling its cytoplasmic
extension to tether chromosomes to cytoplasmic forces (reviewed by Starr and Fisher,
2005). A microtubule and dynein motor protein driven “horsetail” movement plays an
important role in facilitating recombination and alignment between homologous
chromosomes (reviewed by Sawin, 2005). In C. elegans, dynein also plays an important
role in homolog alignment and synapsis; in dynein-depleted animals, alignment kinetics
are disrupted, the SC fails to polymerize between paired homologous chromosomes,
indicating that dynein is required for synapsis initiation between homologs that have
paired (Sato et al., 2009). During meiosis, the C. elegans KASH/SUN proteins ZYG-12
and SUN-1, colocalize and form patches at the nuclear envelope during chromosome
pairing stages (Penkner et al., 2007) which are highly dynamic and movementcompetent (Baudrimont et al., 2010). ZYG-12 interacts with cytoplasmic dynein in yeast
two-hybrid and SUN-1 is required for ZYG-12 recruitment (Malone et al., 2003).
Cytological studies have shown that HIM-8 and ZIM-3 overlap with SUN-1 aggregates
during leptotene and zygotene stages, demonstrating that the PC ends of the
chromosomes localize to the nuclear periphery at the sites of SUN-1 aggregation and
these sites are likely represent the chromosome attachment points to the nuclear
envelope (Penkner et al., 2007; 2009; Sato et al. 2009). It has also been shown that
23
proper chromosome axis assembly is required for the wild-type PC protein localization
and for formation of SUN-1 aggregates (Penkner et al., 2009; Baudrimont et al., 2010).
The current model suggests that local enrichment of SUN-1 and ZYG-12 in the
vicinity of PC ends of chromosomes during early meiosis provides a platform for
connecting the chromosome ends to the cytosolic microtubule network. Through this
connection, the force generated by the microtubules is used for the chromosome
movement that facilitates homolog alignment and prevents nonhomologous synapsis. It
is proposed that homology is assessed at PC ends in the context of dynamic SUN-1
patches; when the right partner is found, synapsis is initiated. Through this highly
dynamic process homologous chromosome are brought together while nonhomologs are
separated (Baudrimont et al., 2010) (Figure 1.3). Evidence for this model was first
revealed by a missense mutation in the SUN domain of SUN-1 that disrupts localization
of ZYG-12; sun-1(jf18) mutants fail to form SUN-1 or ZYG-12 patches and exhibit
defects in chromosome movement that are accompanied by defects in homolog pairing
and nonhomologous synapsis (Penkner et al., 2007; Baudrimont et al., 2010). Meiosisspecific modification of the N-terminus of SUN-1 has also been found to occur; 7
phosphorylated serine sites (Ser8, Ser12, Ser24, Ser35, Ser43, Ser58, and Ser63) and 1
threonine (Thr36) have been identified (Penkner et al., 2009). Cytological studies using
antibodies against the phosphoepitope Ser8 (S8-Pi) showed that it is localized in foci and
patches on the nuclear envelope during leptotene and zygotene, and the signal gradually
becomes weaker and eventually disappears at late pachytene; Ser12 and Ser24 also
exhibit similar phosphorylation pattern (Penkner et al., 2009). The phosphorylation of
SUN-1 at these sites corresponds to the time window of homology search when SUN-1
24
aggregates are formed, suggesting that SUN-1 phosphorylation has a role in patch
formation and homologue pairing (Penkner et al., 2009).
HIM-3 family functions during meiosis
The C. elegans him-3 gene encodes a meiosis-specific protein that localizes to
chromosome axes from the onset of meiotic prophase until the metaphase I to anaphase I
transition; it is essential for homologue alignment, spatial reorganization of germline
nuclei, synapsis, and chiasmata formation (Couteau et al., 2004, Zetka et al., 1999).
Furthermore, SUN-1 patches are not properly formed at early meiosis in him-3 null
allele gk149, likely due to the failure to appropriately localize PC proteins (Penkner et al,
2009). Despite these defects, him-3(gk149) mutants show wild-type levels kinetics of
appearance and disappearance of RAD-51-marked early recombination intermediates
(Couteau et al., 2004).
Since homologous chromosomes are not available as
recombination partners, this observation suggests that sister chromatids are used as
repair templates in the absence of HIM-3.
This result implies that HIM-3 is a
component of the barrier to using sister chromatids as recombination partners and
functions in establishing the preference for non-sister chromatids as templates for repair
(Couteau et al., 2004).
Alignment of proteins functioning in synapsis, DNA repair and mitotic
checkpoints in yeast has revealed the presence of a common structural domain, the
HORMA domain (for Hop1p, Rev7p, and Mad2p, Figure 1.4) that encompasses the
majority of the HIM-3 sequence (Aravind and Koonin, 1998). Hop1p is a component of
the axial element that is essential for pairing, synapsis and recombination of meiotic
25
chromosomes (Hollingsworth and Byer, 1989; Hollingsworth et al., 1990); Rev7p is a
subunit of the yeast DNA polymerase involved in DNA sythesis (Nelson et al., 1996);
and Mad2p is a component of the spindle assembly checkpoint and is conserved in yeast
and vertebrates (Li and Benezra, 1996; He et al, 1997). The precise function of the
HORMA domain is still unclear but it is believed to be an adaptor domain involved in
mediating protein-protein interaction (Aravind and Koonin, 1998). Recent studies have
identified two new HORMA-domain containing proteins in mouse called HORMAD1
and HORMAD2; similar to HIM-3 they are localized to the chromosome axes during
leptotene/zygotene, and HORMADs deficiency results in disruption of a number of
meiotic processes including SC formation, recombination and segregation (Fukuda et al.,
2009; Wojtasz et al., 2009; Shin et al., 2010).
Two hypomorphic alleles of him-3, me80 and vv6 result in a substitution of a highly
conserved residue of the HORMA domain (Figure 1.4). HIM-3 levels are decreased in
him-3(me80) mutant germlines, suggesting that protein is unstable; however in him3(vv6) mutant germlines, HIM-3 levels are not detectably altered and the protein is
loaded normally to chromosome axes (Couteau et al., 2004) (Figure 1.5). Despite this,
him-3(vv6) mutants still exhibit severe defects in homologue alignment, synapsis, and
chiasma formation (Couteau et al, 2004); resulting in a high embryonic lethality (emb)
and a him phenotype due to X-chromosome missegegration. In addition, cell cycle
progression is delayed in him-3(vv6) mutants as indicated by an extension of the
leptotene/zygotene stages that is characterized by nuclear polarization, as the
chromosomes are polarized to one end of the nuclei (Francis et al., 1995) (Figure 1.6B).
One possible explanation for this delay is that the homologous chromosomes are
26
defective in chromosome pairing and require more time for the homology search.
Furthermore, vv6 mutant germlines are marked by extensive nonhomologous synapsis
between autosomes, indicating that HIM-3vv6 retains the ability to support SC
polymerization; the level of nonhomologous synapsis reaches 70% of pachytene nuclei
and is thought to contribute to the delay in the disappearance of RAD-51-marked
recombination intermediates (Couteau et al., 2004).
The C. elegans genome encodes three paralogs of HIM-3 in C. elegans, called
HTP-1, HTP-2, and HTP-3 (him-three paralog). Like HIM-3, they contain a HORMA
domain and localize to the chromosome axes during meiosis (Martinez-Perez and
Villeneuve, 2005; Couteau and Zetka, 2005; Goodyer et al., 2008; Martinez-Perez et al.,
2008) (Figure 1.4). HTP-1 and HTP-2 are two highly homologous proteins sharing 82%
protein identity, and less than 30% with HTP-3 and HIM-3 (Couteau and Zetka, 2005).
htp-1 mutants are defective in chromosome pairing and crossover formation, indicating
that it is required for these processes; however despite the pairing defect, synapsis is not
inhibited and forms between nonhomologous chromosomes (Couteau and Zetka, 2005;
Martinez-Perez and Villeneuve, 2005). Cytological studies of htp-1 mutants showed that
the SC component SYP-1 is loaded precociously onto immature chromosome axes
(Couteau and Zetka, 2005). These results suggest that htp-1 is part of the surveillance
system to prevent synapsis from happening until alignment between homologs has been
attained (Martinez-Perez and Villeneuve, 2005; Couteau and Zetka, 2005). In contrast,
depletion of both HTP-1 and HTP-2 causes synapsis to be completely abolished,
suggesting that they have an antagonistic function in SC formation and that HTP-2 is
required for SC assembly (Couteau and Zetka, 2005). HTP-3 interacts with HIM-3 in
27
yeast-two-hybrid screen (M. Zetka unpublished results) and HTP-3 immuoprecipitated
complexes contain HIM-3 (Goodyer et al., 2008), suggesting that the two proteins
physically interact. Consistent with this interaction, HTP-3 is required to recruit HIM-3,
and HTP-1/HTP-2 onto chromosome axes (Goodyer et al., 2008; Severson et al., 2009).
HTP-3 association to the axes is dependent on SCC-3 (Goodyer et al., 2008), but
interestingly, REC-8 and SMC-1 are not detectable in htp-3 mutants, indicating that
HTP-3 is in turn required for cohesin loading during meiosis (Severson et al., 2009).
These results together showed that cohesins and HTP-3 are inter-dependable for their
association with meiotic chromosomes, and together with HIM-3, HTP-1 and HTP-2
form a functional axes that mediates essential meiotic processes. Like HIM-3, HTP-3 is
required for homolog alignment and synapsis (Goodyer et al., 2008). In contrast to him3 null mutants, RAD-51 is not detected in the absence of HTP-3 (Couteau et al., 2004;
Goodyer et al., 2008). However, RAD-51 is recruited to DSBs that are artificially
provided by irradiating htp-3(RNAi) germlines, indicating that DSBs can be efficiently
processed without HTP-3 (Goodyer et al., 2008). This evidence collectively suggests
that HTP-3 has an additional role in recombination initiation at the level of DSBs
formation (Goodyer et al., 2008).
The biological significance of chaperonin complex and its relevance to meiosis
Molecular chaperones are required to assist in the folding of proteins in the cells
(Melki et al., 1997; Bukau and Horwich, 1998; Martin, 1998; Kusmierczyk and Martin,
2001) by recognizing and binding newly synthesized polypeptides to prevent premature
folding and aggregation (Ellis and van der Vies, 1991; Gething and Sambrook, 1992).
28
Defects in this process lead to protein misfolding or formation of protein aggregates that
could have serious consequences such as toxicological diseases: for examples Alzheimer,
Parkinson, and Huntington diseases (Slavotinek and Biesecker, 2001; Stefani et al., 2003;
Broadley et al., 2009). Different chaperone classes are defined by molecular size,
cellular compartment and function (Slavotinek and Biesecker, 2001). There are many
chaperon families, including Hsp100, Hsp90, Hsp70, Hsp60, Hsp40(DnaJ) (Slavotinek
and Biesecker, 2001; Kubota et al., 1995). The Hsp60 class of chaperones is also
referred to as chaperonins, they are complexes that composed of two stacked rings of
symmetrically arranged subunits of approximately 60 kDa each (Slavotinek and
Biesecker, 2001; Bukau and Horwich, 1998). Chaperonins assist newly synthesized
proteins to reach their native forms by binding and folding them inside a large central
channel within each ring (Bukau and Horwich, 1998). Chaperonins are divided into two
groups: Group I chaperonins include GroEL in prokaryotes, Hsp60 in mitochondria, and
Rubisco subunit binding protein (RBP) in plants and require a separate co-chaperonin or
capping molecule for function (Slavotinek and Biesecker, 2001). Group II chaperonins
are found in archaebacteria and eukaryotic cells and are highly related to each other,
sharing up to 40% amino acid identity (Kubota et al., 1995). Unlike the archaebacterial
chaperonins TF55 (Trent et al., 1991) and the thermosome (Phipps et al., 1993) that
consist of only two subunits α and β, (Phipps et al., 1991, Knapp et al., 1994), the
eukaryotic chaperonin containing T-complex protein 1 (CCT, also called TRiC) is made
with up to nine subunits. These multiple subunits are arranged into two stacked rings
and have a central cavity larger than Group I chaperonins (Slavotinek and Biesecker,
2001). Each subunit shares a common architecture: an apical domain for substrate
29
recognition and binding, an intermediate domain and a central domain for ATP binding
(Kusmierc and Martin, 2001). The binding and hydrolysis of ATP are essential for
folding and release of substrate proteins (Martin, 1998).
Pulse-chase analysis has
demonstrated that up to 15% of newly synthesized eukaryotic proteins interact with CCT
(Slavotinek and Biesecker, 2001). CCT is involved in the folding of actin and tubulin in
eukaryotes (Gao et al., 1992; Yaffe et al., 1992; Lundin et al., 2007) and also some key
cell cycle regulators such as Cdc20 and Plk-1 (Camasses et al., 2003, Liu et al., 2005),
indicating that CCT substrates are not limited to cytoskeletal proteins.
Substrate
interaction with CCT is done by subunit-specific and geometry-dependent manner
(Llorca et al., 1999).
Studies on the crystal structure of thermosome from T.
acidophilum have shown that while in open state waiting for substrate binding, the
helical protrusion of the apical domain can assume several context-dependent
conformations, and such structure plasticity may help the interaction between the
chaperonin and various types of substrates (Bosch et al., 2000).
The translocation of proteins with complex folding arrangements across nuclear
pores into the nucleus presumably involves their refolding once imported; however,
nuclear chaperones are still poorly characterized with the exception nucleoplasmin
which promotes nucleosome assembly (Laskey et al., 1978; Earnshaw et al., 1980).
Interestingly, a potential nuclear localization signal similar to the one described for the
nuclear protein NuMa (Yang et al., 1992), is also found in the T-complex protein 1
(TCP-1) (Horwich and Willision, 1993), suggesting that CCT may be localized to the
nucleus. Furthermore, studies in mice have shown that a significant amount of TriC-P5,
a protein that shares extensive homology with TCP-1, is found associated with the
30
nuclear matrix (Joly et al., 1994). However, it is unclear how TriC-P5 is organized in
the nuclear matrix, and whether TriC-P5 is also a part of the complex or is in a
monomeric form (Joly et al., 1994). In addition, a subset of CCT chaperonin is localized
to the nucleus in meiotic rat spermatocytes and associates with condensed chromosomes
(Soues et al., 2003), suggesting a nuclear role. While it has been suggested that CCT
may assist in the folding of players that organize heterochromatin (Soues et al., 2003),
its function inside the meiotic nucleus is essentially unknown.
C. elegans as a model to study meiosis
The nematode system serves as a powerful genetics tool for the study of
chromosome-segregation. C. elegans has two visibly distinguishable genders, males
(XO) and hermaphrodites (XX).
Spontaneous non-disjunction is rare in wild-type
(Hodgkin et al., 1979), and mutations that result in meiotic chromosome missegregation
can be easily identified by a high incidence of male, or him phenotype.
The adult germline is spatiotemporally organized such that the distal end contains
immature mitotically proliferating nuclei germ cells and the proximal end contains
mature gametes (Kimble and Crittenden, 2007). The germline nuclei in the gonad are
matured in a syncytium (Schedl, 1997) and upon meiotic entry, nuclei at different stages
of meiotic prophase can be easily distinguished by DAPI staining (Francis et al., 1995)
(Figure 1.6A), thus making time-course analysis of meiotic processes and cytological
investigations in the germline possible.
Tools and antibodies to specifically monitor homologue alignment, synapsis and
recombination have been developed. Initial homologue alignment can be monitored by
31
fluorescence in-situ hybridization (FISH) using probes that can detect specific
chromosomal loci in nuclei (Dernburg et al., 1998); unpaired homologous chromosomes
are evident as two separated FISH signals while paired homologous chromosomes result
in one or two closely juxtaposed FISH signals.
Alignment between homologous
chromosomes is stabilized by SC formation and peaks at the pachytene stage when
synapsis is completed (MacQueen and Villeneuve, 2001) and almost all nuclei have
paired FISH signals. Antibodies against SUN-1, HIM-8, ZIM-3, HIM-3, HTP-3, HTP1/HTP-2, SYP-1/2 are all available and can be used to monitor PC association with the
nuclear envelope as well as meiotic axes assembly (Zetka et al., 1995; Phillips et al.,
2005, Phillips and Dernburg, 2006; Goodyer et al., 2008; Martinez-Perez et al., 2008;
Penkner et al., 2009). In addition, antibodies against the SYP proteins, such as SYP-1, a
component of the central region of the SC (MacQueen, et al., 2002) can be used to
examine the level and timing of synapsis. Recombination can be monitored using
antibodies against RAD-51; anti-RAD-51 marked early recombination intermediates
appear as chromosomal foci at the beginning of leptotene stage that peak in number at
early pachytene and gradually disappear towards the end of pachytene, thereby
permitting a time-course analysis of the progression of recombination (Colaiacovo et al.,
2003). In cases where mutants are unavailable, gene function can be analyzed using
RNA interference (RNAi) by introducing double-strand RNA (dsRNA) into the worms
by either injecting, soaking or feeding bacteria that engineered to express dsRNA, to
trigger degradation of mRNA resulting in the knock down of corresponding gene leading
to phenotypes associates with loss of protein function (reviewed by Maine, 2008).
Transgenes can be expressed by Mos1-mediated single copy insertion (MosSCI), a
32
recently developed technique designed to insert a single copy of the transgene into a
specific site that provides efficient expression in tissues that frequently silence
transgenes, including the germline (Frokjaer-Jenson et al., 2008).
Rationale for study
Despite many years of previous studies, the mechanistic basis of how homologous
chromosomes initially find each other, and how alignment and synapsis are coordinated
are still not fully understood. In him-3(vv6), HIM-3 localization and expression level are
normal, but nevertheless the initial alignment is impaired and synapsis occurs between
nonhomologous chromosomes, indicating that alignment and synapsis are decoupled in
this mutant. In this study, I use him-3(vv6) as tool to identify factors regulating SC
formation between homologous chromosomes and functioning in HIM-3-dependent
processes. The goal of this study is to further understand the mechanisms regulating
chromosome pairing and synapsis.
33
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44
Appendix for Chapter I
45
STAGE
CHROMOSOME AND SC MORPHOLOGY
BOUQUET FORMATION
leptotene
axial elements form
telomeres begin to cluster
zygotene
chromosome synapsis initiates
telomeres tightly clustered
pachytene
chromosome fully synapsed
telomeres disperse
diplotene
SC disassembled
diakinesis
further chromosome compaction
Table 1.1: Chromosome and SC morphology during different stages in meiotic prophase I. Adapted from the review by Roeder,
1997.
(from Page and Hawley, 2004)
Figure 1.2: Diagram of the synaptonemal complex. LE: lateral elements. CE: central element. The four slinkys
represent chromatin.
A.
B.
(Adapted from Baudrimont et al., 2010)
Figure 1.3: Schematic representation of chromosome ends shuffling between SUN-1 aggregates. A) Chromosome axes (green)
supports the binding of PC proteins that connects chromosome ends to SUN-1/ZYG12 to bridge chromosome to cytoplasmic forces
(orange arrows) used to move chromosomes. B) When the homologous chromosomes are found, synapsis overcomes the cytoplasmic
forces and synapsis can be established. However when nonhomologous chromosomes are encountered, cytoplasmic forces overcome
the attempt to synapse and shuffling continues until all the homologs find their partners.
gk149
vv6(S→F)
me80(R→A)
HIM-3-Ce
HTP-1-Ce
HTP-2-Ce
HTP-3-Ce
Hop1p-Sc
Mad2p-Sc
Rev7p-Sc
---MATKEQIVEHRESEIPIASQWK--ATFPVDLEIEKNSEMFALRYIKCASAFILDRRGILDEKCFKTR-TIDKLLVTAFQSSVPA
MAPLETIYDESLNKSADSIDDKKWS--KLFPRIVSDPDRSSNFMTRAIYVAFSAVLRNRNILGQEYFTKNYITEKLKCMTLCFRNPR
MAPLENNYNESLNKSKDAIDDKTWS--KLFPSIVSDPDRSSNFMIRAIYVVFSAVLRQRNILEKEYFSKNYITENLSCMTLSFKNLR
---MDESFDSSVVPGSLTSDDRAIFNEQTLKNGDENSKSSLEVMANCVYLANSTILRERKVIPAEYFQDFQVYGDVSGYTLRQDIPE
-----MSNKQLVKPKTETKTEIT-------------TEQSQKLLQTMLTMSFGCLAFLRGLFPDDIFVDQRFVPEKVEKNYNKQNTS
-----MS-—QSISL-----------------------KGSTRTVTEFFEYSINSILYQRGVYPAEDFVT------VKKYDLTLLKTH
-----------------------------------MNRWVEKWLRVYLKCYINLILFYRNVYPPQSFDYT----TYQSFNLPQFVPI
81
85
85
84
69
51
48
Figure 1.4: N-terminal portion of the HORMA domain inside HIM-3. ClustalW multiple alignment of HORMA-domain containing
proteins showing the N-terminal portion of the HORMA domain (underlined in black). htp-1, htp-2 and htp-3 are three him-3 paralogs in C.
elegans. The predicted amino acids substitutions him-3(vv6) and him-3(me80) are shown by arrows and the amino acids deleted in the him-3
null allele gk149 are underlined in red. Ce, C. elegans and Sc, S. cerevisiae.
DAPI
HIM-3
WT
him-3(vv6)
Figure 1.5: HIM-3vv6 localizes to meiotic chromosome axes. Immunofluorescence micrographs
showing DAPI and HIM-3 antibody staining of pachytene nuclei in wild-type and him-3(vv6) germlines.
Scale bar, 5µm.
A
Mitotic/
Premeiotic
B
Late
Pachytene
WT
Early
Pachytene
LeptoteneZygotene
(Transition
Zone)
Mid
Pachytene
Diakinesis
Diplotene
him-3(vv6)
Figure 1.6: him-3(vv6) mutants have a meiotic cell cycle delay. DAPI-stained hermaphrodite
germlines of the indicated genotypes showing the nuclei at different stages of meiotic prophase. A)
wild-type hermaphrodites showing the asymmetrically positioned chromatin cluster typical of
leptotene-zygotene stages. B): him-3(vv6) mutant germlines show an extended region (red line) in
which nuclei exhibit chromosome clustering, indicating a delay in progression through
leptotene/zygotene. Scale bars, 5µm.
Chapter II:
Isolation of him-3(vv6) Suppressors
46
Summary of Chapter II
This chapter will cover the EMS-based suppressor screen using the him-3(vv6)
alleles and the identification and preliminary characterization of the suppressor
candidates. I screened for candidates that suppressed the embryonic lethality phenotype
of him-3(vv6) as indicated by an increase in the progeny number. From the screen, I
have isolated 4 dominant (vv38, vv39, vv41, vv50) suppressors and 1 semi-dominant
(vv52) suppressor. vv38 was identified as an intragenic suppressor that affects another
residue inside the HORMA domain, while vv39 and vv52 are both extragenic. In these
suppressor strains, the number of progeny increased 3- to 6-fold, suggesting that the
autosomal nondisjunction phenotype of vv6 mutants is rescued. Furthermore, these
suppressor strains exhibit varying levels of X chromosome non-disjunction, suggesting
that the suppressors differentially affect the segregation of the sex chromosome. DAPI
staining of the germline nuclei shows a less extended leptotene/zygotene region in the
suppressor strains, indicating that meiotic cell cycle progression is better compared to
him-3(vv6). Furthermore, there is a rescue of chiasma formation defects in both vv39
and vv52 suppressor mutants as shown by the presence of more bivalents at diakinesis.
There are no embryonic lethality and him phenotypes associated with the vv39 or vv52
mutations alone, suggesting that these mutations do not cause a complete functional
disruption of their corresponding genes.
47
Materials and Methods
EMS mutagenesis/screening
EMS mutagenesis was performed according to the standard procedures (Brenner S,
1974). him-3(vv6)unc-24(e138) hermaphrodites of L4 stage were treated with 25mM
EMS (Sigma) in M9 for 4 h at 20 °C. Worms were washed 4X in M9 media, and plated
onto seeded NGM plates. Staged collections were taken of the F1 generation, and these
were plated onto NGM plates at 20 °C. Staged L4 collections of the F2 generation were
plated onto NGM plates and grew until adulthood. They were allowed to lay eggs and
the progeny numbers were screened. Candidates were outcrossed three times with wildtype males to eliminate background mutations generated by EMS.
Scoring of brood size
Staged L4 F2 generation was plated individually onto NGM plates, transferred
daily for three days, and their total progeny numbers were scored. Individuals exhibiting
a significantly increased brood size compared to him-3(vv6) were isolated and retested.
Cytological Preparation of gonads and staining
To stain chromatins, hermaphrodite gonads were dissected 20-24h post L4 stage in
M9 and fixed with 3% paraformaldehyde for one hour. The samples were washed with
M9 three times, and were mounted I Vectashield antifading medium (Vector
Laboratories, Burlingame, CA) containing 2µl/ml 4’,6-diamidino-2-phenylindole
(DAPI).
48
Measuring the length of transition zone
Germline nuclei were first stained with DAPI as mention above. Pictures of the
entire gonad were then taken and an entire image of the gonad was assembled using
Photoshop 6.0. Transition zone nuclei have distinct polarized appearance (Francis et al.,
1995). The region extending between the first polarized nucleus and the last polarized
nucleus was measured to obtain the length of transition zone.
Linkage analysis
Six strains were constructed in which him-3(vv6) was coupled to a visible dumpy
(dpy) marker that corresponds to each of the six chromosomes: dpy-5(e61) for
chromosome I, dpy-2(e8) for chromosome II, dpy-17(e164) for chromosome III, dpy13(e184) for chromosome IV, dpy-11(e224) on chromosome V, and dpy-3(e27) for
chromosome X. Each suppressor line was then crossed with all of these six strains to
determine the linkage group of the suppressor. If the suppressor and the dpy marker did
not map to the same chromosome, a quarter of the dpy F3 would be suppressed if the
suppressor is recessive while three-quarter would be suppressed if the suppressor is
dominant. Alternatively, if the suppressor and the dpy marker mapped to the same
chromosome, none of the dpy F3 will show suppressed phenotype. The crosses and
procedures are shown in Chart 2.1.
Single Nucleotide Polymorphism (SNP) mapping
Mutations were first assigned linkage by linkage analysis as describe above. Once
the linkage group is known, SNP mapping was performed as described by Wicks et al.
49
(2001). Hawaiian (CB4856) males were crossed with him-3(vv6)unc-24(e138); vv39
and him-3(vv6)unc-24(e138); vv52 suppressor mutant hermaphrodites, F1 generation
animals were allowed to self-fertilize to generate F3 that were homozygous for the
suppressor locus as shown in. The suppressed lines were isolated and they were lysed to
obtain their genomic DNA. The DNA was then subjected to PCR using primers that
were specific for each SNP and restriction digest to detect the presence of polymorphism.
The proximity of the SNP to the mutation was estimated by the relative proportion of the
CB4856-specific band and the wild-type-derived-specific band. If the SNP is close to
the suppressor locus, the less likely that there is recombination between them, resulting
in an almost exclusively wild-type-derived-specific band for that SNP. Alternatively if
the SNP is far away from the suppressor locus, recombination between them is very
likely, resulting in an almost equal proportion of CB4856-specific band and wild-typederived-specific band (Chart 2.2).
PCR and restriction digest were performed as the followings:
PCR-mix:
15.1 µl H2O
2.5 µl 10x Taq PCR buffer
0.2 µl 25 mM dNTPs
2.5 µl 2 mM Forward primer
2.5 µl 2 mM Reverse primer
0.2 µl Taq polymerase
2 µl template
PCR – program:
Step 1 denaturation 94°C, 5 min
Step 2 denaturation 94°C 40 sec
Step 3 annealing 58°C 40 sec
Step 4 elongation 72°C 1 min
35 cycles (step 2 to step 4)
Step 5 final elongation 72°C 5 min
50
Restriction Digest:
Restriction Enzyme was added to PCR vial; each mix contains 5 U of restriction enzyme.
Digest mix :
2 µl NEB Buffer
(3 µl BSA)
10 µl PCR product
1 µl Enzyme
fill to 20 µl H2O
Digested PCR reactions were loaded on 2 % agarose gels in 1XTBE. A 1kb+ DNA
ladder was used as a marker.
51
Results
Isolating the suppressors from screen
him-3(vv6) was first linked to a visible marker, unc-24(e138) (him-3 and unc-24 are
0.26 m.u. apart) and him-3(vv6)unc-24(e138) hermaphrodites were treated with 25mM
EMS, a potent mutagen that generates single point mutations (Johnsen and Baillie, 1997).
The F1 progeny, heterozygous for any putative suppressor mutation, were allowed to
self-fertilize to generate F2 progeny, and the resulting F3 progeny was screened for an
increase in the brood size compared to him-3(vv6)unc-24(e138) homozygotes (16±8).
Thus in the suppressor screen, a brood size larger than 25 was considered as suppressed,
and five suppressors (vv38, vv39, vv41, vv50, and vv52) were isolated following the
screening of 2382 mutagenized genomes. The suppressed him-3(vv6)unc-24(e138)
hermaphrodites were crossed with wild-type males to generate heterozygous F1 progeny
and the him-3(vv6)unc-24(e138) homozygotes were isolated from the F2 population and
rescreened for suppression of embryonic lethality in the next generation. Following
three outcrosses the level of suppression was assessed by scoring viable progeny. I
found that in these suppressor strains, brood sizes were increased 3-6 fold (Figure 2.3),
indicating the embryonic lethality of vv6 mutants was significantly suppressed. To
determine if the suppression effect on vv6 was dominant or recessive, suppressor strains
were crossed with vv6 males and the F2 was screened for the suppression of embryonic
lethality. If the suppressor is dominant, the F2 progeny would show a suppressed
phenotype; however, if the suppressor is recessive, the F2 progeny would exhibit
embryonic lethality and him phenotype at vv6 levels. vv38, vv39, vv41, and vv50 behave
as dominant suppressors of embryonic lethality while vv52 acts semi-dominantly.
52
Furthermore, they exhibited varying levels of X chromosome non-disjunction as
indicated by the differences in the frequency of male progeny. The ones that had the
lowest frequencies were him-3(vv6; vv38)unc-24(e138) and him-3(vv6)unc-24(e138);
vv50 with 1.6% (0.9-2.5, 95% C.I.) and 2.5% (1.5-4, 95% C.I.) respectively. On the
other hand, him-3(vv6)unc-24(e138); vv39, him-3(vv6)unc-24(e138); vv41 and him3(vv6)unc-24(e138); vv52 had high frequencies with 6.5% (5.2-8, 95% C.I.), 4.7% (3.26.4, 95% C.I.) and 8.3% (6.5-10.4, 95% C.I.) respectively.
In comparison to the
frequency in him-3(vv6)unc-24(e138) (6.9% (3.4-11.3, 95% C.I.)),
vv38 and vv50
suppressed both emb and him phenotypes of him-3(vv6), while vv39, vv41 and vv52
suppressed the level of embryonic lethality but did not suppress the him phenotype of
him-3(vv6).
This result showed that these suppressors differentially affected the
segregation of autosomes and X chromosomes.
Previous studies of him-3 have
demonstrated that X-chromosome pairing is abolished in him-3 null mutants (Couteau et
al., 2004), indicating that HIM-3 is required for X chromosome alignment. In him-3(vv6)
mutants, X chromosome aligns with wild-type kinetics and undergoes homologous
synapsis, but nevertheless these mutants still exhibit a him phenotype as a result of
reduced crossing over (Couteau et al., 2004). These results together suggest that HIM-3
function is also required for processes occurring after homologue alignment and
synapsis. It is possible that vv39, vv41 and vv52 function with HIM-3 in promoting the
homologue alignments or synapsis alone, hence in these suppressed strains on the
autosomal non-disjunctions are restored but the X chromosome remains unaffected.
53
Cytological studies of the suppressors
DAPI staining of the germline nuclei of the suppressor strains revealed that they
have less extended transition zones in comparison to him-3(vv6)unc-24(e138) germlines
(Table 2.4). In comparison to the length of him-3(vv6)unc-24(e138) (122±18.5µm), him3(vv6)unc-24(e138);
vv39
(73.6±12µm)
and
him-3(vv6)unc-24(e138);
vv50
(99.4±24.9µm) had the shortest and the longest average lengths among the suppressors.
Consistent with these observations, vv39 strongly suppressed the embryonic lethality
phenotype of him-3(vv6)unc-24(e138) while vv50 had a weaker effect. Furthermore, the
strongest suppressors of embryonic lethality vv38, vv39 and vv52 also showed the
strongest suppression of the extended transition zone, suggesting that the meiotic cycle
progression were better in these strains. Interestingly, all the suppressor strains showed
a tighter clustering of chromosomes in the transition zone, while the germline nuclei at
transition zone in him-3(vv6) germline showed only partial clustering (Couteau et al.,
2004). This observation suggests that the nuclear reorganization that occurs at the onset
of homologous chromosome alignment is partially restored in the suppressed strains,
raising the possibility that the suppressors partially rescue the homologue alignment
defects in him-3(vv6) mutants.
Mapping and analysis of the suppressors
I chose to focus my primary efforts on vv38, vv39 and vv52 because they are the
strongest suppressors. First I wanted to find out the linkage group of vv38. I took the
him-3(vv6; vv38)unc-24(e138) mutants and crossed them with each of the six him-3(vv6);
dpy strains that correspond to all of the six chromosomes. dpy-5(e61) for chromosome I,
dpy-2(e8) for chromosome II, dpy-17(e164) for chromosome III, dpy-13(e184) for
54
chromosome IV, dpy-11(e224) for chromosome V, and dpy-3(e27) for chromosome X. If
the suppressor and the dpy marker were linked, none of the F2 dpy progeny would show
suppressed phenotype (refer to Chart 2.1). At the end of the experiments, 19/24 F2 dpy5(I); 18/24 F2 dpy-2(II); 15/21 F2 dpy-17(III); 17/22 F2 dpy-11(V); 19/23 F2 dpy-3(X),
were suppressed. However, none of the 22 F2 dpy-13(IV) showed suppressed phenotype.
These results further confirmed that vv38 is a dominant suppressor and it is positioned
on chromosome IV. Since him-3 is also on chromosome IV, him-3 was sequenced in
vv38 mutants; vv38 is an intragenic suppressor, that corresponds to an A to G mutation
that results in a glutamic acid to lysine substitution at residue 36 inside the HORMA
domain of the predicted protein sequence (refer to Figure 2.5), in addition to the vv6
mutation. The vv6 mutation results in the nonconservative substitution of small and
polar serine with large and non-polar phenyalanine at residue number 35, corresponding
to the serine at position 16 in the HORMA domain of human MAD2 that belongs to α1
(Lo et al., 2000) that binds to MAD1 by a parallel intermolecular coil-coil formation (Lo
et al., 2000, reviewed by Nasmyth, 2005). Consequently the vv6 mutation might be
expected to disrupt HIM-3 folding or interaction with other proteins. In vv38, serine at
residue number 35 is still substituted with phenyalanine, but in addition, glutamic acid at
residue number 36 is substituted with lysine. Similar to serine, lysine is polar, and it has
the longest side chain in all amino acids raising the possibility that the polar group in
lysine extends beyond the non-polar benzene group of phenylalanine and restores the
hydrophilicity to that part of the protein, thus restoring HIM-3 structure or protein
interaction, and once again making the conformational change of the protein possible.
To find out which chromosome vv39 is on, I performed linkage group analysis as
55
described for vv38. At the end, 18/22 F2 dpy-5(I); 13/19 F2 dpy-17(III); 13/18 F2 dpy13(IV); 17/20 F2 dpy-11(V); 15/18 F2 dpy-3(X), were suppressed. However, none of the
18 F2 dpy-2(II) showed suppressed phenotype.
These results confirmed that the
suppression effect of vv39 over him-3(vv6) is dominant and it is positioned on
chromosome II.
To further narrow down the genetic position of vv39, two separate three-factor
crosses were performed. First, I constructed a him-3(vv6) strain containing two visible
markers on chromosome II sqt-1(sc1) and unc-4(e26), located at 3.43 m.u. and 1.77 m.u.,
respectively, so the suppression effect of vv39 could be followed. The results showed
that none of the 12 unc-4 F2 recombinant was linked to the vv39 suppression, while all
of the 14 sqt-1 F2 recombinants were linked to the vv39, suggesting that vv39 was
positioned to the left of unc-4. For the second cross, I constructed another him-3(vv6)
strain containing two visible markers unc-4(e26) and dpy-2(e8), located at 1.77 m.u. and
0.05 m.u. on chromosome II respectively. In this case, 12/14 dpy-2 recombinants were
linked to vv39, while 7/15 unc-4 recombinants showed linkage, indicating that vv39 was
located between dyp-2 and unc-4. Since there were no more visible markers in this
genetic interval, SNP mapping was performed. SNP mapping in C. elegans is based on
crosses between wild-type-derived mutants and Hawaiian CB4856, a strain that shows
uniformly high density of single nucleotide polymorphisms compared with the wild-type
strain (Wicks et al., 2001). The tightly linked visible marker unc-24(e138) was used to
identify him-3(vv6) homozygotes in the F2, and the homozygous suppressor mutation
was identified by screening F3 for the presence of a suppressed phenotype as indicated
by a high progeny number. All mapping experiments were carried out in him-3(vv6)
56
background so that the suppressed phenotype could be followed. Once the F2 lines that
carried the homozygous suppressor mutations were identified, they were used as
material for PCR and restriction digests to reveal the presence of SNPs.
To confirm that vv39 is indeed on chromosome II, I first performed a SNP bulk
analysis using the DNA lysis of suppressed and non-suppressed F2 worms. A wild-typespecific band appeared in both suppressed and non-suppressed lysis when a SNP located
at the middle of chromosome IV was used, indicating that vv6 mutation was present in
both cases. In addition, the suppressed bulk showed another wild-type-specific pattern
when a SNP in the middle of chromosome II was used, suggesting tthe presence of
another mutation in the suppressed lines that was not present in the non-suppressed lines,
consistent with previous linkage group analysis.
To do SNP fine mapping, three
separated crosses between the vv39 suppressor strains and CB4856 were performed. 86
F2 suppressed lines were isolated from the first cross, however the mapping results were
inconsistent. vv39 is a dominant suppressor since vv6 unc-24/vv6 unc-24; vv39/vv39
individuals have an average brood size of 95±29 in comparison to 71±22 in vv6 unc24/vv6 unc-24; vv39/+ animals and consequently it is possible that some of the isolated
F2 suppressed lines were heterozygous for the suppressor mutation. To address this
problem, only F2 individuals that gave more than 110 progeny were subjected to SNP
mapping in the following experiments. 47 F2 suppressed lines were isolated and the
mapping data were consistent; SNPs ranging from –4.93 m.u to 20.7 m.u were tested
and vv39 was mapped between 0.09 m.u. and 2.65 m.u. on chromosome II. A fourth
cross was performed to further narrow down its genetic position;
19 F2 suppressed
lines were isolated and the results indicated that vv39 is positioned between 0.5 m.u. and
57
1.32 m.u. This 0.82 m.u. interval contains 470 predicted genes, two of which (C18E9.6
and zyg-11), were previously found to interact with HIM-3 in a yeast-two-hybrid screen
(M. Zetka, unpublished data), and pch-2, which plays a role in the pachytene checkpoint
that prevents chromosome segregation when synapsis or crossover formations are
defective (Bhalla and Dernburg, 2005; Joyce and McKim, 2009). Because vv39 is a
dominant suppressor, I could not perform complementation tests on these three genes to
screen for suppression effect. Alternatively, genomic DNA from the vv39 suppressor
mutant was sent for sequencing for these three genes, but no mutation was detected.
Further SNP mapping was attempted but the suppressed lines generated from the crosses
were not informative. As an alternative method, the genomic DNA of vv39 mutants was
sent to Michael Smith Genome Science Centre in Vancouver for Illumina Sequencing, a
sequencing method based on solid phase amplification followed by sequencing-bysynthesis of randomly fragmented DNA. The DNA fragments are attached to solid
surface called the flow cell, and they are amplified by PCR to generate clusters on the
surface of flow cell. Clusters are then sequenced and imaged with each reaction step.
The Illumina Genome Analyser System uses dNTPs that have 3’-reversible fluorescently
labeled terminator, and each emits a different fluorescent signal. As sequencing reaction
occurs, all four fluorescent-labeled dNTPs are added to the sequencing reaction, imaged,
and the 3’-terminator is removed to allow for the next sequencing step, and this process
is repeated for multiple cycles (Fox et al., 2009). Once the sequences were determined,
the exonic regions inside the interval of interest (between 0.5m.u. and 1.32m.u. on
chromosome II) were assembled in collaboration with the group of Steve Jones at the
UBC Genome Science Centre using bioinformatics (Rose et al., 2010), and then
58
compared to other known polymorphisms in the C. elegans wild-type strain (N2 Bristol).
This analysis revealed the presence of single mutation in the interval that mapped to the
cct-4 gene, corresponds to a C to T mutation that results in a proline to serine
substitution at residue 382 in the predicted protein sequence (refer to Figure 2.6).
I wanted to verify if vv39 is able to suppress the him-3 null allele gk149, which can
give me an indication if vv39 allows the bypass of him-3 function. I crossed him3(vv6)unc-24(e138); vv39 hermaphrodites with him-3(gk149) males and screened the F3
that did not contain Unc worms for suppression of embryonic lethality.
15 such
individuals were isolated and they had an average progeny number of 5±3 and a 26.9%
(18.6-36.1, 95% C.I.) percent of males, in comparison to him-3(gk149) that had an
average progeny number of 8±4 and a 30.8% (23.6-38, 95% C.I.) percent of males. This
result showed that vv39 was not able to suppress him-3(gk149), and the function of vv39
requires him-3.
For vv52, I performed linkage group analysis as described for vv38. 8/19 F2 dpy5(I); 9/18 F2 dpy-17(III); 8/18 F2 dpy-13(IV); 8/16 F2 dpy-11(V); 9/19 F2 dpy-3(X),
were suppressed. However, none of the 17 F2 dpy-2(II) showed suppressed phenotype.
These results confirmed that the suppression effect of vv52 over him-3(vv6) is semidominant and it is positioned on chromosome II.
SNP mapping was also performed on vv52 as described for vv39.
Since the
suppression effect of vv52 behaves semi-dominantly, only F2 animals that gave 100
progeny or more were used for mapping. Three separate crosses of him-3(vv6)unc24(e138); vv52 with the Hawaiian strain were performed and 127 F2 suppressed lines
isolated; bulk analysis was performed using the DNA lysis of suppressed lines. A
59
similar pattern as vv39 was obtained indicating that vv52 is also located on chromosome
II, which was consistent with the linkage group analysis. Further fine mapping with
individual SNPs determined that vv52 is positioned between -6 m.u. and 1.32 m.u.; since
the gene cct-4, C18E9.6, zyg-11, and pch-2 also map within this interval, they were
sequenced in vv52 mutants, but no mutation was detected suggesting that it is an
independent event.
To determine if the suppressors vv39 and vv52 result in a phenotype on their own,
him-3(vv6)unc-24(e138); vv39 and him-3(vv6)unc-24(e138); vv52 were crossed with
wild-type males to generate F1 that was heterozygous for vv6, unc-24 and the suppressor;
the F3 were screened for loss of the him-3(vv6)unc-24(e138) chromosome as indicated
by the absence of Unc individuals. 30 of such F3 individuals were identified in each the
vv39 and vv52 line, although a quarter of these individuals were predicted to be
homozygous for the suppressor, no him or emb phenotype could be detected suggesting
that the suppressor itself does not cause these phenotypes. However from the same
experiment, I separated vv6 from unc-24 as a consequence of a rare recombination event,
permitting an investigation into whether the suppressor mutation is recessive lethal. A
construct was made in which the vv39 mutation in him-3(vv6) background was linked to
a recessive visible marker on chromosome II, dpy-2(e8). I crossed this strain with wildtype males and generated F1 progenies that were heterozygous for him-3, dpy-2 and the
suppressor mutations. The F1 were individually picked, and the F2 individuals were
screened for embryonic lethality. If the suppressor mutant is embryonic lethal, all the
worms that are homozygous for the dpy-2 marker would be dead and only three quarters
of the F2 progenies would survive. At the end of this experiment, I recovered F2 dpy
60
worms, indicating that the suppressor mutation itself did not cause lethality.
61
References
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chromosme synapsis in Caenorhabditis elegans. Science 310: 1683-1686.
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Couteau, F., Nabeshima, K., Villeneuve, A., and Zetka, M. (2004). A component of C.
elegans meiotic chromosome axes at the interface of homolog alignment, synapsis,
nuclear reorganization, and recombination. Current Biology 14: 585592.
Fox, S., Fillichkin, S., and Mockler, T.C. (2009). Applications of Ultra-highThroughput sequencing. Methods in Molecular Biology 553: 79-108.
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Laboratory Press, Cold Spring Harbor, New York.
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Nasmyth, K. (2005). How do so few control so many? Cell 120: 739-746.
Rose, A.M., O’Neil, N.J., Bilenky, M., Butterfield, Y.S., Malhis, N., Flibotte, S., Jones,
M.R., Marra, M., Baillie, D.L., and Jones, S.JM. (2010). Genomic sequence of a mutant
strain of Caenorhabditis elegans with an altered recombinaton pattern. BMC Genomics
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62
Appendix for Chapter II
63
Scenario 1: suppressor and dpy marker are unlinked
vv6 unc-24/vv6 unc-24 ; s/s
X
vv6/vv6 ; dpy/+ ♂
↓
F1
vv6 unc-24/vv6 + ; s/+
;
½ dpy/+
½ +/+
↓
F2
¼ vv6 unc-24/vv6 unc-24 ;
½ vv6 unc-24/vv6 +
¼ vv6/vv6
¼ s/s
½ s/+
¼ +/+
;
¼ dpy/dpy
½ dpy/+
¼ +/+
↓
F3 Dominant ¾ Dpy progeny are suppressed
Recessive ¼ Dpy progeny are suppressed
Scenario 2: suppressor and dpy marker are linked
vv6 unc-24/vv6 unc-24 ; s/s
X
vv6/vv6 ; dpy/+ ♂
↓
F1
vv6 unc-24/vv6 + ;
½
½
s +/+ dpy (1)
s/+ (2)
↓
F2
¼ vv6 unc-24/vv6 unc-24 ;
½ vv6 unc-24/vv6 +
¼ vv6/vv6
(1) ¼ dpy/dpy
½ s +/+ dpy
¼ s/s
↓
F3
None of the Dpy progeny shows suppressed phenotype
Chart 2.1: Flow chart of linkage analysis for the suppressors. s refers to the
suppressor mutation, dpy refers to a dumpy marker.
F1
F2
S
S
S
Chart 2.2: A schematic representation for SNP mapping for the suppressor
mutation with 2 SNPs. s refers to suppressor mutation. The Hawaiian (CB4856) allele
is in red and the wild-type derived allele is in black. Black squares refer to SNP sites at
the wild-type allele and red squares refer to SNP sites at the CB4856 allele. After the
cross between the suppressor strain and CB4856, F1 animals were heterozygous for
wild-type and CB4856 alleles. This example shows when the suppressor locus is more
proximal to second SNP site. The crossover is unlikely to occur in between the
suppressor locus and the second SNP, resulting heterozygous for wild-type and CB4856
at first SNPs but homozygous wild-type at the second SNP.
200
180
160
140
Average
progeny
number
120
100
80
60
40
20
0
unc-24
him-3(vv6)
him-3(vv6; vv38)
unc-24
unc-24
him-3(vv6)unc-24; him-3(vv6)unc-24; him-3(vv6)unc-24; him-3(vv6)unc-24;
vv39
vv41
vv50
vv52
Figure 2.3: Partial rescue of embryonic lethality of him-3(vv6) in suppressor mutants. Histogram showing average progeny number of
individuals of indicated genotypes. Error bars represent standard deviations. Suppressed mutants showed 3- to 6-fold increase in average
progeny number compared to him-3(vv6)unc-24(e138) homozygotes.
Genotypes
unc-24(e138)
him-3(vv6) unc-24(e138)
him-3(vv6; vv38); unc-24(e138)
him-3(vv6) unc-24(e138); vv39
him-3 (vv6) unc-24(e138); vv41
him-3(vv6) unc-24(e138); vv50
him-3(vv6) unc-24(e138); vv52
Length of Transition Zone (µm)
59.2 ± 10 (n=10)
122 ± 18.5 (n=9)
75.6 ± 21 (n=10)
73.6 ± 12 (n=9)
85.7 ± 20.8 (n=9)
99.4 ± 24.9 (n=10)
78.7 ± 21.3 (n=9)
Table 2.4: Transition zones are less extended in the suppressor strains compared to unc-24(e138) him-3(vv6). Table showing the
length of transition zone (nuclei in leptotene/zygotene stages of meiotic prophase) of indicated genotypes. Transition zone length was
determined by measuring the distance between the first proximally positioned and last distally positioned polarized nuclei. Sample sizes and
standard deviations are shown.
vv38(E→K)
vv6(S→F)
HIM-3-Ce
HTP-1-Ce
HTP-2-Ce
HTP-3-Ce
Hop1p-Sc
Mad2p-Sc
Rev7p-Sc
---MATKEQIVEHRESEIPIASQWK--ATFPVDLEIEKNSEMFALRYIKCASAFILDRRGILDEKCFKTR-TIDKLLVTAFQSSVPA
MAPLETIYDESLNKSADSIDDKKWS--KLFPRIVSDPDRSSNFMTRAIYVAFSAVLRNRNILGQEYFTKNYITEKLKCMTLCFRNPR
MAPLENNYNESLNKSKDAIDDKTWS--KLFPSIVSDPDRSSNFMIRAIYVVFSAVLRQRNILEKEYFSKNYITENLSCMTLSFKNLR
---MDESFDSSVVPGSLTSDDRAIFNEQTLKNGDENSKSSLEVMANCVYLANSTILRERKVIPAEYFQDFQVYGDVSGYTLRQDIPE
-----MSNKQLVKPKTETKTEIT-------------TEQSQKLLQTMLTMSFGCLAFLRGLFPDDIFVDQRFVPEKVEKNYNKQNTS
-----MS-—QSISL-----------------------KGSTRTVTEFFEYSINSILYQRGVYPAEDFVT------VKKYDLTLLKTH
-----------------------------------MNRWVEKWLRVYLKCYINLILFYRNVYPPQSFDYT----TYQSFNLPQFVPI
81
85
85
84
69
51
48
Figure 2.5: vv38 corresponds to a mutation inside the HORMA domain of HIM-3. ClustalW multiple alignments of HORMA domain
containing proteins showing the N-terminal portion of the HORMA domain (underlined in black). htp-1, htp-2 and htp-3 are three him-3
paralogs in C. elegans. The predicted amino acids substitutions of him-3(vv6) and him-3(vv38) is shown by arrows. Ce, C. elegans and Sc,
S. cerevisiae.
vv39
(P/S)
CCT-4
CCT-1
CCT-2
CCT-3
CCT-5
CCT-6
CCT-7
CCT-8
KVIKVTGVQNPGHAVSILLRGSNKLVLEEADRSIHDALCVIRCLVKKKALLPGGGAPEM
ELILIKGPKS-RTASSIILRGANDVMLDEMERSVHDSLCVVRRVLESKKLVAGGGAVET
RLLRFSGVKL-GEACSVVLRGATQQILDESERSLHDALCVLVTHVKESKTVAGAGASEI
EYYTYVTAET-TTACTVVLRGPSKDVINEVERNLQDSLHVVRNIMINPKLVPGGGALEM
RMLSIEQCPN-NKAVTIFVRGGNKMIIDEAKRALHDALCVIRNLVRDSRIVYGGGSAEL
KTTFIEECRA-PKSVTLLIKGPNKHTITQIKDAIHDGLRAVFNTIVDKAVLPGAAAFEI
RYNFFEDCSK-AQACTLLLRGGAEQFIAETERSLHDAIMIVRRAKKNDSIVAGGGAIEM
NVVVFDKKSETGKVATIIIRGSSQSRIDDVERAVDDAVNTYKALTKDGKLLAGAGAVEI
430
420
409
418
426
420
412
450
Figure 2.6: vv39 mutation results in an amino acid substitution in CCT-4. Alignment of CCT-4 with all the other seven CCT subunits in
C. elegans. vv39 results in a proline to serine substitution of amino acid 382 (marked in red) of CCT-4.
Chapter III:
Characterization of the Suppressor cct-4(vv39)
64
Summary of Chapter III
vv39 has been identified as a novel allele of cct-4, which encodes the delta subunit
of the type II chaperonin complex.
This chapter will focus on the detailed
characterization and functional analysis of cct-4(vv39).
CCT-4 is localized to the
cytoplasm and to the nucleus in wild-type germlines. In addition, a portion of CCT-4 is
colocalized with chromatin during leptotene/zygotene, indicating that it may have a role
during chromosome pairing.
cct-4(RNAi) germlines show defects in axes
morphogenesis, ZIM-3 recruitment and SC formation, indicating that CCT-4 is required
for these processes. cct-1(RNAi) and cct-3(RNAi) germlines also show similar defects in
axes morphogenesis and SC formation, suggesting that CCT-4 function is mediated
through the chaperonin complex. In cct-4(vv39) mutants the axes morphogenesis is
delayed during early leptotene, but the recruitment of ZIM-3 and SYP-1 is still sustained
at later meiotic stages.
In him-3(vv6) mutants there is also a delay in axes
morphogenesis, but unlike cct-4(vv39) mutants the loading of ZIM-3 is impaired
throughout the germline. In him-3(vv6); cct-4(vv39) mutants, there is rescue of axes
morphogenesis and recruitment of ZIM-3. Interestingly, CCT-4vv39 can also suppress the
pairing defect of htp-1(gk174) null mutants. It is possible that CCT-4vv39 prevents the
precocious synapsis in this mutant by delaying axes morphogenesis. Therefore the
homologous chromosome can properly paired without the interference of premature SC
formation. All of this suggests that the CCT chaperonin complex may play a role in
mediating meiotic chromosome axes morphology through folding of axes component
HIM-3.
65
Material and Methods
Cytological Preparation of gonads and staining
Hermaphrodite gonads were dissected 22-24h post L4 stage in 1XPBS and fixed
with 1% paraformaldehyde for 5 minutes. Samples were frozen in dry ice for at least 10
minutes then placed in methanol at −20°C for 1 minute. Slides were washed with 1 X
PBST (1 X PBS, 0.1% Tween 20) three times and were blocked in 0.7% BSA/1 X PBS
for 1 hour. The primary antibodies were applied overnight at 4 oC. Antibodies were
diluted in 1 X PBS as follows: anti-rabbit-CCT-4 (1:50), anti-rabbit-HIM-3 (1:200), antiguinea pig-HTP-3 (1:700), anti-rabbit-HTP-3 (1:200), anti-rabbit-HTP-1 (1:300), antirabbit-HIM-8 (1:500), , anti-rabbit-PLK-2 (1:10), anti-rabbit-RAD-51 (1:200), antiguinea pig-SYP-1 (1:800), anti-guinea pig-ZIM-3 (1:100), anti-rabbit-GFP (1:2000),
anti-guinea pig-SUN-1 (against serine 12, 1:1300).
After three washes in 1 X PBST, secondary antibodies were applied (anti-rabbitAlexa 488 1:1000, anti-guinea pig-Alexa 488 1:600, anti-guinea pig-Alexa 555 1:1000,
anti-rat-Cy3 1:300) for 2 hours at room temperature. After three more washes with 1 X
PBST, the samples were mounted with Vectashield antifading medium (Vector
Laboratories, Burlingame, CA) containing 2µl/ml 4’,6-diamidino-2-phenylindole
(DAPI).
Fluorescence In Situ Hybridization (FISH)
PCR-amplified 5S rDNA was used as probe for the right end of chromosome V.
The probe was labeled with digoxigenin-11-dUTP. FISH was performed as described in
Couteau et al. (2004). Hybridized digoxigenin-labeled probe was detected with Cy3-
66
conjucated anti-digoxigenin antibody (1:100). Samples were mounted with DAPI in
Vectashield.
FISH and anti-SYP-1 staining for evaluation of nonhomologous synapsis
FISH was first performed with the 5S rDNA probe as above. Instead of adding the
Cy3-conjucated anti-digoxigenin antibody, anti-SYP-1 was applied overnight at 4 oC.
After four washes with 1 X TBST-BSA (1 X TBS, 0.1% Tween 20, 1% BSA), Cy3conjucated anti-digoxigenin antibody and anti-guinea pig-Alexa 488 were applied for 2
hours at room temperature. After three more washes with 1 X PBST, the samples were
mounted with DAPI in Vectashield as above.
Time-course analysis of pairing and appearance of RAD-51 foci
Data for three complete gonads were collected for each genotype and/or each probe
used. For each gonad, stacks of 30 optical sections were collected in increments of 0.2
µm covering the entire thickness of one layer of nuclei, and an image of the entire gonad
was assembled using Photoshop 6.0. The region extending between the first mitotic
nuclei and the last pachytene nuclei was divided into five equal-sized zones. FISH
signals or RAD-51 foci were then scored by examination of each single nucleus through
its volume. FISH signals were considered paired if the distance between the signals was
≦0.7 µm (MacQueen and Villeneuve, 2001). Data for each zone of the three gonads
were pooled together, giving a total number of nuclei with paired/unpaired signals, or a
total number of nuclei having zero, one, two to six, seven to ten, or more than eleven
RAD-51 foci.
67
Production and Testing of Antibodies
Antibody
against
CCT-4
was
generated
using
synthetic
peptide
CKIINSENDSNVNLKM according to standard protocol (Genescript Coporation,
Piscataway, NJ). It was then purified by immunoaffinity chromatography with activated
immunoaffinity supports (Affi-Gel 10, Bio-Rad), according to the manufacturer’s
instructions.
The specificity of the antibody was verified by failure to detect
immunofluorescence staining signals in cct-4(RNAi) germlines.
RNA interference
RNA interference experiments were performed as described in Fire et al (1998)
using
the
following
oligos
for
dsRNA
templates
TAATACGACTCACTATAGGAGCCAGAGAGTGTTCGCAAT-3’
amplification:
and
5’-
5’-TAATA
CGACTCACTATAGGCTTCAACCGTGTCTCCCATT-3’ for cct-4(RNAi), 5’-TAATA
CGACTCACTATAGGAAACGCCGCATTGATAAAAT-3’
and
5’-TAATACGACTC
ACTATAGGGGGATCAATTGAGCTTTGGA-3’ for cct-1(RNAi), 5’-TAATACGAC
TCACTATAGGAATCATGCGTGAAGAGGACA-3’
and
5’-TAATACGACTCACT
ATAGGCCTTTGATGGTCCACGAAGTA-3’ for cct-3(RNAi), 5’-TAATACGACTCAC
TATAGGGCTCATTTGTTTCCCTCACC-3’ and 5’-TAATACGACTCACTATAGGG
TCCGTTTCTTCAAAACTTGATGT-3’ for him-8(RNAi).
Stage adults (20-24h post L4) were injected with ~500 ng/µl dsRNA and
cytologicical analysis were performed 68-72h post-injection. The worms were dissected,
fixed, and stained as described above.
68
In-vitro Mutagenesis
cct-4(vv39) mutation was generated by in-vitro mutagenesis using the PCR-based
Site-Directed Mutagenesis System kit (Invitrogen, Carlsbad, CA), and the mutagenesis
primers
5’-AGGTGACTGGTGTTCAAAATTCAGGACATGCT-3’ and
AGTAGTTCCACTGACCACAAGTTTTA-3’.
5’-TTTCC
Mutation was verified by PCR and
sequencing.
Three-Fragment Vector Construction
Commercial One Shot TOP10 Chemically Competent E. coli was used as host
strain for high frequency transformation, plasmid DNA amplification and plasmid
construction was performed with the MultiSite Gateway Three-Fragment Vector
Construction kit (Invitrogen) using the primers 5’-GGGGACAACTTTGTATAG
AAAAGTTGAGATCTCTAAAAGTTACATAAAATT-3’ and 5’-GGGGACTGCTTT
TTTGTACAAACTTGCTGGAAAAGAAAATTTGATTTTTAA-3’ for amplifying the
promoter region of pie-1 gene. The amplified PCR product was cloned into pDONR P4P1R using BP clonase creating the 5’ Gateway entry clone.
The primers 5’-
GGGGACAAGTTTGTACAAAAAAGCAGGCTACATGCCACCAGCAGTTCCAGCC
-3’
and
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTATTAGCGGACAGC
CATGACAAT-3’ were used to amplify cct-4(vv39) made previously by in-vitro
mutagenesis. The amplified gene was cloned into pDONR 221 to generate internal
Gateway
entry
clone.
The
primer
pairs
5’-GGGGACAGCTTTCTTGTA
CAAAGTGGAAATTTTCAGCATCTCGCGCCCGTG-3’ and 5’-GGGGACAACTTT
GTATAATAAAGTTGGACTAGTAGGAAACAGTTATGTTTG-3’ were used to amplify
69
the 3’ untranslated region of unc-54. The amplified product was cloned into pDONR
P2R-P3 to create 3’ Gateway entry clone. By combining all three entry clones into the
Gateway compatible pCFJ150, a destination vector was made with respectively pie-1
promoter, cct-4(vv39) gene, and unc-54utr.
Mos Single Copy Insertion (MosSCI)
MosSCI were performed as described in Frokjaer-Jenson et al (2008). 20-24h post
L4 stage EG4322 (ttTi5605 II; unc-119(ed3) III) adult nematodes were injected with
pCFJ150 containing pie-1p::cct-4(vv39)::unc-54utr, together with two another
constructs: pMR910 containing the GFP marker under the control of pharyngeal muscle
promoter myo-2 (myo-2::GFP), and pJL44 containing Mos1 transposase under the
control of the heat shock promoter (Phsp::transposase). The injected animals were
individually plated and were left at 20oC overnight for recovering. On the next day the
worms were heat shocked at 33-34oC for one hour then left in 15 oC for 4 hours for
recovering, and were moved to 20oC overnight. The worms were heat shocked again on
the second and third day, and then were left at 20oC until starvation. Once they were
starved, the plates were screened for insertion animals that showed rescue of the unc119(ed3) phenotype and did not have the GFP marker. The transgenic animals were
isolated, and the presence of insertion was verified by PCR and sequencing.
70
Results
vv39 corresponds to a mutation in the CCT chaperonin subunit CCT-4
As described in the previous chapter, the result generated by Illuminar Sequencing
in collaboration with Steven Jones’ group revealed the presence of a mutation in the
third exon of the open reading frame corresponding to the cct-4 gene, resulting in a
P382S substitution of the predicted protein. cct-4 encodes the delta subunit of the CCT
chaperonin complex and alignment of all CCT subunits indicates that P382 is present
only in CCT-4 (Figure 3.1A), whereas alignment of CCT-4 in C. elegans, C. briggsae, C.
remanei, H. sapiens, M. musculus, S. cerevisae, and D. melanogaster indicates that this
residue is conserved in all Caenorhabditis species and in mammals. In S. cerevisae, and
D. melanogaster, this amino acid is replaced by alanine and methionine, respectively
(Figure 3.1B); since both alanine and methionine are non-polar and hydrophobic, the
hydrophobicity of this residue is universally conserved. By substituting proline with a
polar hydrophilic residue like serine in cct-4(vv39) mutants, it may have an effect on the
conformation and the tertiary structure of the protein; based on the known crystal
structure of thermosome and its sequence homology to the corresponding CCT-4 subunit
(Ditzel et al., 1998), the cct-4(vv39) mutation lies within the apical domain between S19
and S20, and may affect the substrate recognition and binding abilities of CCT-4.
To ultimately confirm that cct-4(vv39) corresponds to the suppressor mutation, I
first performed in-vitro mutagenesis to artificially generate the vv39 mutation in the cct4 cDNA using site-directed mutagenesis and then generated a germline expression
construct of cct-4(vv39) using the 5’ promoter region of the germline specific gene pie-1
and the unc-54 3’untranslated region for stable germline expression (Merritt et al., 2008).
Afterwards, I performed MosSCI by introducing the destination plasmid into the
71
germline of the ttTi5605 II; unc-119(ed3) III hermaphrodites, along with two other
constructs: one containing the GFP marker under the control of pharyngeal muscle
promoter myo-2, and another containing Mos1 transposase under the control of the heat
shock promoter. Excision of the Mos1 transposon creates a single double-strand break
in non-coding DNA, which is repaired by copy DNA from extrachromosomal template
into the genome (Frokjaer-Jenden et al., 2008). The loss of the myo-2::GFP co-injection
marker accompanied by the rescue of the unc-119(ed3) phenotype were used to
distinguish worms that carried the MosSCI insert. I recovered a single insertion line and
confirmed the integration on chromosome II by PCR: vvIs17[pie-1p::cct-4(vv39)::unc54utr]. vvIs17 was then introduced into the him-3(vv6) background to determine if the
germline expression of cct-4(vv39) would be able to suppress the embryonic lethality of
him-3(vv6). A quarter of the him-3(vv6) homozygotes segregated from him-6(vv6)/+;
vvIs17/+ heterozygotes were predicted to be homozygous for the insertion and would
suppress the embryonic lethality associated with him-3(vv6), resulting in an increase in
progeny number: since him-3(vv6) mutants had an average progeny number of 23+11,
worms that gave more than 35 progeny were considered to be suppressed. An F2 line
that produced 118 progeny was individually plated, retested, and the presence of the
insert was verified by PCR. him-3(vv6); vvIs17 homozygotes gave an average progeny
number of 131+34 and 7.2% (6.1-8.3, 95% C.I.) of males, a value not significantly
different from the average progeny number and frequency of males observed for the
original him-3(vv6); cct-4(vv39) suppressed line (103+42 and 6.9% (5.6-8.3, 95% C.I.)
of males). I next examined him-3(vv6); vvIs17 germlines cytologically and observed
that the leptotene/zygotene region of the germline was less extended in comparison to
72
him-3(vv6), indicating that the cell cycle progression defect of him-3(vv6) was rescued.
Furthermore, examination of the diakinesis nuclei of him-3(vv6); vvIs17 mutant
germlines revealed fewer DAPI-stained bodies in comparison to him-3(vv6) mutants and
similar to him-3(vv6); cct-4(vv39) germlines, suggesting an increase in the number of
bivalents (Figure 3.2).
These results collectively suggest that transgenic CCT-4vv39
expression in the germline is able to suppress the defects in chiasma formation and
embryonic lethality associated with him-3(vv6) mutants and indicate that cct-4(vv39) is
the suppressor mutation. Since many new meiotic markers had been developed since the
original description of the him-3(vv6) phenotype, I used them to investigate more
precisely the vv6 defect, the phenotype of cct-4(vv39), the basis for cct-4(vv39)-mediated
suppression, and the function of cct-4 in germline processes.
CCT-4 colocalizes with chromatin throughout the germline
Since CCT-4 is a component of the CCT chaperonin complex, I next investigated
the functional context of CCT-4vv39 suppression of the pairing defects and associated
nonhomologous synapsis observed with axis mutants.
To investigate its possible
functions in the germline, I raised antibody against CCT-4 to determine its localization
in the germline (Figure 3.3). As expected I could detect CCT-4 marked foci within the
cytoplasm, consistent with its known role in cytosolic protein folding. However, a strong
nuclear CCT-4 signal was detected that was strongly reduced in cct-4(RNAi), indicating
it was specific (Figure 3.4). In nuclei at leptotene/zygotene, CCT-4 appeared colocalized
with the polarized chromatin while at pachytene its presence is more diffuse within the
nuclei.
These observations suggested that CCT-4 has potential nuclear roles and
functions during meiotic chromosome pairing stages.
73
cct-4(RNAi) results in germline defects
To investigate the functions of CCT-4 in the germline, I first examined nuclear
morphology in DAPI-stained cct-4(RNAi) germlines using the rrf-1(pk1417) background
that is defective in somatic RNAi, but sensitive in the germline (Sijen et al., 2001). I
found that the germlines of cct-4(RNAi) animals were much smaller and had fewer
nuclei; macronuclei and micronuclei were observed and distributed throughout the
germline with irregular spacing (Figure 3.5). Since CCT is required for the proper
folding and function of actin and tubulin (Gao et al., 1992; Yaffe et al., 1992; Lundin et
al., 2007), and Cdc20 (Camasses et al., 2003), these defects might reflect the
requirements of microtubules and cell cycle proteins in the germline mitotic divisions.
Furthermore, the polarization of chromatin characteristic of leptotene/zygotene nuclei
was lost, suggested that early meiotic processes may be disrupted by cct-4(RNAi).
CCT-4 is required to assemble axes competent for PC protein recruitment and SC
assembly
To further investigate which processes were affected by cct-4(RNAi), I first
examined HIM-3 and ZIM-3 localization. In the early leptotene/zygotene region of the
germline, HIM-3 could be detected on axes, indicating that nuclei entered meiosis in the
absence of cct-4; however, HIM-3 localization on axes was discontinuous and punctuate
and meiotic chromosome axis morphology appeared to be immature. Furthermore, no
ZIM-3 could be detected in these nuclei (Figure 3.6) similar to him-3(vv6) mutants. To
examine SC formation in cct-4(RNAi) germlines, I did an immunostaining using antiHIM-3 and anti-SYP-1 antibodies.
In contrast to him-3(vv6) mutants, cct-4(RNAi)
pachytene nuclei HIM-3-marked chromosome axes still had a discontinuous appearance
74
and SYP-1 failed to associate with chromosomes and remained diffuse (Figure 3.7).
These results suggest that CCT-4 is required for the assembly of meiotic chromosome
axes competent for PC-mediated pairing and SC assembly.
Loss of CCT-1 and CCT-3 recapitulates cct-4(RNAi) axes morphogenesis defects
The C. elegans genome encodes at least seven subunits corresponding to
subunits of the highly conserved CCT chaperonin complex, including the CCT-4-like
subunits CCT-1 and CCT-3 (Leroux and Candido, 1995; 1997). To investigate whether
it is the chaperonin complex function or a complex-independent CCT-4-specific function
that mediates axes morphogenesis and SC formation, I performed cct-1(RNAi) or cct3(RNAi) in rrf-1(pk1417) and examined the resulting germline phenotypes (Figure 3.7).
Interestingly in both cct-1(RNAi) and cct-3(RNAi) germlines, the HIM-3-marked axis
morphology at pachytene appeared to be immature, similar to cct-1(RNAi) and SYP-1
also failed to localize to the chromosome axes and remained diffuse within the nucleus.
Since cct-1(RNAi), cct-3(RNAi) and cct-4(RNAi) showed defects in axes morphogenesis
and SYP-1 recruitment, it is likely that the CCT chaperonin complex is responsible for
mediating axes assembly or individual CCT subunits have complex-independent
functions in this process.
cct-4(vv39) mutants exhibit meiotic cell cycle delay
As mentioned in the previous chapter, cct-4(vv39) homozygotes were not
embryonic lethal, and did not segregate dead embryos or exhibit him phenotype. Since
cct-4 deletion mutants are lethal (Wormbase), this suggests that vv39 does not disrupt the
essential function of CCT-4 in cytosolic protein folding and that the major meiotic
75
processes are largely unaffected. However, close examination of axis morphogenesis
and SC formation in cct-4(vv39) mutant germlines revealed that the leptotene/zygotene
region was populated by both polarized and non-polarized nuclei, while in wild-type the
same region was populated exclusively with polarized nuclei (Figure 3.8). In these
nuclei, HIM-3-marked axes appeared thinner and more discontinuous and showed less
colocalization with SYP-1 in comparison to wild type nuclei at the same stage,
suggesting a delay in axis morphogenesis. Consistent with this interpretation, ZIM-3
was only localized to those cct-4(vv39) nuclei in the leptotene/zygotene region showing
a polarized chromatin morphology nuclei in which robust loading of HIM-3 could be
detected (Figure 3.9).
These results suggest that in cct-4(vv39) mutants, axis
morphogenesis required for proper PC protein recruitment and SC formation is delayed,
but ultimately achieved. Axis morphology and SC formation at pachytene appeared to
be normal and bivalents were observed at diakinesis.
him-3(vv6) mutants have defects in axes morphogenesis and synapsis progression
that are suppressed by CCT-4vv39
Since several axis markers were not available at the time of the initial
characterization of him-3(vv6) mutants, I examined meiotic chromosome axis
morphogenesis with antibodies against 1) HTP-3, which is required for the recruitment
of all other known axis components, including HIM-3 and HTP-1/2 (Goodyer et al.,
2008; Severson et al., 2009) and 2) HTP-1/2 (Martinez-Perez et al., 2008). HTP-3
localization to developing meiotic chromosome axes appeared to be on time and at wildtype levels in him-3(vv6) mutants; however the HTP-3-marked meiotic chromosome
axes at early leptotene/zygotene stages, appeared punctuate and discontinuous in
76
comparison to WT nuclei at the same stage (Figure 3.10). Similarly, HIM-3 and HTP1/2 were both recruited to the axes of him-3(vv6) mutants (Figure 3.10 and Figure 3.11),
but the early leptotene/zygotene stage nuclei showed also showed a discontinuous and
immature axis morphology. Consistent with the interpretation that axis morphogenesis is
delayed in him-3(vv6) mutants, SYP-1 localization also appeared to be delayed at early
leptotene/zygotene;
SYP-1
appeared
in
very
few
short
stretches
in
early
leptotene/zygotene nuclei, in comparison to the long stretches of SYP-1 observed in
wild-type nuclei of the same stage (Figure 3.12). In him-3(vv6); cct-4(vv39) suppressed
germlines, the delayed axis morphogenesis defect was rescued; HTP-3-marked
chromosome axes appeared to be more contiguous in comparison to him-3(vv6), and
longer SYP-1 stretches were formed (Figure 3.12). These results suggest that HIM-3vv6
localization to meiotic chromosome axes affects their timely morphogenesis and that this
defect is rescued by CCT-4vv39.
cct-4(vv39) restores autosomal PC protein recruitment in him-3(vv6) mutants
The correct morphogenesis of meiotic chromosome axes is also required for
appropriate localization of PC proteins mediating meiotic chromosome pairing to the
pairing centers (Phillips et al., 2005; Phillips and Dernburg, 2006). Because him-3(vv6)
mutants are specifically defective in autosomal pairing (Couteau et al., 2004), I
examined the localization of the PC protein ZIM-3, which localizes to the PC of
chromosomes I and IV (Phillips and Dernburg, 2006).
In wild-type, ZIM-3 was
localized to the nuclear periphery during leptotene/zygotene stages and two foci can
often be observed, corresponding to the paired chromosomes I and IV (Phillips and
Dernburg, 2006) (Figure 3.13).
However in him-3(vv6), no ZIM-3 was detectable
77
throughout the germline (Figure 3.13), indicating that ZIM-3 recruitment to the PC is
defective in this mutant. However, in him-3(vv6); cct-4(vv39) suppressed germlines,
ZIM-3 recruitment was restored (Figure 3.13) and the appearance and disappearance of
ZIM-3 followed wild-type kinetics: it appeared in the first polarized leptotene/zygotene
nucleus, and disappeared at pachytene stages where the nuclei were depolarized (Phillips
and Dernburg, 2006). These results indicated that ZIM-3 recruitment to the PC of
autosomes I and IV is defective in him-3(vv6) mutants, and is restored by CCT-4vv39.
Since a previous study had demonstrated that X chromosome pairing is not affected in
him-3(vv6) mutants (Couteau et al., 2004), I examined the localization of the X
chromosome PC protein HIM-8 (Phillips et al., 2005). Consistent with the pairing
proficiency of this chromosome, HIM-8 localized to discrete foci at the nuclear
periphery in leptotene/zygotene stage nuclei in both him-3(vv6) and him-6(vv6); cct4(vv39) mutant germlines (Figure 3.14). Despite HIM-8 recruitment and pairing
proficiency, him-3(vv6) mutants exhibit defective X chromosome crossing over and
concomitant X chromosome nondisjunction resulting in a him phenotype (Couteau et al.,
2004), indicating that HIM-3vv6 disrupts X-chromosome recombination following
successful chromosome pairing. Since him-3(vv6); cct-4(vv39) mutants do not rescue the
him phenotype of him-3(vv6), we conclude that the basis of cct-4(vv39) suppression is
mediated through restoration of ZIM-3 loading (and perhaps loading of all autosomal PC
proteins), and a corresponding increase in autosomal pairing and crossing over.
Defective ZYG-12/SUN-1 patch formation in him-3(vv6) mutants is restored by
CCT-4vv39
Previous studies have shown that the inner and outer and nuclear envelope proteins
78
SUN-1/ZYG-12 colocalized and formed patches at the nuclear periphery during
leptotene/zygotene stages (Penkner et al., 2007). The local enrichment of SUN-1 and
ZYG-12 in the vicinity of PC ends of chromosomes at leptotene/zygotene provides a
platform for connecting the chromosome ends to the cytosolic microtubule network
required for meiotic chromosome movement during pairing stages (Sato et al. 2009;
Baudrimont et al., 2010).
In wild-types, ZYG-12 patches appeared at the nuclear
envelope at the same time of the appearance of polarized leptotene/zygotene nuclear
morphology (Sato et al., 2009). In individual nuclei, one to nine large ZYG-12 patches
could be observed (Sato et al., 2009) (Figure 3.15); upon completion of synapsis, ZYG12 redistributes throughout the nuclear envelope except for one small focus that remains
associated with the paired HIM-8 signals at pachytene (Sato et al., 2009). SUN-1 also
formed patches at leptotene/zygotene and these patches overlapped with the PC proteins
ZIM-3 and HIM-8, indicating that these patches represent variable numbers of PC
chromosome ends being brought together for homology assessment (Penkner et al., 2007;
2009; Sato et al. 2009). It has also been shown that the N-terminus of SUN-1 is
subjected to meiotic-specific post-translational modification (Penkner et al., 2009); the
phosphorylation of Ser8, Ser12 and Ser24 corresponds to the time window of homology
search when SUN-1 patches are formed, suggesting that SUN-1 phosphorylation at these
residues has a role in patch formation and homologue pairing (Penkner et al., 2009).
Loss of Ser12 by substituting it with a nonphosphorylatable residue caused defects in
SUN-1 patch formation, chromosome pairing and nonhomologous synapsis (Penkner et
al., 2009), indicating that it is required for these SUN-1 mediated functions.
Furthermore, previous studies have shown that the him-8(me4) missense mutation
79
disrupts HIM-8 association with SUN-1/ZYG-12 patches, but HIM-8 is still bound to the
X chromosome PC. This result showed that HIM-8me4 is defective in the activity of
promoting patch formation or association (Sato et al. 2009). It is proposed that SUN1/ZYG-12 patches are nucleated by association of high density of PC proteins HIM-8 or
ZIMs (Sato et al. 2009). Consistent with this idea, patches are never observed that lack
a closely apposed focus of HIM-8 or ZIMs (Sato et al. 2009), and high-copy arrays
consist of HIM-8 or ZIMs binding sites are consistently associated with SUN-1/ZYG-12
patches (Phillips et al., 2009).
Immunostaining of him-3(vv6); zyg-12::GFP and him-3(vv6); cct-4(vv39); zyg12::GFP germlines using anti-GFP antibody detected only a single ZYG-12 patch per
nucleus in him-3(vv6), while in him-3(vv6); cct-4(vv39) suppressed germlines, multiple
ZYG-12 patches were formed, similar to wild-types (Figure 3.15).
Similarly,
immunostaining of him-3(vv6 and him-3(vv6); cct-4(vv39) mutant germlines using
phospho-epitope specific antibodies to Ser12 of SUN-1 (S12-Pi) showed that only one
SUN-1S12-Pi patch per nucleus was observed in him-3(vv6) mutant germlines
corresponding to the X chromosome PC protein HIM-8 (Figure 3.14). In contrast,
multiple patches SUN-1S12-Pi were observed in him-3(vv6); cct-4(vv39) suppressed
germlines (Figure 3.14). These results indicated that links between the autosomal PCs
and cytoskeletal forces required for homolog pairing is defective in him-3(vv6) and is
restored by CCT-4vv39.
him-3(vv6) mutants have extensive nonhomologous synapsis
Previous reports have shown that pairing and synapsis on each chromosome is
mediated by its corresponding PC protein (Phillips et al., 2005; Phillips and Dernburg,
80
2006). It is believed that synapsis initiates at the PC in association with PC proteins,
where the homology assessment takes place (Sato et al., 2009). Interestingly, him-3(vv6)
mutants have only one homologously paired PC (HIM-8) and extensive nonhomologous
synapsis, suggesting that synapsis initiation between nonhomologous chromosomes was
being initiated outside of the context of homologously paired PCs. I was interested to
know if the homologous pairing of a single PC would be sufficient to license synapsis
globally, irrespective of homology. To investigate this possibility, I depleted him-8 in
him-3(vv6) by RNAi to eliminate the only PC protein present in this mutant, and
abrogate X chromosome pairing and synapsis. In him-3(vv6); him-8(RNAi) mutant
germlines, leptotene/zygotene nuclei lost their polarized appearance suggesting that the
presence or pairing of a single functional PC is sufficient to drive the partial nuclear
polarization observed in him-3(vv6) mutants. Furthermore, SC initiation was abolished
in him-3(vv6); him-8(RNAi) mutant germlines since SYP-1 was not detected on
chromosomes and instead formed one or two nuclear aggregates (Figure 3.16). These
results suggest that the pairing of a single PC is sufficient to license global synapsis
irrespective of homology.
Defective initial pairing of him-3(vv6) mutants is partially rescued by CCT-4vv39
Since cct-4(vv39) suppressed the timely axis morphogenesis and ZIM-3
recruitment defects of him-3(vv6), I next investigated if these events correlate with
suppression of the defect in homolog pairing using FISH to target the 5S rDNA locus
located on the right third of chromosome V (Figure 3.17). In wild-type, the percentage
of nuclei with paired FISH signals significantly increased to 55% upon entry into
leptotene/zygotene (p<0.0001, Fisher’s Exact Test) and increased to 91% by early
81
pachytene (p<0.0001) and 99% by late pachytene, indicating stabilization by synapsis.
In him-3(vv6) mutant germlines, the level of pairing was 9% at the beginning of
leptotene/zygotene, not significantly different in comparison to the premeiotic region
(p=0.25); however, the pairing level significantly increased to 18% by the end of
leptotene/zygotene (p=0.02) and did not change significantly upon pachytene entry
(p=0.85). These results for wild-type and him-3(vv6) mutants were consistent with
previously published reports (Couteau et al., 2004).
In him-3(vv6); cct-4(vv39)
germlines, the level of pairing was 10.7% at the beginning of leptotene/zygotene, a value
not significantly different in comparison to the premeiotic region (p=0.3) or to the
pairing level observed in him-3(vv6) mutants in at the same stage (p=0.81), but
significantly lower than wild-type (p<0.0001). However, pairing level increased to
31.4% by the end of leptotene/zygotene (p=0.0008) and was significantly higher than
the level observed in him-3(vv6) mutant germlines at the same stage (p=0.03). By the
end of pachytene, pairing levels reached 47% in him-3(vv6); cct-4(vv39) mutant
germlines, a value that was also significantly higher than that observed in him-3(vv6)
mutants (p<0.0001), but still significantly lower than that observed in wild-type
germlines at the same stage (p<0.0001). These results together indicated that him3(vv6)-associated pairing defects are significantly, but not fully restored by CCT-4vv39mediated suppression of axis morphogenesis and ZIM-3 recruitment.
CCT-4vv39 partially suppresses nonhomologous synapsis in him-3(vv6) mutants
Because the homolog pairing defect of him-3(vv6) mutants was partially
suppressed in him-3(vv6); cct-4(vv39) mutants, I next investigated if the nonhomologous
82
synapsis defect in him-3(vv6) was also suppressed by cct-4(vv39). I performed FISH
followed by immunostaining with anti-SYP-1, which allowed me to monitor the pairing
status of Chromosome V and its participation in synapsis simultaneously. The results of
this experiment are shown in Figure 3.18.
The levels of homologously synapsed
chromosomes were significantly higher in him-3(vv6); cct-4(vv39) than in him-3(vv6) in
all stages (p<0.0001, <0.0001, 0.0001) while at the same time, the levels of
nonhomologously synapsed chromosomes were significantly lower in him-3(vv6); cct4(vv39) than in him-3(vv6) in all stages (p<0.0001, <0.0001, <0.0001). These results
collectively suggest that cct-4(vv39)-mediated increases in homolog pairing lead to an
increase
in
homologously
synapsed
chromosomes
at
the
expense
of
the
nonhomologously synapsed classes. I also observed an increase in the unpaired and
unsynapsed class in suppressed germlines in comparison to him-3(vv6) at all stages
(p=0.04, p=0.005, p=0.01), consistent with the appearance of more HIM-3-marked axes
without SYP-1 localization and more single unpaired tracts of DAPI-stained
chromosomes (Figure 3.19). These results suggested that the nonhomologous synapsis
observed in him-3(vv6) mutants is suppressed by CCT-4vv39.
CCT-4vv39 restores RAD-51 kinetics to wild-type levels in him-3(vv6) mutant
germlines
Previous studies have shown that recombination is initiated normally in him3(vv6) mutants, but the kinetics is delayed as RAD-51-marked recombination
intermediates persist into inappropriately late stages in comparison to wild-types
(Couteau et al., 2004).
The extensive nonhomologous synapsis and cell-cycle
progression delay in him-3(vv6) mutants may contribute to the delay in the
83
disappearance of RAD-51-marked early recombination intermediates, since homologous
chromosomes are not spatially available as recombination partners and recombination
intermediates may not be resolved on time. Since cell cycle progression as evidenced by
a less extended leptotene/zygote region is partially restored in the suppressed line, and
more chromosomes are homologously synapsed, I next determined if cct-4(vv39) also
suppressed the defects in progression of meiotic recombination observed in him-3(vv6)
mutants. I performed an immunostaining in him-3(vv6) and in him-3(vv6); cct-4(vv39)
using antibody against RAD-51, and the results are shown in Figure 3.20. Consistent
with previously published data, recombination initiation as assessed by the appearance
of RAD-51-marked recombination intermediates at early leptotene/zygotene was not
significantly different between him-3(vv6) mutants and wild types (p=0.82), but the
RAD-51 foci persisted into late pachytene in the former (p=0.003). In him-3(vv6); cct4(vv39) mutant germlines, the distribution of nuclei with the indicated number of RAD51 foci was not significantly different than in wild-type at all stages (p=0.75, 0.21, 0.59,
0.42), but was significantly different than in him-3(vv6) at late pachytene (p=0.008).
These results demonstrate that the defect in RAD-51-marked recombination kinetics
observed in him-3(vv6) mutants is suppressed CCT-4vv39.
CCT-4 partially rescues the homolog pairing defects of htp-1(gk147) mutants
As described in the previous chapter, cct-4(vv39) could not suppress the
embryonic lethality and him phenotype of the him-3 null allele gk149, indicating that
suppression required the presence of HIM-3. To investigate if the CCT-4vv39-mediated
suppression of meiotic defects is specific to him-3(vv6), I also tested the ability of CCT4vv39 to suppress other mutants that exhibit pairing defects and nonhomologous synapsis:
84
the missense mutant sun-1(jf18) unable to retain ZYG-12 at the nuclear envelope
(Penkner et al., 2007) and the htp-1 null mutant htp-1(gk174) (Couteau and Zetka 2005;
Martinez-Perez and Villeneuve 2005). In sun-1(jf18) mutant germlines, PC proteins are
recruited to the PC, but SUN-1/ZYG-12 patches failed to form at the nuclear periphery
during leptotene/zygotene, leading to defects in chromosome movement that are
accompanied by defects in homolog pairing and nonhomologous synapsis (Penkner et
al., 2007; Baudrimont et al., 2010).
In htp-1(gk174) mutants, homolog pairing is
defective and synapsis formed between nonhomologous chromosomes (Couteau and
Zetka, 2005); however, SUN-1 patches are formed albeit in reduced numbers and ZIM-3
loading is unaffected (Baudrimont et al., 2010).
In the case of sun-1(jf18), cct-4(vv39) was not able to suppress the associated
embryonic lethality and him phenotype suggesting that CCT-4 and SUN-1 are not
operating through the same pathway. However, cct-4(vv39) was able to significantly
suppress the embryonic lethality of htp-1(gk174) null mutants; htp-1(gk174) mutants had
an average progeny number of 4+3 and while htp-1(gk174); cct-4(vv39) mutants showed
a significant increase to 27+13. However the him phenotype was not suppressed since
htp-1(gk174); cct-4(vv39) mutants had 17.3% (13.2-21.5, 95% C.I.) of males among
surviving progeny, a value not significantly different from the 17.6% (10.4-25.4, 95%
C.I.) observed in htp-1(gk174) mutants alone. To investigate in more detail the basis of
CCT-4vv39 suppression of htp-1(gk174), I determined the number of DAPI-stained bodies
in diakinesis nuclei of in mutant germlines. htp-1(gk174); cct-4(vv39) mutants exhibited
significantly less nuclei with 12 DAPI bodies than htp-1(gk174 mutants (p<0.0001); in
addition htp-1(gk174); cct-4(vv39) suppressed germlines had significantly more nuclei
85
with 7 and 10 DAPI bodies than htp-1(gk174) (p=0.007 and p<0.0001 respectively)
(Figure 3.21), collectively indicating a significant, but partial restoration of chiasma
formation. Consistent with previously published results, examination of pairing levels in
htp-1(gk174) mutant germlines revealed that they were significantly lower than in wild
types at all meiotic stages (p<0.0001 in all cases) and did not increase significantly at
any stage (Couteau and Zetka 2005; Martinez-Perez and Villeneuve 2005) (Figure 3.22).
In htp-1(gk174); cct-4(vv39) suppressed germlines however, pairing levels significantly
increased at leptotene/zygotene in comparison to htp-1(gk174) mutants (p=0.03), but
was still very low in comparison to wild type germlines (p=0.04) at the same stage. The
level of pairing in htp-1(gk174); cct-4(vv39) mutants was also significantly higher than
in htp-1(gk174) single mutants at all pachytene stages (p=0.03 at early pachytene,
p=0.01 at mid-pachytene, p=0.0004 at late pachytene), but were still significantly lower
than in wild type germlines (p<0.0001 in all cases). These results indicated that the
pairing defects of htp-1(gk149) mutants are significantly, but not fully suppressed by
CCT-4vv39.
To determine if the increase in pairing levels observed in htp-1(gk174); cct-4(vv39)
was accompanied by a reduction in nonhomologous synapsis, I examined axis
morphogenesis and SC formation (Figure 3.23). In htp-1(gk174) mutant germlines,
unsynapsed chromosomes axes as indicated by the presence of HIM-3 without
colocalization of SYP-1 could be detected at pachytene. Similarly, unsynapsed axes
were also observed in htp-1(gk174); cct-4(vv39) mutants, but they were more abundant
in the suppressed germlines than in htp-1(gk174) mutants alone. These results suggest
that the homolog pairing defect observed in the absence of HTP-1 mutants is partially
86
suppressed by cct-4(vv39), leading to more homologously paired chromosomes and a
reduction in the global number of chromosomes engaging in nonhomologous synapsis.
87
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90
Appendix for Chapter III
91
A.
B.
CCT-4
CCT-1
CCT-2
CCT-3
CCT-5
CCT-6
CCT-7
CCT-8
KVIKVTGVQNPGHAVSILL
ELILIKGPKS-RTASSIIL
RLLRFSGVKL-GEACSVVL
EYYTYVTAET-TTACTVVL
RMLSIEQCPN-NKAVTIFV
KTTFIEECRA-PKSVTLLI
RYNFFEDCSK-AQACTLLL
NVVVFDKKSETGKVATIII
390
380
361
370
378
380
364
402
CE-CCT-4
CBR-CCT-4
TRA8P6V8
HS-TCP1-delta
MM-TCP-1-delta
SC-Cct-1p
DM-CG5525
KVIKVTGVQN--PGHAVSILL
KVIKVTGVQN--PGHAVSILL
KIIQVTGVQN--PGQAVSVLI
KLLKITGCAS--PGKTVTIVV
KLFKITGCTS--PGKTVTIVV
KIVRVTGIRNNNARPTVSVVI
KFVKITGIQN--MGRTVSIIC
390
141
366
390
331
379
383
Figure 3.1: vv39 mutation results in an amino acid substitution in CCT-4. vv39 corresponds to a proline to serine substitution of amino
acid 382 (marked in red) of CCT-4. A) Alignment of CCT-4 with all the other seven CCT subunits in C. elegans; proline is only present in
CCT-4 and not in other subunits. B) Alignment of CCT-4 in C. elegans, C. briggsae, C. remanei, H. sapiens, M. musculus, S. cerevisae, and
D. melanogaster respectively. Proline is conserved in all Caenorhabditis species and in mammals. In S. cerevisae, and D. melanogaster,
this amino acid is replaced by alanine and methionine, respectively. Proline, alanine and methionine are all non-polar and hydrophobic
residues, thus the hydrophobicity of this residue is conserved.
100
WT (n=40)
90
him-3(vv6) (n=38)
80
him-3(vv6); cct-4(vv39) (n=39)
% nuclei
70
him-3(vv6); vvIs17[pie-1p::cct-4(vv39)::unc-54utr]
(n=40)
60
50
40
30
20
10
0
6
7
8
9
10
11
# of DAPI stained bodies at diakinesis
Figure 3.2: cct-4(vv39) partially restores chiasma formation in him-3(vv6) mutants. Histogram showing the
percentage of diakinesis nuclei with the indicated number of DAPI-stained bodies. Wild types consistently exhibit six
DAPI figures representing the six chromosome pairs joined by chiasmata, while him-3(vv6) mutants rarely exhibit
these levels (p<0.0001). cct-4(vv39) increases the frequency of appearance of bivalents in him-3(vv6) mutant nuclei
(p<0.0001 for 6 DAPI bodies and p=0.033 for 8 DAPI bodies), and a similar effect is exhibited by germlines
expressing vvIs17-driven CCT-4vv39 (p<0.0001 for 6 DAPI bodies and p=0.013 for 8 DAPI bodies). The differences
between him-3(vv6); cct-4(vv39) and him-3(vv6); vvIs17 are not statistically significant in all cases.
HTP-3
CCT-4
MERGE
Diakinesis
Pachytene
Leptotene/
Zygotene
DAPI
Figure 3.3: CCT-4 localizes to the cytoplasm and to the nucleus. Immunofluorescence micrographs showing
DAPI, anti-HTP-3 and anti-CCT-4 staining of indicated meiotic stages. CCT-4 is localized to the germline nuclei
and the cytoplasm in the leptotene/zygotene and pachytene regions of the germline. At leptotene/zygotene, a
portion of CCT-4 is colocalized with chromatin indicated by white arrows while during diakinesis, its presence is
more diffuse within the nuclei. Scale bars, 5µm.
HTP-3
CCT-4
MERGE
cct-4(RNAi)
WT
DAPI
Figure 3.4: The CCT-4 antibody is specific to CCT-4. Immunofluorescence micrographs showing DAPI, antiHTP-3 and anti-CCT-4 of pachytene nuclei in WT and cct-4(RNAi). In cct-4(RNAi) germlines, CCT-4 is not
detectable in the nuclei and is severely reduced in cytoplasm. Scale bars, 5µm.
rrf-1(pk1417)
rrf-1(pk1417); cct-4(RNAi)
Figure 3.5: cct-4(RNAi) disrupts germ cell proliferation and nuclei polarization. Immunofluorescence micrographs showing DAPIstained hermaphrodite germ lines. rrf-1(pk1417); cct-4(RNAi) germlines are smaller and contain less germ cells and are marked by the
appearance of micronuclei (white arrows in the inset). The chromatin polarization characteristic of leptotene/zygotene nuclei is lost in
rrf-1(pk1417); cct-4(RNAi). Scale bars, 5µm.
HIM-3
ZIM-3
MERGE
rrf-1(pk1417);
cct-4(RNAi)
rrf-1(pk1417)
DAPI
Figure 3.6 : cct-4 is required for recruitment of ZIM-3 to pairing centers. Immunofluorescence micrographs showing
DAPI, anti-HIM-3 and anti-ZIM-3 staining of leptotene/zygotene nuclei in rrf-1(pk1417) and rrf-1(pk1417); cct-4(RNAi).
ZIM-3 failed to localize to the PCs at the nuclear periphery in cct-4(RNAi) germlines. Scale bars, 5µm.
HIM-3
SYP-1
MERGE
rrf-1(pk1417)
cct-3(RNAi)
rrf-1(pk1417);
cct-1(RNAi)
rrf-1(pk1417);
cct-4(RNAi)
rrf-1(pk1417)
DAPI
Figure 3.7:
cct-1(RNAi) and cct-3(RNAi) recapitulate cct-4(RNAi) phenotypes. Immunofluorescence
micrographs showing DAPI, anti-HIM-3 and anti-SYP-1 staining of pachytene nuclei in the indicated genotypes.
HIM-3-marked chromosome axes appear to be discontinuous and SYP-1 fails to localize to the axes in rrf-1(pk1417);
cct-4(RNAi), rrf-1(pk1417); cct-1(RNAi), and rrf-1(pk1417); cct-3(RNAi). Scale bars, 5µm.
HIM-3
SYP-1
MERGE
cct-4(vv39)
WT
DAPI
Figure 3.8: cct-4(vv39) mutants have a meiotic cell cycle delay. Immunofluorescence micrographs showing DAPI, antiHIM-3, anti-SYP-1 staining of leptotene/zygotene nuclei. In WT, the chromatin is polarized and the chromosome axes show
robust loading HIM-3 and of SYP-1. In cct-4(vv39) mutant germlines, the leptotene/zygotene region of the germline is
populated by both polarized and non-polarized nuclei (white arrows); HIM-3-marked axes appear thinner and more
discontinuous and show less colocalization with SYP-1. Scale bars, 5µm.
HIM-3
ZIM-3
MERGE
cct-4(vv39)
WT
DAPI
Figure 3.9: ZIM-3 is localized to the polarized nuclei in cct-4(vv39) mutants. Immunofluorescence micrographs
showing DAPI, anti-HIM-3, anti-SYP-1 staining of leptotene/zygotene nuclei. In WT, the chromatin is polarized and ZIM-3
is localized to the nuclear periphery. In cct-4(vv39) mutant germlines, the leptotene/zygotene region is populated by both
polarized and non-polarized nuclei (white arrow); and ZIM-3 is localized exclusive to the polarized nuclei with robust
loading of HIM-3. Scale bars, 5µm.
HIM-3
HTP-3
MERGE
him-3(vv6)
WT
DAPI
Figure 3.10: Chromosome axes morphogenesis is immature in him-3(vv6). Immunofluorescence micrographs showing
DAPI, anti-HIM-3 and anti-HTP-3 stainings of early leptotene/zygotene nuclei. In WT, HIM-3 and HTP-3 formed
continuous tracks indicated by white arrows, while in him-3(vv6) HIM-3 and HTP-3 appeared to be punctuate and noncontiguous. Scale bars, 5µm.
DAPI
HTP-1
HTP-3
MERGE
WT
him-3(vv6)
Figure 3.11: HTP-1 localizes to chromosome axes in him-3(vv6) mutants. Immunofluorescence micrographs of
DAPI, anti-HTP-1 and anti-HTP-3 stainings of leptotene-zygotene nuclei. HTP-1 localizes to the HTP-3-marked
chromosome axes in him-3(vv6) germ line similar to wild types. Scale bars, 5µm.
HTP-3
SYP-1
MERGE
him-3(vv6);
cct-4(vv39)
him-3(vv6)
WT
DAPI
Figure 3.12: cct-4(vv39) restores chromosome axis morphogenesis in him-3(vv6) mutants. Immunofluorescence micrographs
showing DAPI, anti-HTP-3 and anti-SYP-1 staining of early leptotene/zygotene stage nuclei. Chromosome axes appear thin and
discontinuous in him-3(vv6) and the morphogenesis is restored in him-3(vv6); cct-4(vv39). SYP-1 loading is more robust in him3(vv6); cct-4(vv39) in comparison to him-3(vv6). Scale bars, 5µm.
HTP-3
ZIM-3
MERGE
him-3(vv6);
cct-4(vv39)
him-3(vv6)
WT
DAPI
Figure 3.13: ZIM-3 loading is defective in him-3(vv6) but is restored in him-3(vv6); cct-4(vv39).
Immunofluorescence micrographs showing DAPI, anti-HTP-3 and anti-ZIM-3 stainings of leptotene/zygotene nuclei.
ZIM-3 was not detectable in him-3(vv6) but its localization is restored in him-3(vv6); cct-4(vv39). Scale bars, 5µm.
DAPI
HIM-8
SUN-1S12-Pi
MERGE
WT
him-3(vv6)
him-3(vv6);
cct-4(vv39)
Figure 3.14: him-3(vv6) mutants are competent for HIM-8 localization. Immunofluorescence micrographs
showing DAPI, anti-SUN-1S12-Pi and anti-HIM-8 stainings of leptotene/zygotene nuclei. HIM-8 signals are
colocalized with SUN-1 in him-3(vv6,) similar to WT and him-3(vv6); cct-4(vv39). Scale bars, 5µm.
ZYG-12
MERGE
him-3(vv6);
cct-4(vv39);
zyg-12::GFP
him-3(vv6);
zyg-12::GFP
zyg-12::GFP
DAPI
Figure 3.15: ZYG-12 patch formation is defective in him-3(vv6) and is restored in him-3(vv6); cct-4(vv39).
Immunofluorescence micrographs showing DAPI and anti-GFP stainings of leptotene/zygotene nuclei. zyg-12::GFP and
him-3(vv6); cct-4(vv39); zyg-12::GFP nuclei have multiple patches of ZYG-12 at the nuclei periphery while only one
ZYG-12 patch is detectable at the nuclei periphery in him-3(vv6) mutants. Scale bars, 5µm.
HTP-3
SYP-1
MERGE
him-3(vv6);
him-8(RNAi)
him-8(RNAi)
WT
DAPI
Figure 3.16: Synapsis initiation is abolished in him-3(vv6); him-8(RNAi). Immunofluorescence
micrographs showing DAPI, anti-HTP-3 and anti-SYP-1 staining of leptotene/zygotene nuclei in indicated
genotypes. The asymmetrical clustering of nuclei at this stage is lost in him-3(vv6); him-8(RNAi) and SYP-1
forms nuclear aggregates and fails to localize to chromosome axes. Scale bars, 5µm.
100
90
80
% nuclei
with
paired
FISH
signals
70
60
WT
50
him-3(vv6)
40
him-3(vv6); cct-4(vv39)
30
20
10
0
Premeiotic
Leptotene/Zygotene
Premeiotic
Leptotene/Zygotene
Premeiotic
Leptotene/Zygotene
Pachytene
Pachytene
Pachytene
Figure 3.17: cct-4(vv39) partially suppresses the homologue pairing defects of him-3(vv6) mutants. Histograms showing the pairing levels
of WT, him-3(vv6) and him-3(vv6); cct-4(vv39) during indicated stages. Pairing levels are monitored by using a probe targeting the 5S rDNA
locus, representing the right half of chromosome V. The pairing levels of him-3(vv6) and him-3(vv6); cct-4(vv39) are both significantly lower
than WT (p<0.0001, Fisher’s Exact Test), but are not significantly different from each other (p=0.81) upon entry into leptotene/zygotene.
Pairing levels rise significantly in him-3(vv6); cct-4(vv39) by the end of leptotene/zygotene in comparison to him-3(vv6) (p=0.03). Pairing
levels in him-3(vv6); cct-4(vv39) suppressed germlines is also significantly higher than him-3(vv6) (p<0.0001) at late pachytene.
% nuclei
WT
pachytene
% nuclei
% nuclei
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
him-3(vv6); cct-4(vv39)
100
90
80
70
60
50
40
30
20
10
0
him-3(vv6)
leptotene/zygotene
pachytene
homologously synapsed
unpaired and unsynapsed
paired and not synapsed
nonhomologously synapsed
and unsynapsed
nonhomologously synapsed
leptotene/zygotene
pachytene
Figure 3.18: cct-4(vv39) suppresses the nonhomologous synapsis defects of him-3(vv6). Histogram showing percentage of nuclei with
paired and unpaired chromosomes V in association with synapsis marker SYP-1. Homologously synapsed identifies paired FISH signals in
association with SYP-1 tracts while unpaired and unsynapsed identifies paired FISH signals without SYP-1 tracts. Nonhomologously
synapsed and unsynapsed identified separated FISH signals, one of which is in association with a SYP-1 tract while nonhomologously
synapsed identifies separated FISH signals of which both are associated with SYP-1 tracts. him-3(vv6); cct-4(vv39) mutants have significantly
less nonhomologously synapsed chromosomes at all the indicated stages compared to him-3(vv6) (p<0.0001, <0.0001, <0.001), while they
still have significantly more nonhomologously synapsed chromosome compared to WT at all indicated stages (p<0.001, <0.0001, <0.001).
HIM-3
SYP-1
MERGE
him-3(vv6);
cct-4(vv39)
him-3(vv6)
WT
DAPI
Figure 3.19: Partial synapsis in him-3(vv6) and him-3(vv6); cct-4(vv39) mutants. Immunofluorescence
micrographs showing DAPI, anti-HIM-3 and anti-SYP-1 stainings of pachytene nuclei in germlines of the indicated
genotypes. White arrows indicate unsynapsed chromosome axes marked by the presence of HIM-3 without SYP-1.
Scale bars, 5µm.
% nuclei
WT
pachytene
leptotene/
zygotene
% nuclei
% nuclei
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
him-3(vv6)
leptotene/
zygotene
pachytene
0 RAD-51 focus
him-3(vv6); cct-4(vv39)
1 RAD-51 focus
2-6 RAD-51 foci
7-10 RAD-51 foci
11+ RAD-51 foci
leptotene/
zygotene
pachytene
Figure 3.20: cct-4(vv39) restores recombination progression in him-3(vv6) mutants. Histograms showing percentage of nuclei
with the indicated number of RAD-51 foci during different meiotic prophase stages. RAD-51 foci appear on time and at normal levels
in him-3(vv6) mutant germlines, but their disappearance is delayed compared to WT (p=0.82 at the beginning of leptotene/zygotene,
p=0.003 at the end of pachytene). In him-3(vv6);cct-4(vv39) suppressed germlines, both appearance and disappearance of RAD-51
foci are not different from wild-type kinetics at all stages.
100
90
80
% nuclei
70
60
WT (n=40)
50
htp-1(gk174)(n=38)
htp-1(gk174); cct-4(vv39) (n=31)
40
30
20
10
0
6
7
8
9
10
11
12
# of DAPI stained bodies at diakinesis
Figure 3.21: cct-4(vv39) partially restores chiasma formation in htp-1(gk174) mutants. Histogram showing the percentage of
diakinesis nuclei with the indicated number of DAPI-stained figures. Wild-type diakinesis nuclei are invariably marked by the
presence of six DAPI-stained figures representing the 6 chromosome pairs joined by chiasmata. htp-1(gk174); cct-4(vv39) has
significantly more nuclei with 7 and 10 DAPI bodies than htp-1(gk174) (p=0.007 and p<0.0001 respectively). Also htp-1(gk174);
cct-4(vv39) has significantly less nuclei with 12 DAPI bodies than htp-1(gk174) (p<0.0001).
WT
% nuclei with paired FISH signals
100
htp-1(gk174)
90
80
htp-1(gk174); cct-4(vv39)
70
60
50
40
30
20
10
0
premeiotic
leptotene/
zygotene
early
pachytene
mid
pachytene
late
pachytene
Figure 3.22: cct-4(vv39) partially suppressed the initial homologue alignment defects of htp-1(gk174) mutants.
Histogram showing pairing levels in germline nuclei of the indicated stages. Pairing is monitored using a probe
targeting the 5S rDNA locus. Pairing levels are significantly increased in htp-1(gk174); cct-4(vv39) mutants at
leptotene/zygotene in comparison to htp-1(gk174) (p=0.03), but are still very low in comparison to WT (p=0.04) at
the same stage. The level of pairing attained by late pachytene is also significantly different in htp-1(gk174) and htp1(gk174); cct-4(vv39) (p=0.0004).
HIM-3
SYP-1
MERGE
htp-1(gk174);
cct-4(vv39)
htp-1(gk174)
WT
DAPI
Figure 3.23: Reduced and partial synapsis in htp-1(gk174); cct-4(vv39). Immunofluorescence micrographs
showing DAPI, anti-HIM-3 and anti-SYP-1 stainings of mid-pachytene nuclei. Unsynapsed axes (white arrows
indicated by unsynapsed chromosome axes marked by HIM-3 without colocalization of SYP-1) are more prominent
in htp-1(gk174); cct-4(vv39) than in htp-1(gk174). Scale bars, 5µm.
Chapter IV:
Discussion and Conclusion
92
Isolation of genetic suppressors of meiotic defects
In this study, I was able to successfully isolate five suppressors of the embryonic
lethality of him-3(vv6), indicating that the autosomal nondisjunction defects of him3(vv6) mutants was partially rescued.
In addition, the suppressor mutants also
exhibited a less extended leptotene/zygotene region that correlated with the level of
suppression of the embryonic lethality, suggesting the increased fidelity of autosomal
segregation originated in a rescue of early meiotic prophase defects. The suppressor
mutants showed differing X chromosome nondisjunction frequencies as estimated by
the frequency of (X0) male progeny. In vv38 and vv50 suppressor mutants, both the
autosomal and X chromosome non-disjunction were suppressed. However in vv39,
vv41 and vv52, the X chromosome nondisjunction was not rescued. In him-3(vv6)
mutants, the X chromosomes paired and synapsed normally, but nevertheless there is a
dramatic reduction in crossing over suggesting that him-3(vv6) also disrupts crossover
formation on the homologously synapsed X chromosomes(Couteau et al., 2004). It is
possible that in vv39, vv41 and vv52, the suppressors rescued only the (autosomal)
pairing and synapsis defects of him-3(vv6) mutants, resulting in the suppression of
autosomal non-disjunction but not the X chromosome defect in crossing over. In the
case of vv38 and vv50, the suppressors might function with HIM-3 in promoting
pairing, synapsis and in crossover formation, therefore in these suppressed lines both
the autosomal and X chromosome nondisjunction showed some rescue.
Genetic mapping was performed on the three strongest suppressors: vv38, vv39 and
vv52; vv38 was found to be an intragenic suppressor with a mutation within the HORMA
domain. Since the HORMA domain is believed to be involved in mediating protein-
93
protein interactions (Aravind and Koonin, 1998) and the vv38 mutation is also located
inside the HORMA domain similar to the vv6 mutation, it is possible that vv38 restored
the appropriate protein-protein interaction between HIM-3 and a not yet identified target
protein or HIM-3 structure per se. vv39 and vv52 were dominant and semi-dominant
intergenic suppressors, and were both mapped to chromosome II. Because of their
dominant suppressions over him-3(vv6), it was not possible to do complementation test
to determine if they were allelic. The frequency of generating dominant suppressor
mutations after EMS mutagenesis appears to be relatively low (Greenwald and Horvitz,
1980; 1982).
A previous suppressor screen on the uncoordinated and egg-laying
defective mutant unc-93(e1500) had isolating dominant suppressors at a frequency of 4
X 10-5 or 1/2500 (Greenwald and Horvitz, 1980). In comparison, this suppressor screen
isolated 5 dominant suppressors following the screen of 2382 genomes. Therefore this
screen had a frequency of 1/476 in isolating dominant suppressors.
It has been
previously proposed that the frequent occurrence of dominant mutations in certain genes
may reflect structural and functional features of their gene products (Park and Horvitz,
1986). Examples of other mutations with dominant effects in C. elegans include unc-1
(Park and Horvitz, 1986), unc-54 (Macleod et al., 1977) and unc-93 (Waterston et al.,
1984), all encoding structural proteins of muscle; these gene products function in
multimeric forms, suggesting mutability to dominant phenotypes may be a characteristic
of genes encoding proteins that function in complexes and have many levels of
interaction between proteins (Waterston et al., 1984; Park and Horvitz, 1986).
Furthermore, several alleles of sup-11 have been shown to dominantly suppress unc93(e1500) (Greenwald and Horvitz, 1982). It was proposed that one possible way that
94
these sup-11 alleles might suppress unc-93(e1500) was by altering SUP-11 to restore
function to a protein complex that consisted of at least SUP-11 and UNC-93 (Greenwald
and Horvitz, 1982).
Because HIM-3 is involved in different conserved meiotic
processes and may function or interact with various proteins in mediating these events, it
is possible that there is a similar higher probability of isolating suppressors with
dominant effects.
A role for CCT function in meiotic prophase I
The cct-4(vv39) mutation did not cause embryonic lethality or him phenotype
and cct-4(vv39) mutants were not recessive lethal, indicating that vv39 did not disrupt an
essential function of cct-4.
Careful examination of cct-4(vv39) mutant germlines
revealed that a delay in meiotic chromosome axis morphogenesis as indicated by
appearance of thin and punctuate HIM-3-marked axes that had less colocalization with
SYP-1 in early leptotene/zygotene nuclei. Unlike him-3(vv6) mutants, the axes were
managed finally assemble at later leptotene/zygotene stage and ZIM-3 was localized
appropriately (Figure 4.1B), consistent with the absence of autosomal and X
chromosome non-disjunction. In the germline, CCT-4 localized to both the cytoplasm
and to the nuclei, suggesting it both cytosolic and nuclear functions in this compartment.
Furthermore, a robust CCT-4 signal was found colocalized with the polarized chromatin
of leptotene/zygotene stage nuclei, suggesting that CCT-4 has a role during chromosome
pairing stages. RNAi experiments showed that the chromosome axes had a
discontinuous appearance in cct-4(RNAi) germlines and that the PC protein ZIM-3 and
the SC component SYP-1 failed to localize to chromosomes, despite the fact that nuclei
95
had entered meiotic prophase. These results implied that CCT-4 functions in promoting
meiotic chromosome axis assembly essential for the recruitment of PC proteins and SC
formation. Since RNAi against two other CCT chaperonin subunits (CCT-1 and CCT-3)
resulted in similar defects in axis morphogenesis and SC assembly, the simplest
interpretation is that CCT-4 function meiotic functions are mediated through a nuclear
CCT chaperonin complex; however, the possibility that these subunits have CCT
complex-independent meiotic functions cannot be excluded.
Some evidences for a nuclear function for the CCT complex have been described
in other systems. Previous studies in meiotic rat spermatocytes found a subset of CCT
chaperonin was localized to the nucleus and associated with condensed chromosomes
(Soues et al., 2003). TriC-P5, a protein that shares extensive homology with TCP-1, is
found in the nucleus in mice where it is attached to the nuclear matrix (Joly et al., 1994).
It was proposed that it may assist in the folding of nuclear matrix proteins (Joly et al.,
1994). Furthermore, a potential nuclear localization signal was identified in TCP-1
subunit (Horwich and Willision, 1993), suggesting that CCT may be localized to the
nucleus and have a nuclear function. Despite the evidence for a nuclear localization of
the CCT complex, its precise function is still not known. It is possible that certain
proteins with complex folding patterns may need to be folded within the nucleus
following nuclear import or that some nuclear proteins may require chaperonin activity
to adopt conformations required for timely assembly of nuclear structures or to interact
appropriately with their interacting partners. In the case of CCT regulates meiotic
chromosome axes morphogenesis through the folding of HIM-3 by making it
conformationally capable to carry on its function.
96
HIM-3vv6 results in delayed axes morphogenesis
It has been previously reported that him-3(vv6) mutants have pairing defects
accompanied by autosomal nonhomologous synapsis, and defects in recombination
progression and crossover formation (Couteau et al., 2004). In this study, I documented
several previously unrecognized defects in him-3(vv6) mutants: immature axis
morphology at early leptotene/zygotene and defective recruitment of autosomal, but not
X chromosome PC proteins. In addition SUN-1/ZYG-12 only formed one patch at the
nuclear periphery during leptotene/zygotene, and this patch corresponded to HIM-8marked X chromosomes, consistent with the interpretation that X chromosomes retain
competency for pairing in the presence of HIM-3vv6. him-3(vv6) mutants share many
phenotypic features previously described for him-3(gk149) null mutants including the
defective recruitment of autosomal PC proteins (Baudrimont et al., 2010), competency
for HIM-8 localization (Phillips et al., 2005), and SUN-1 patch formation in association
with the X chromosome PC at leptotene/zygotene (Baudrimont et al., 2010). In contrast
to him-3(vv6) mutants, however, synapsis is abolished in the absence of HIM-3 at axes
(Zetka et al. 1999; Couteau et al., 2004), indicating that HIM-3 is required to establish a
functional chromosome axis structure necessary for both the recruitment of autosomal
PC proteins and SC formation. The fact that him-3(vv6) behaves like the null allele
except there is robust SC formation between nonhomologous chromosomes can be
explained by a delay in chromosome axis morphogenesis in the presence of HIM-3vv6.
In this scenario, the immature state of chromosome axes during leptotene/zygotene
results in defects in autosomal PC protein recruitment and in chromosome pairing;
however, since HIM-3 is structurally present at chromosome axes to support SC
97
polymerization, unpaired autosomes engage in nonhomologous synapsis (Figure 4.1C).
CCT-4vv39 suppresses the axis morphogenesis defects of him-3(vv6) mutants
This study has shown that the pairing, nonhomologous synapsis, recombination
progression and chiasma formation defects in him-3(vv6) mutants were at least partially
suppressed by cct-4(vv39).
In him-3(vv6); cct-4(vv39) mutants, chromosome axis
morphogenesis at early leptotene/zygotene appeared to be more contiguous than in him3(vv6) or in cct-4(vv39) mutants alone. These results suggest that CCT-4vv39 dominantly
compensates for a HIM-3vv6-related defect that disrupts timely axis assembly, and
consequently downstream processes such as chromosome pairing and crossing over.
CCT complex targets are known to be enriched in beta sheets and/or low in alpha helices.
(Yam et al., 2008), suggesting that proteins with such secondary structures require the
activity of this chaperonin complex to assemble into their functional conformation. The
predicted him-3 protein is almost entirely comprised of the HORMA domain (Aravind
and Koonin, 1998) based on structural investigations of the HORMA-domain containing
Mad2 protein, it consists of three alpha helices, a large six-stranded beta sheet on one
side, and a short beta hairpin on the other side (Lo et al., 2000; reviewed by Nasmyth,
2005). One possible explanation is that the CCT chaperonin complex located in the
nucleus is required for the folding of HIM-3 into a conformation competent for
chromosome recruitment and for interactions required for axis formation. The him3(vv6) mutation results in the substitution of polar serine with non-polar phenyalanine at
residue number 35, corresponding to the serine at position 16 in the HORMA domain of
human MAD2 that belongs to α1 (Lo et al., 2000) that is essential for its interaction with
98
MAD1 (Lo et al., 2000; reviewed by Nasmyth, 2005). In him-3(vv6; vv38), in addition
to the vv6 mutation, glutamic acid at residue number 36 is substituted with lysine.
Similar to serine, lysine is polar, and it has the longest side chain in all amino acids
raising the possibility that the polar group in lysine extends beyond the non-polar
benzene group of phenylalanine and restores the hydrophilicity to that part of the protein.
It is possible that HIM-3vv6 disrupts its interaction with the CCT complex. In the case of
cct-4(vv39), the mutation affects a conserved residue at the apical domain which is
responsible for substrate recognition and binding (Kusmierczyk and Martin, 2001); this
may result in a converse loss of interaction with HIM-3, resulting in the impaired axis
assembly observed in this mutant.
In this scenario, the suppression of the axis
morphogenesis defects observed in him-3(vv6); cct-4(vv39) mutants can be explained by
a restoration of the interaction between HIM-3 and CCT-4 through compensatory
changes in protein structure, resulting in timely HIM-3 folding, partial restoration of
axes morphogenesis and rescue of the downstream meiotic defects.
Mechanisms of suppression of nonhomologous synapsis during meiotic prophase
Previous studies on pairing and synapsis have revealed evidence that the PCs
have two distinct functions: the stabilization of pairing interactions in the absence of
synapsis, followed by the initiation of synapsis at these sites (MacQueen et al., 2005).
Each PC is bound by one of the four zinc-finger proteins and mutants in them result in
the disruptions of pairing and synapsis specific for their corresponding chromosomes,
indicating that these proteins are required for PC function (Phillips et al., 2005; Phillips
and Dernburg, 2006). In PC-homozygous deletion backgrounds, synapsis between the
99
two X chromosomes is dramatically reduced in comparison to the heterozygous situation,
suggesting that the PCs promote synapsis (MacQueen et al., 2005); however, synapsis in
these deletion homozygotes still occurs at low frequencies, indicating that other
chromosome locations can support synapsis initiation (reviewed by Colaiácovo, 2006).
It was previously shown that the autosomal PC proteins (ZIM-1, -2, and -3) localized to
discrete foci at the nuclear envelope during leptotene/zygotene stages, at the time when
pairing and synapsis are initiated (Phillips et al., 2005). Results from the same study also
showed that individual loss of zim-1, zim-2, and zim-3 results in unsynapsed phenotypes
of their corresponding chromosomes (Phillips et al., 2005). In this context, the
substantial amount of synapsis observed in him-3(vv6) mutants is surprising given that
ZIM recruitment is defective. One possible explanation is that the HIM-8-mediated
pairing and SC initiation at the X chromosome PCs is sufficient to trigger synapsis
initiation outside the context of the PCs on the autosomes. Consistent with this
interpretation, loss of him-8 in him-3(vv6) mutants resulted in loss of SC initiation and
instead SC component SYP-1 localized to a nuclear aggregate. This result suggested that
synapsis initiation at a single PC (or the X chromosome specifically) is sufficient to
license synapsis globally for the other chromosomes, irrespective PC pairing status or of
homology.
Several other mutants in C. elegans have been observed to cause nonhomologous
synapsis. In sun-1(jf18) missense mutants, SUN-1/ZYG-12 patches do not form and are
accompanied by defects in chromosome movement and pairing and by the appearance of
nonhomologous synapsis (Penkner et al., 2007; Baudrimont et al., 2010). In sun-1(jf18)
mutants, homolog pairing is not fully disrupted (Sato et al., 2009) despite the failure to
100
form SUN-1/ZYG-12 patches, suggesting that chromosomes are structurally competent
for meiotic pairing and synapsis. ZYG-12 also functions in the sun-1 pathways since it
loss of zyg-12 also results in loss of patch formation, pairing defects and nonhomologous
synapsis (Sato et al., 2009), suggesting that ZYG-12 promotes chromosome pairing and
prevents nonhomologous synapsis similar to SUN-1. However, CCT-4vv39 was not able
to suppress sun-1(jf18) defects, suggesting that they are not operating through the same
pathway. In htp-1(gk174) null mutants, HIM-3 is recruited to chromosome axes and
ZIM-3 is localized normally (Baudrimont et al., 2010), but SC components are loaded
precociously during leptotene/zygotene stages (Couteau and Zetka, 2005). htp-1 has
been proposed to be part of a surveillance system preventing synapsis from occurring
until alignment between homologs has been attained (Couteau and Zetka, 2005;
Martinez-Perez and Villeneuve, 2005), but the defect leading to the observed
nonhomologous synapsis is not understood. It is possible that in htp-1(gk174) mutants
chromosomes are prematurely stabilized by synapsis before homolog pairing takes place,
resulting synapsis between nonhomologs (Figure 4.1D). Interestingly CCT-4vv39 is able
to partially suppress the pairing defects of htp-1(gk174), consistent with the
interpretation that the delay in axis morphogenesis and synapsis in cct-4(vv39) mutants
causes a delay in the loading of SC components in htp-1(gk174) mutants. As a result,
the homologous chromosomes have an extended window of opportunity for achieving
pairing in htp-1(gk174); cct-4(vv39) mutants without the interference of premature SC
initiation and consequently higher levels of pairing accompanied by a reduction in
nonhomologous synapsis are observed.
101
Conclusion and future directions
This study is the first that describes a functional role of chaperonin during meiotic
prophase and opens a complete new area for investigation. The chromosome axes
maturation mediated by the chaperonin is critical for the recruitment of meiotic
components onto the axis, leading to homologous chromosome pairing, synapsis and the
accurate homologous chromosome segregation at meiosis I. This study showed that the
meiotic chromosome axes component HIM-3 is a potential substrate of the CCT
chaperonin complex and that the CCT complex may play a role in mediating axis
morphogenesis through the folding of HIM-3. In the future, it would be interesting to
study the nuclear CCT chaperonin complex and its clients to learn more about their
involvement in meiotic processes and the role of protein folding in the assembly of the
complex structures that are the hallmark of meiotic prophase.
102
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Appendix for Chapter IV
105
A
Axes morphogenesis
requires CCT-4
WT
Zygotene
Early Leptotene
Meiotic Entry
Pairing stabilizes
by synapsis
Folding HIM-3
B
cct-4
(vv39)
C
him-3
(vv6)
D
htp-1
(gk174)
ZYG-12
Axes morphogenesis
is delayed
Delay in
SC formation
Axes morphogenesis
is delayed
Defects in ZIMs
recruitment
Synapsis between
nonhomologs
Precocious SC
formation
SUN-1
PC Proteins (ZIMs)
HTP-3
HIM-3
SC component (SYP-1)
Figure 4.1: Illustrative model of CCT-4 mediated timely axes assembly. The
homolog pairs are shown in red and green, and different proteins are shown with the
indicated colours.
A: Homolog pairing and synapsis require a well-formed
chromosome axes necessary for the proper recruitment of the ZIMs, formation of
SUN-1/ZYG-12 patches at nuclear periphery, and the formation of SC. B: In
cct-4(vv39), axes morphogenesis is delayed at early meiosis in which SC formation is
not as robust in comparison to wild-type, but the recruitment of the ZIMs and synapsis
are still sustained at later stages. C: In him-3(vv6), the chromosome axes morphology
is defective. ZIM loading is defective even in later stages, resulting in autosomal
alignment and nonhomologous synapsis defects. cct-4(vv39) rescues the him-3(vv6)
associated meiotic defects by restoring axes morphology and ZIMs recruitment. D: In
htp-1(gk174), SC components are loading precociously onto immature axes, resulting
pairing and nonhomologous defects. cct-4(vv39) suppresses the pairing defects of
htp-1(gk174) by delaying the loading of SC components. Therefore the homologs can
paired appropriately without the interference of premature synapsis.
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Glossary
Centromere: Region of DNA where two sister chromatids are in contact, also acts as the
point of spindle attachment.
Chaperone: Protein that binds to other polypeptides, preventing their aggregations and
promoting their folding and/or assembly into multimeric complexes.
Chaperonin: Members of the Hsp60 class of chaperones.
Chromosome axes: A proteinaceous structure formed between sister chromatids, which
have been referred to as axial elements or chromosome cores. They are referred to as
lateral elements in the context of SC.
Chiasma (plural, chiasmata): Specific point of attachment between homologous
chromosomes observed at the diplotene stage of prophase I. It is the site on the
chromosome at which genetic exchange during crossing over had previously occurred.
Chromatid: One of the two side-by-side replicas produced during DNA replication.
Chromatin: The substance of chromosomes; including DNA and chromosomal proteins.
Crossing-over: The exchange of corresponding chromosome parts between homologs by
breakage and reunion.
Conformation: The three-dimensional arrangement of the atoms within a molecule.
Domain: Region within a protein that folds and functions in a semi-independent manner.
Gamete: Cell type in diploid organisms that carries only one set of chromosomes and is
specialized in sexual reproduction.
Germline: The cell lineage in a multi-tissued eukaryote from which the gametes derive.
FISH (fluorescence in situ hybridization): In situ hybridization using a probe coupled to
a fluorescent molecule.
Homolog: A member of a pair of homologous chromosomes.
Homologous chromosomes: Paired chromosomes of diploid cells, each carrying one of
the two copies of the genetic material carried by that chromosome.
Homologous alignment/pairing: close juxtaposition of homologous chromosomes during
early meiosis before the initiation of synapsis.
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Homologous recombination: A type of genetic recombination in which nucleotide
sequences are exchanged between homologous chromosomes. Homologous
recombination in meiosis results in either chromosomal crossover or non-crossover.
Linkage Group: Group of genes that reside on the same chromosome.
Nondisjunction: The failure of homologs or sister chromatids to separate properly to
opposite poles.
Nonhomologous chromosomes: Chromosomes that are not members of the same pair.
Nonhomologous synapsis: A phenomenon in which synapsis is occurring in between
nonhomologous chromosomes.
Pairing Center (PC): cis-acting regions located near the end of each chromosome that are
involved in homologous pairing in organisms such as flies, maize and worms. In C.
elegans, these regions are located at near one end of each chromosome and have been
implicated in promoting pairing, initiation of synapsis and recombination.
Synapsis: A term used to describe the connection of homologues via the SC.
Synaptonemal complex (SC): A ladder-like protein structure that holds each pair of
homologous chromosomes together. It aligns along the axis of each homologous
chromosome forming the “lateral elements”. Additional proteins involved in the
formation of transverse filaments interconnecting the lateral elements, forming the
“central region” of the SC.
Telomere: Region of repetitive DNA found at the end of chromosomes.
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