Characterization of a novel htp-3(vc79) mutant
in Caenorhabditis elegans
Camille Delouche
Department of Biology
McGill University, Montréal
July 19th 2016
A thesis submitted to McGill University in partial fulfillment of the
requirements of the degree of Master of Science.
© Camille Delouche, 2016
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Table of contents
Abstract……………………………………………...………………………………………3
Résumé……………………………………………..……………………………………….4
Acknowledgments…………………………………..………………………………………6
List of figures………………………………………..……………………………………...7
Chapter 1: Introduction
Meiosis overview…………………………………………………………..……………..…9
Caenorhabditis elegans as a model organism……………………………..……………….12
Chromosome cohesion, pairing and synapsis in C. elegans………………..……………...13
Recombination in C. elegans………………………………………………..……………..16
HORMA domain.……………………………………………………………..……………18
HTP-3 functions………………………………………………………………..…………..20
Chapter 2: Characterization of htp-3(vc79)
General meiotic characteristics…………………………………………………………….24
Meiotic protein localization in htp-3(vc79) ……………………………………………….27
Double Strand Break formation in htp-3(vc79) ……………………………………...……29
HTP-3 expression levels……………………………………………………………...……30
Suppressor screen………………………………………………………………….………31
Chapter 3: Materials and Methods
Caenorhabditis elegans culture…………………………………...………………………..33
Staining……………………………………………………………..…………………...…33
Microinjection…………………………………………………………………………..….34
RNAi by feeding and by injection…………………………………………………………34
Western blot……………………………………………………………………………..…35
Chapter 4: Future direction, discussion and conclusion
Future direction…………...………………………………………………………………..36
Discussion….……….………..…………………………………………………………….38
Conclusion.………….………...……………………………………………………………41
References……………….…………………………………………………………………42
Figures…………….………………………………………………………………………..48
2
Abstract
The stereotyped meiotic pairing and the subsequent synapsis that keeps the
homologous chromosomes together for the shuffling of genetic material to take place is
central to meiosis. In the nematode Caenorhabditis elegans, HTP-3 holds a vital role in the
assembly of the synaptonemal complex (SC) that polymerizes throughout the length of the
homologs during synapsis. HTP-3 has established roles in pairing and synapsis, but also in
double strand break (DSB) formation, a necessary first step in the aforementioned exchange
of genetic material. HTP-3 contains a HORMA domain, which is a protein-protein interaction
domain conserved amongst proteins with roles ranging from apoptosis to the spindle
assembly checkpoint (Aravind, 1998; Jao, 2013). We know that HTP-3 has a central role in
meiosis, but the molecular mechanism by which it performs its functions is unclear because
the protein is involved in so many processes. Characterization of the htp-3(vc79) allele, which
contains a mutation in the conserved HORMA domain of HTP-3, has allowed me to dissect
the functions specifically of the HORMA domain regarding the numerous roles of HTP-3.
HTP-3 loading on the SC is abrogated in the htp-3(vc79) mutant, where instead meiotic
aggregates of HTP-3 can be observed in pachytene nuclei. Analysis of this mutant uncovered
that an intact HORMA domain in HTP-3 is necessary for its co-loading on the axis with
REC-8, a component of the cohesion complexes that keep the sister chromatids together until
meiosis II. However, DSBs still occur in the mutant indicating that its HORMA domain is
dispensable for this particular function. Further experiments could investigate how the
mutation in htp-3(vc79) affects the binding partner landscape of HTP-3 in order to shine light
on meiotic processes such as pairing, synapsis and DSB formation.
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Résumé
L'appariement méiotique suivi de la synapse qui maintiennent les chromosomes homologues
ensemble pour que le brassage de matériel génétique puisse avoir lieu sont au cœur de la
méiose. Chez le nématode Caenorhabditis elegans, HTP-3 tient un rôle essentiel dans
l'assemblage du complexe synaptonémal (synapton) qui polymérise sur toute la longueur des
homologues lors de la synapse. HTP-3 possède des rôles établis dans l'appariement et la
synapse, mais aussi dans la formation de cassures double brin, une première étape nécessaire
pour l'échange de matériel génétique mentionné ci-dessus. HTP-3 contient un domaine
appelé HORMA, qui est un domaine d'interaction protéine-protéine conservé parmi des
protéines dont les rôles vont de l'apoptose a une fonction dans le point de contrôle
d’assemblage des fuseaux (Aravind, 1998; Jao, 2013). Nous savons que HTP-3 joue un rôle
central dans le processus de la méiose, mais le mécanisme moléculaire par lequel il exerce
ses fonctions est encore inconnu parce que la protéine est impliquée dans de nombreuses
étapes. Caractérisation de htp-3(vc79), qui contient une mutation dans le domaine HORMA
conservé de HTP-3, m'a permis de disséquer les fonctions spécifiques de ce domaine. Le
chargement de HTP-3 sur le synapton est abrogé dans le mutant htp-3(vc79); des agrégats de
HTP-3 sont observés a la place dans les noyaux de pachytènes. L'analyse de ce mutant a
révélé qu'un domaine HORMA intact est nécessaire pour la coordination du chargement de
HTP-3 avec REC-8 sur l'axe, un composant des complexes de cohésion qui maintiennent les
chromatides sœurs ensemble jusqu'à la méiose II. Cependant, des cassures double brin se
produisent encore dans le mutant indiquant que son domaine HORMA n’est pas nécessaire
pour cette fonction particulière. Des expériences futures vont étudier comment la mutation
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dans htp-3(vc79) affecte le paysage des partenaires de liaison de HTP-3 afin de faire la
lumière sur les processus de la méiose, tels que l’appariement, la synapse et la formation de
cassures double brins.
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Acknowledgements
I want to start by thanking my supervisor Monique Zetka for giving me the
opportunity to immerse myself into meiotic research and her caring disposition. Thanks to
professors Richard Roy, Gary Brouhard and Christian Rocheleau for their time and advice.
Hvala najlepše to Aleks, my best friend luckily before, thankfully during and
hopefully after my graduate studies, without whom I would not be where I am today.
I am very thankful for the presence at my side of fellow lab mates and dear friends
Anja and Hanh, from the very beginning to the very end. I also enjoyed the CRISPR and
diaper talks with Annina and Sara *pat*
For knowing me better than I know myself and for not hesitating to talk about
anything (or write letters), I owe the world to my parents and Magigi! I am also grateful to
my adoptive family on rue Drolet!
Special thanks to some wonderful people who supported/guided me one way or
another: Kaan, Kevin, the birds, Alex, and Gabriel :)
I think, tonight, I’m gonna get Kantapia.
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List of figures
Figure 1. C elegans gonadal organization
Figure 2. Summary of htp-3(vc79) phenotype
Figure 3. Analysis of diakinesis nuclei in htp-3(vc79) via DAPI stainings
Figure 4. Analysis of DSB formation in htp-3(vc79) via RAD-51 stainings
Figure 5. Pachytene localization of HTP-3 and REC-8 in htp-3(vc79) germlines Figure
6. Pachytene localization of HTP-3 and SYP-1 in htp-3(vc79) germlines
Figure 7. Pachytene localization of REC-8 and SYP-1 in htp-3(vc79) germlines
Figure 8. Western blot of wild type, htp-3(vc79) and htp-3(tm3655)
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Chapter 1: Introduction
Meiosis is a type of cellular division conserved within sexually reproducing
organisms that produces the highly specialized, haploid gametes that transmit the genetic
information to offspring. It is similar to mitosis, which occurs in somatic cells, in that both
require highly regulated chromosome segregation to the daughter cells. In both meiosis and
mitosis, the genetic material is first doubled, so that the cell contains two homologous
chromosomes (maternal and paternal), consisting of two sister chromatids each. While the
only division in mitosis generates two diploid cells, meiosis consists of two successive
divisions that generates four haploid cells. In the first meiotic division, the homologous
chromosomes segregate to the daughter cells; in the second, the chromatids are separated.
The second major difference between mitosis and meiosis is that meiosis often yields
novel combinations of maternal and paternal genetic material through the mechanism of
meiotic recombination, ultimately allowing for genetic diversity. Double stranded breaks are
generated throughout the chromosomes and some are selected to be repaired as cross overs
using the nearby homolog, which generates recombinant chromosomes. This is accompanied
by the creation of a physical linkage between them called chiasmata, which is necessary for
accurate segregation later in meiosis. Meiotic recombination is a regulated process that relies
on proteins that are highly conserved from yeast to mammals. Errors occurring in the
execution, timing and regulation of the steps of meiosis often lead to chromosome
missegregation (reviewed by Hassold, 2001). Aneuploidy, or the abnormal number of
chromosomes in a cell, very often gives rise to a lethal or diseased phenotype and is in fact
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the leading genetic cause of miscarriages as well as the most common reason behind mental
retardation (reviewed by Hassold, 2001; reviewed by Lui, 2013). Despite the importance of
this process for the continued survival of sexually reproducing organisms, an extensive
amount of research is still necessary to decode the exact mechanism of this process.
Meiosis overview
Meiosis is a multistep process that relies on faithfully controlled changes made to the
structure of the chromatin. These structural changes are induced by proteins that are loaded
onto and unloaded from the chromatin in a highly regulated, timely fashion. An important
step in chromatin reorganization is chromosome condensation. Two classes of Structural
Maintenance of Chromosomes (SMC) proteins are particularly important for controlled
reorganization of the chromatin: condensin and cohesin (Miyazaki, 1994; Chan, 2004; Lee,
2013). Overall chromosome condensation is regulated by the amount of condensin
complexes associated with the chromatin at a particular moment of time, while Sister
Chromatid Cohesion (SCC) is regulated by cohesin complexes (Chan, 2004; reviewed by
Díaz-Martínez, 2008). Condensin and cohesin complexes load and unload from chromatin
both before and throughout meiosis, in manners dictated by the necessities of the DNA at a
specific point in time. DNA needs to be more condensed to facilitate chromosomal movement
for example, and sister chromatids need to be held together sometimes (meiosis I) but not at
other times (meiosis II) [reviewed by Miyazaki, 1994; Chan, 2004].
Restructuring of the chromatin occurs within two distinct phases: meiosis I and
meiosis II, each made up of 4 distinct phases, defined by chromatin state or DNA position in
the nucleus. In prophase, chromosomes condense and, in prophase I, chromosomes move
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through the nucleus to find their homologous partners. In metaphase, chromosomes are
visible as condensed bodies congressing to the center of the division plane, called the
metaphase plate. In anaphase, the chromosomes are pulled to opposite poles of the cell
thanks to a proteinaceous complex controlling microtubule shortening and elongation.
Division ends in telophase, with the actual separation of the DNA and the cell material into
two daughter cells (cytokinesis). Each of these four stages are repeated twice in each cell
without any additional DNA replication, which halves the number of chromosomes from the
starting material, thereby producing haploid gametes. Changes to the nuclear envelope such
as dissolution and reformation occur in parallel to the DNA modifications (Barer, 1960;
reviewed by Greenstein, 2005). Because the first meiotic division concerns homologous
chromosomes whereas the second concerns sister chromatids, prophase I and II are especially
distinct. Amongst the differences is that prophase II is significantly shorter; consisting mostly
of the nuclear membrane break down in a mitosis-like manner. The synaptonemal complex
holding the homologs together needs to dissolve before they separate during anaphase I; and
similarly, the cohesin complexes holding the sister chromatids together are removed before
anaphase II (Schild-Prüfert, 2011).
At the beginning of prophase I, homologs move around in the nucleus until they have
found their correct homologous partner chromosome (reviewed by Page, 2003). They then
undergo pairing followed by a more intimate association along their entire length in a process
called synapsis (reviewed by Walker, 2000; Peoples, 2002). Homologous chromosomes
create crossover points where genetic material is exchanged, and the physical bridges
(chiasmata) that result from crossover event hold the homolog together until they are
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dissolved (reviewed by Roeder, 1997 and Page, 2003). The paired homologs align at the
metaphase plate and are pulled apart during anaphase via microtubules attached to
kinetochores, proteinaceous structures which assemble on the centromeres (Albertson, 1982).
In the second round of cell division, the sister chromatids are already held together by cohesin
complexes and no genetic exchange should occur, so prophase II is less complex. The sister
chromatids align at the metaphase plate and are pulled apart by a similar mechanism as the
homologs; kinetochore microtubules (reviewed by Hillers, 2015).
A key event in meiosis is the exchange of genetic material between homologous
chromosomes, which occurs only in prophase I. Prophase I comprises 5 stages, which were
named after the cytological appearance of the chromosomes. The first stage is leptotene,
where the chromosomes can be seen as thin threads, and the sister chromatids are held
together by the cohesin complex (reviewed by Zickler, 1998). Synapsis, via the
polymerization of the synaptonemal complex (SC) between the homologs, starts during
zygotene (paired threads) and is fully complete at pachytene, where the chromosomes can
be seen as thick threads. During diplotene, the synaptonemal complex disassembles to form
two threads of chromosomes until diakinesis when the condensed chromosomes are seen
moving through the nucleus to align at the division plane.
In conclusion, meiosis is a highly coordinated process made up of numerous steps
during which DNA is accurately reorganized various times. This results in the generation of
gametes with half the starting number of chromosomes; as well as added genetic variation.
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Caenorhabditis elegans as a model organism
Caenorhabditis elegans (C. elegans) is an excellent model organism for studying
meiosis as their gonads are arranged in a spatiotemporal way, which allows you to monitor
the temporal progression of meiosis in the same animal (Francis, 1995). The five
chronological steps of prophase I of meiosis are spatially arranged from the distal tip
(premeiotic zone) through transition zone [leptotene/zygotene], followed by pachytene until
the diplotene zone towards the proximal end, just before diakinesis and the spermatheca
(Figure 1). Additionally, C. elegans has a short generation time of three days (Brenner, 1974)
and state-of-the art methods of interfering with the genetic or expression levels of proteins
are well established (RNAi and CRISPR [Fire, 1998; Friedland, 2013), making C. elegans
ideally suited for genetic studies. Lastly, this animal is highly amenable to microscopic
analysis, thanks to its translucent tissues and the relative ease of access to the gonads that
maintain the spatiotemporal organization of the different stages of meiosis even after
dissection.
C. elegans has six chromosomes, including five autosomes (I-V) and one sex
chromosome (X) which is either present in two copies (in hermaphrodites) or in one copy (in
males) [Brenner, 1974]. Males rarely occur in natural conditions, but mutations leading to
non-disjunction of the X chromosome lead to a drastic increase in the proportion of males in
the progeny, which is one of the indications of a meiotic phenotype (Him or high incidence
of males) [Ward, 1979; Hodgkin, 1979]. Non-disjunction of autosomes at meiosis leads to
high embryonic lethality (Emb), another phenotype that can be indicative of a meiotic defect
(Hodgkin, 1979).
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The C. elegans germline develops together with the somatic part of the gonad
(Sulston, 1977). The primordial germ-cells Z2 and Z3 are present in the first larval stage of
the animal. They develop mitotically throughout the larval stages L2 and L3 until mid-L4,
when the meiotic program is initiated (Kadyk, 1998). First, sperm is produced and stored
distally in the gonads, then oocytes are produced and pass the spermatheca to be fertilized
before their exit through the vulva. Adult animals have a reproductive lifespan of about 3
days, after which they stop laying eggs but live for up to 2-3 weeks after (Brenner, 1974).
Chromosome cohesion, pairing, synapsis in C. elegans
A well described exchange of genetic material between homologous chromosomes
occurs during prophase I. Several well-orchestrated mechanisms ensure that the homologous
chromosomes are held together long enough for recombination to be facilitated: cohesion of
sister chromatids as well as pairing and synapsis of homologous chromosomes (reviewed by
Walker, 2000 and Zetka, 2009).
In C. elegans, Sister Chromatid Cohesion (SCC) is mediated by a complex made up
of 4 components, all of which are necessary for a functional cohesin ring to firmly hold the
sisters next to each other (Onn, 2008). Two of the components are of the family of Structural
Maintenance of Chromosomes proteins (SMC-1 and SMC-3), the third component is a
kleisin (there are 3 partially redundant meiotic kleisins in C. elegans: REC-8, COH-3 and
COH-4; and one mitotic kleisin: SCC-1) and the last component is SCC-3 (Pasierbek, 2001;
Pasierbek, 2003; Brar, 2009). These assembled SCC complexes have ATPase activity and
their subunits contain hinge domains that allow dimerization and oscillation between an open
and closed conformation (Anderson, 2002). This is thought to be the mechanism by which
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cohesin complexes are assembled, presumably around adjoining sister chromatids, in order
to hold them close by until anaphase II when they segregate to daughter cells.
Cohesion via REC-8 is usually established during DNA replication, before
recombination and synapsis (Pasierbek, 2001). However, cohesion mediated by COH-3 and
COH-4 is independent of replication, but rather depends on meiotic recombination (Severson,
2014). Equational sister chromatid separation occurs during diakinesis in both hpt-3 and rec8 mutants, as can be explained most simply if COH-3 and COH-4 are sufficient for partial
cohesion (Pasierbek, 2001; Severson, 2009). The kleisins are partially redundant for cohesion
formation, as a complete loss of cohesion and random sister chromatid separation is observed
only in the triple kleisin mutant. However, COH-3 and COH-4 are not sufficient to provide
the cohesion necessary for DSB repair, as shown by the chromosome fragmentation
indicating unrepaired DSB in the rec-8 null allele rec-8(ok978) reported in 2009 by Severson
et al (Severson, 2009). In conclusion, cohesion via COH-3 and COH-4 seems to be dependent
on meiotic recombination but this established cohesion is only partial as it does not seem to
be sufficient for repair of these meiotic DSBs.
Cohesion must first be fully functional and sister chromatids must be able to act as
one entity before starting to look for their homolog partners throughout the nucleus in the
transition (leptotene and zygotene) stage of prophase I. Instead of moving randomly in a large
3D space, the chromatin is secluded to a section of the nucleus and one of the chromosome
ends is attached to the inner nuclear envelope, which is thought to render their special search
more efficient (Scherthan, 1996). Chromosome attachment to the inner nuclear membrane is
mediated through pairing center proteins that localize to the chromosome ends and connect
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to SUN-1 and ZYG-12 proteins, nuclear membrane spanning proteins that link the
chromosomes to the cytosolic microtubule network with motor proteins that is generating the
force for chromosome movements during pairing (Dernburg, 1998; Sato, 2009). The
movement transduction that results facilitates the chromosomal nuclear search until
homologs find each other as well as creates force to separate homologs that would otherwise
illegitimately synapse (Sato, 2009).
Pairing occurs if the chromosomes recognize each other as homologous, and
legitimate synapsis quickly follows via the polymerization of a heteromeric complex
throughout the length of the paired homologs, the synaptonemal complex (reviewed by
Walker, 2000). As opposed to other organisms, synapsis in C. elegans is not dependent on
the formation of DSBs and recombination initiation (Dernburg, 1998). The SC is made up of
lateral elements that load first on the cohesive sister chromatids (HTP-1,2,3 and HIM-3), and
of transverse elements that are dependent on the lateral elements for loading (SYP-1,2,3,4)
(Zetka, 1999; Couteau, 2005). The assembly of this complex occurs during the leptotene and
zygotene stages of prophase I, while the chromatin is segregated to one side of the nucleus
in a crescent shape, cytological characteristic of this region. A crucial meiotic protein called
HTP-3 holds a key structural role in the hierarchical assembly of proteins at the synaptonemal
complex by offering binding sites to other lateral elements of the SC at both its N-terminal
HORMA domain and its C-terminal tail (Kim, 2014). It is known that a number of proteins
partially co-localize with HTP-3 on chromosome tracks at different time points during
prophase I, including the C. elegans kleisins REC-8, COH-3/4 and the rest of the cohesin
complex proteins as well as the SC component proteins SYP-1 through SYP-4 (Severson,
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2009; Schild-Prüfert, 2011). In fact, REC-8 and HTP-3 are partially co-dependent for their
loading onto the axis, more specifically, REC-8 does not load in the absence of HTP-3, but
COH-3/4 can partially load HTP-3 on the chromosomal axes when REC-8 is absent
(Severson, 2009). Additionally, COH-3/4 load on the axis independently of HTP-3,
mediating an HTP-3 and REC-8-independent cohesion (Severson, 2009; Severson, 2014).
HTP-3’s recruitment to DNA has been thought to differentiate mitosis from meiosis, as it is
vital for a number of meiosis-specific processes including pairing, synapsis and double strand
breaks (DSBs) formation.
Recombination in C. elegans
DSBs are made during prophase I as the first step in homologous recombination
(Zickler, 2006; Lemmens, 2011). They are also necessary to produce chiasmata, the
cytological manifestations of the points of genetic exchange between non-sister homologous
chromosomes. Because these crossovers are the structures that keep the homologs together
towards the end of prophase I, wild type (WT) diakinesis nuclei contain six bivalents, or
twelve paired homologous chromosomes. The C. elegans protein responsible for creating
programmed meiotic DSBs is SPO-11 (Keeney, 1997), which explains why spo-11(ok79)
exhibits 12 DAPI stained bodies at diakinesis; this is because when double strand breaks are
not made, no genetic exchange occurs which prevent chiasma formation and disrupt the
physical linkage between the homologs which are then seen as unpaired univalents (Dernburg,
1998; Lemmens, 2011). The programmed meiotic double strand breaks introduced by SPO11 can be selected to become crossovers, in which case all other DSBs on that chromosome
are repaired in a process called crossover interference (Meneely, 2002).
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Since germ cells will eventually give rise to entire organisms, there is an increased
incentive to tightly control repair of the programmed meiotic double strand breaks in these
cells. This is done by favoring homologous recombination (HR) (using preferably the
homologous chromosome as a way to later favor the formation of crossing over) over non
homologous end joining (NHEJ) for example, a very error prone DSB repair mechanism that
essentially consists in ligating the cut ends (Clejan, 2006; Hayashi, 2007). On the other hand,
homologous recombination includes the intertwining of DNA strands via RAD-51, a protein
that mediates strand invasion by bringing three DNA strands together (one from the damaged
homolog and two from the other homolog acting as the repair template) to bring a repair
template close to the DSB site to facilitate accurate repair (Alpi, 2003). This is the reason
why the C. elegans community uses RAD-51 foci as a marker for DSBs. In mutants such as
spo-11(ok79) where DSBs are not formed, no RAD-51 can be detected in meiotic gonads
(Alpi, 2003). RAD-51 foci reappear when these mutants are irradiated, indicating that
RAD51 not only labels programmed meiotic DSBs but also naturally occurring double strand
lesions such as those irradiation-induced (Alpi, 2003). Accurate repair of these meiotic
double strand breaks is crucial for faithful chromosome segregation as well as to avoid cell
cycle checkpoint arrest. When DSBs are not efficiently repaired, due to the lack of repair
template within close proximity for example, this usually results in numerous instances of
chromosome fragmentation (more than 12 DAPI stained bodies) such is the case for
rec8(ok078)[Pasierbek, 2001].
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HORMA domain
The HORMA domain is a protein-protein interaction domain, conserved amongst
multiple organisms including 3 Saccharomyces cerevisiae proteins (Hop1p, Rev7p and Mad2)
after which it was named as well as the four C. elegans SC components: HTP-1, 2, 3 and
HIM-3 (Aravind, 1998; Muniyappa, 2014; Vader, 2014; reviewed by Rosenberg, 2015). The
three original chromatin-associated yeast proteins in which the HORMA domain was first
identified are involved in meiotic synaptonemal complex assembly (Hop1p), in translesion
DNA synthesis (Rev7p) and in the mitotic spindle assembly checkpoint (Mad2) (Aravind,
1998). REV7 has also been associated with choice of repair pathway in double strand break
repair in mouse and human cell lines, indicating a conserved function of this domain in DSB
formation and repair (Xu, 2015). A HORMA domain was discovered in two additional yeast
proteins with clear roles in autophagy: Atg13 and Atg101 (Jao, 2013; Hegedűs, 2014). All in
all, this domain has been involved in diverse cellular pathways only occasionally related.
In MAD2, the most studied HORMA domain containing protein (both functionally
and structurally), the HORMA domain acts via a structural mechanism that comprises a
switch between a closed conformation where the interacting peptide is bound and an open
conformation where the safety belt replaces the interacting peptide (Sironi, 2002).
Additionally, Mad2 is known to utilize a significant amount of energy to convert from its
open conformation to bind motifs on its target protein Cdc20, thereby becoming active and
signaling to prevent anaphase from starting until all chromosomes are attached at the spindle
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(reviewed by Musacchio, 2015). This conformational change of Mad2 is a central aspect to
this checkpoint, without which chromosomes would start segregating without being properly
attached to kinetochores, thereby greatly increasing chances of meiotic errors.
In an attempt to determine how meiotic proteins organize themselves and perform
their function, Kim et al. used the crystal structure of Mad2’s HORMA domain to annotate
the HORMA domain of HIM-3 (Kim, 2014). Kim and coworkers showed that the safety belt
of HIM-3 binds the C-terminus of that same protein, at specific loci now referred to as closure
motifs (Kim, 2014). Based on this, it is hypothesized that the HORMA domain identified in
HTP-3 functions via the same mechanism, being inactive in an open conformation with its
safety belt occupying its binding surface which can transition into an active closed
conformation when the safety belt releases the surface for binding to its interacting peptide.
The peptide that would bind to HTP-3’s safety belt remains to be identified. Synaptonemal
and cohesion proteins would be interesting candidate proteins to test for binding to HTP-3,
since their interaction with HTP-3 may be the mechanism of coordination of cohesion,
synapsis and axis formation.
HORMA domains contain 3 α-helices and 7 β-sheets, which fold into a structure
conserved amongst all proteins with this domain (Sironi, 2002). The nucleotide composition
of all HORMA domains is only conserved to the extent that the resulting folding structure is
the same in all proteins, but there are two specific highly conserved amino acids that stabilize
the HORMA domain structure via a hydrogen bond network (Aravind, 1998). One of these
two, the glutamic acid located in the 4th β-sheet, is mutated in the mutation of interest in this
study: htp-3(vc79).
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HTP-3 functions
HTP-3 mediates programmed meiotic double strand break formation in C. elegans (Goodyer,
2008). Kim and colleagues inquired whether HTP-3 itself or proteins dependent on it to load
on the axis were responsible for this function, so they used a strain in which Cterminus
closure motifs are mutated in an htp-3 null background (Kim, 2014). This rescue does not
bring back pairing and synapsis, nor does it allow proteins such as HTP-1 and HIM3 to load
on the axis, but it does rescue RAD-51 foci appearance and HTP-3 loading on the axis. This
suggests that HTP-3 itself is responsible for DSB formation (Kim, 2014). It is unclear if HTP3’s function in DSB formation is dependent on its localization on the axis though (Goodyer,
2008; Severson, 2014). While fully functional synaptonemal complex formation is
dispensable for loading of RAD-51 and recombination initiation (Colaiácovo, 2003), it
remains to be elucidated if cohesion is required.
Goodyer et al. showed that there are abundant RAD-51 foci in scc-3(RNAi), a strain
in which HTP-3 is unable to load on the chromosomal axis due to the dysfunctional cohesin
complexes (Goodyer, 2008). However, in Severson et al. in 2014, it was shown that there are
almost no RAD-51 foci in a triple kleisin mutant (rec-8; coh-3/4) and the authors
hypothesized that this could be due to the absence of HTP-3 on the axis in this background
since at least one kleisin must be present for appropriate HTP-3 loading (Severson, 2014). It
seems inconsistent that in both scc-3(RNAi) and rec-8; coh-3/4 triple mutant, HTP-3 is in
aggregates at pachytene but only the former is able to generate DSBs (Goodyer, 2008;
Severson, 2014). In fact, there is another similarity between the two backgrounds: both are
defective in cohesion for a similar reason, which is that they are each missing one of the
20
components of the cohesin complex. Due to the absolute requirement for all components of
the cohesin complex for functional cohesion, this results in both strains exhibiting 24 DAPI
stained bodies (Wang, 2003; Severson, 2014). Since these two backgrounds are so similar, it
is therefore unclear why there is a discrepancy in RAD-51 foci formation. A potential
explanation is that a kleisin might be necessary for DSB formation independently of its
requirement for HTP-3 loading. Although REC-8 cannot load on the axis in scc-3(RNAi) due
to the inability of HTP-3 to load, COH-3 and COH-4 are independent of HTP-3 for axis
loading so this might indicate a potential role for these two other meiotic kleisins in DSB
formation. However, as mentioned before, COH-3 and COH-4 are not sufficient to provide
the cohesion necessary for DSB repair (Severson, 2009). This is consistent with the
chromosome fragmentation also observed in htp-3(vc79), where it is presumed COH-3 and
COH-4 protein expression and cellular localization is not affected.
An allele which was isolated in the same mutagenesis experiment as htp-3(vc79) was
has already been examined: htp-3(vc75) [Gilchrist, 2006]. It’s a missense mutation that
doesn’t cause an immediately visible phenotype: the worms do not exhibit the typical
embryonic lethality and high incidence of males that are clear signs of a meiotic mutant
(Couteau, 2011). However, about 30% of germlines exhibit moderate levels of axis
separation in pachytene nuclei that does not seem to be a consequence of DSB formation but
is rather linked to the DNA damage response (Couteau, 2011). These sections of
chromosomes without a proper synaptonemal complex eventually resolve as the
characteristic 6 DAPI stained bodies are present at diakinesis and there is no particular
phenotype after that (Couteau, 2011). This implicates HTP-3 in proper chromatin structure
21
formation necessary for DNA damage response further in pachytene. Major questions that
remain unanswered in the field concern the mechanism of HTP-3 loading onto the
chromosome axis as well as the significance of its co-dependence with the kleisin REC-8.
We previously isolated a strain, htp-3(vc79), that contains a mutation in a highly conserved,
potentially structurally important, residue of the HORMA domain of HTP-3. Interestingly,
HTP-3 protein is present in htp-3(vv79) mutant worms, but does not localize to the DNA, but
in aggregates in the nucleoplasm of pachytene nuclei, making this mutant a powerful tool to
study the mechanism of axis recruitment of HTP-3 and allowing us to dissect the
axisindependent functions of HTP-3. Therefore, this project’s goal is to use htp-3(vc79) to
better understand aspects of meiosis such as the loading and interactions of two central
proteins in C. elegans meiosis: HTP-3 and REC-8. Regarding the latter, there’s been very
recent evidence that REC-8 loading on the axis in mouse could prevent non-homologous
synaptonemal complex formation (Agostinho, 2016). In fact, evidence from the same model
organism indicate that HORMA-domain containing proteins are actually removed from the
axis after a synapsis has been established, via energy provided by an ATPase protein called
TRIP13, that is not part of the cohesin complex despite having the same molecular activity
(Wojtasz, 2009). Research still needs to be done to understand exactly the context in which
HTP-3 and REC-8 are co-dependent and the significance of it. Additionally, although there
is some evidence that HTP-3 does not need to be loaded on the axis to promote DSB
formation, it was thought that an intact HORMA domain was necessary, but this study shows
that a mutation in a conserved residue of the HORMA domain merely decreases incidences
of RAD-51 foci rather than completely abolish them.
22
We know that all of the aforementioned proteins are major players of meiotic
pathways, playing varying roles together with HTP-3 to orchestrate pairing, synapsis and
subsequent processes as accurately as possible. Further experiments are necessary to
determine how a mutated HORMA domain can promote DSB formation.
23
Chapter 2: Characterization of htp-3(vc79)
General meiotic characteristics
The allele htp-3(vc79) was created using the strong mutagen EMS (ethyl
methanesulfonate) at a dose of 25 mM in a screen that identified a total of 71 mutations in
10 genes screened (Gilchrist, 2006). Since EMS induces mutations in an unbiased manner
throughout the genome, the mutagenesis was followed by TILLING (Targeting Induced
Local Lesions IN Genomes) to localize the point mutations generated (Gilchrist, 2006).
TILLING makes use of a single strand DNA nuclease (CEL-1) that cuts mismatched DNA
(i.e. if a mutated and a wild type strand anneal), causing those strands to run at a lower height
on a denaturing gel and therefore making them easily identifiable. This method is often used
to identify mutations in a target gene following a treatment with a nonspecific mutagen like
EMS. The mode of action of EMS is such that it most commonly generates GC to AT
transition mutations. More specifically, the ethyl group of EMS alkylates guanine base pairs,
and as a result of which DNA polymerases subsequently identify the guanines as adenines;
and therefore during DNA replication, a thymine is added opposite the altered guanines.
Sequencing of htp-3(vc79) revealed that the mutation it contains is a G to A transition (GAG
to AAG), which corresponds to glutamic acid (E) to lysine (K) substitution on the protein
level (Figure 2). Both are polar but glutamic acid is negatively charged, while lysine is
positively charged. The substitution occurs at amino acid position 121, located in a β-sheet
of the conserved HORMA domain in the N-terminal of HTP-3.
It was important to start my exploration of this mutant by carefully analyzing the
24
general phenotypes of the worms, specifically to investigate whether there was anything
noticeably different from the wild type. I first noted that htp-3(vc79) animals did not look
healthy and grew slower than wild type on plates; most adult animals were morphologically
affected by this mutation. Additionally, I could observe a significant number of males and
dead embryos, which were all indications that HTP-3 function was affected in this mutant.
To start investigating htp-3(vc79), I decided to quantitatively look at the most common
markers of meiotic defects: embryonic lethality and incidence of males. Both result from
improper chromosomal segregation or non-disjunction; that of autosomes (chromosomes I
to
V) resulting in embryonic lethality and that of the X chromosome in high incidence of males.
As expected, both are markedly higher than wild type (embryonic lethality is 91% and
incidence of males is 16% vs. 0% for both in wild type), indicating a strong meiotic
phenotype (Figure 2). This corroborated my initial observation that these mutants were not
displaying wild type behaviour. Of note is the fact that some meiotic mutants exhibit much
higher percentages of males, in the range of 30-40%, which makes the htp-3(vc79) phenotype
unexpected.
After it became evident that the mutant exhibited a meiotic phenotype, I wanted to
determine the status of the mutated protein by checking the pattern of localization in the adult
germ cells of HTP-3 and a protein often associated with it; the cohesin REC-8. The gonad of
C. elegans is organized into several spatial zones which correspond to specific temporal
points in the pre-meiotic and meiotic program. In wild type adult gonads, HTP-3 can be seen
on the DNA in pre-meiotic/mitotic nuclei from where it transitions to the synaptonemal
complex as early as the transition zone, and it is the first protein to be loaded on the forming
25
axis (Goodyer, 2008). REC-8 can also be seen in the same pre-meiotic/mitotic nuclei; it
eventually transitions to the axis in a manner co-dependent with HTP-3 (Pasierbek, 2001).
Subsequently, HTP-3 stays on the axis throughout pachytene, until it adopts a cruciform
shape on diakinesis bodies, on both the short and long arms of each bivalent (Goodyer, 2008).
In htp-3(vc79) adult gonads, the loading of HTP-3 on the chromatin appears comparable to
wild type in the mitotic zone based on its colocalization with DAPI (data not shown). This
region would normally be followed by the transition zone (corresponding to leptotene and
zygotene in meiosis prophase I), but this region is cytologically indistinguishable in
htp3(vc79). The next and largest segment of the meiotic gonad contains germ cells in
pachytene, where the most striking phenotype of the htp-3(vc79) allele can be observed: the
ubiquitous presence of large aggregates of HTP-3 in all nuclei of this zone. Most often, only
one aggregate per pachytene nucleus is visible, with few exceptions. This indicates a
complete inability of HTP-3 to transition from chromatin to form the base of the axis, as
detected cytologically. In the last section of the meiotic gonad called diakinesis, which
precedes metaphase I, the aggregates disappear and HTP-3 is not detected anymore (Figure
2). Since
HTP-3 usually persists until diakinesis (Goodyer, 2008), it is interesting to note that in
htp3(vc79), the aggregates are resolved by removing the protein altogether.
In diakinesis nuclei, the chromosomes are condensed in such a way that they can
easily be seen cytologically after 4′,6-diamidino-2-phenylindole (DAPI) staining of DNA.
By then, they should be linked to their partner homologs by chiasmata, and be seen as six
distinct DAPI-stained bodies (bivalents), since there are six chromosomes in C. elegans. In
meiotic mutants where crossing over is deficient, such as the null htp-3(tm3655) (Severson,
26
2009), we therefore expect 12 univalents (or unpaired homologs). Mutants of other meiotic
proteins such as HTP-1, HIM-3 and SYP-2 also display 12 univalents at diakinesis
(MartinezPerez 2005, Couteau 2004, Colaiácovo 2003), and the same is true of htp-3(tm3655)
diakinesis nuclei. In htp-3(vc79), the spread of DAPI-stained body count is statistically
different from the null allele, with a flatter distribution and no clear peaks (Figure 2).
Condensation is WT-like in most nuclei of htp-3(vc79) and htp-3(tm3655) (data not shown).
Meiotic protein localization in htp-3(vc79)
So far, it had been established that HTP-3 did not localize in a wild-type pattern
throughout the adult gonad, specifically that it was unable to load on the chromosomal axis
from the chromatin where it is thought to localize prior to the start of the meiotic program.
Since it is also known that numerous major players of meiotic processes also load on that
axis in a manner dependent on HTP-3, one way to approach the analysis of htp-3(vc79) is to
investigate the nature of the protein aggregates. This can be done by testing whether these
aggregates contain meiotic proteins that usually rely on HTP-3 to load on the chromosomal
axis, such as REC-8 and SYP-1 (Goodyer, 2008; Severson, 2009). Since it is known that
REC-8 and HTP-3 colocalize on chromosomal tracks in wild type pachytene nuclei and that
REC-8 does not load on the chromosomal axis in HTP-3 null mutant (Severson, 2009), I
wanted to start with this protein and test whether it would be in the meiotic aggregates with
HTP-3 in htp-3(vc79) gonads. In 90% of stainings, or 57 gonads, REC-8 cannot be detected
in pachytene nuclei, but in 10% of stainings, or in 6 gonads where it is detected, it does not
colocalize with the HTP-3 aggregates (Figure 5). In all gonads stained, there is REC-8
loading onto the pre-meiotic zone chromatin which disappears in the null allele of REC-8
27
called rec-8(ok978), indicating that the antibody is specific and that the REC-8 aggregates
are not an artifact of non-specific staining. To assess the significance of the potential
noncolocalization of REC-8 and HTP-3 in htp-3(vc79), or more specifically, if this pattern is
related to the mutation in HTP-3 or due simply to the displacement of HTP-3 from the axis,
I looked at another condition in which HTP-3 forms aggregates in pachytene nuclei, but is
not directly mutated: scc-3(RNAi) (Pasierbek, 2003). In these worms, the cohesin complexes
are not functional since every component of the complex is necessary for its assembly, and
therefore none of the downstream proteins load on the chromosomal axis, including HTP-3
which instead forms aggregates similar to the ones observed in htp-3(vc79). As expected, the
displacement of HTP-3 from the axis in scc-3 (RNAi) is accompanied by a displacement of
REC-8 from the axis as well, but the two colocalize in aggregates (Figure 5). This indicates
that the specific mutation in htp-3(vc79) is causative of a lack of colocalization between
REC-8 and HTP-3 in nuclei of this strain.
The mutation in the htp-3(vc79) strain is located in the N-terminus of the protein, and
it seems to abrogate an interaction with REC-8, which is consistent with the hypothesis that
the REC-8 interaction happens at the N-terminus of HTP-3, within the HORMA domain. On
the other hand, SYP-1 is expected to load through the recruitment of HIM-3, which is not
likely to be affected by the htp-3(vc79) mutation since it is mediated by the C-terminus (Kim,
2014). To investigate if that was the case, I stained for SYP-1 in both htp-3(vc79) and
scc3(RNAi) and compared its localization with HTP-3. I found that SYP-1 and HTP-3
aggregates colocalize in both backgrounds, indicating that the C-terminus of HTP-3 may
maintain its potential for interaction with other proteins (Figure 6, 7). This implies that the
mutation in the HORMA domain in this mutant may not affect the C-terminus folding and/or
28
interactions of HTP-3. This allele may consequently be a separation of function allele that
could allow us to determine which functions require the N-terminal and which require the Cterminal.
Double Strand Break formation in htp-3(vc79)
It has been previously established that a vital function of HTP-3 is to mediate
programmed meiotic DSB formation (Goodyer, 2008). However, there is conflicting
evidence, discussed in the introduction, about whether HTP-3 loading on the chromosomal
axis is necessary for the protein to perform this particular function. My preliminary analysis
of DSB formation after various manipulations of htp-3(vc79) had two aims: to determine
whether the HORMA domain of HTP-3 is required for DSB formation, and to confirm
whether loading on the SC is also required.
In order to answer these questions, I first compared the appearance of RAD-51 foci
in gonads of wild type, htp-3(tm3655) and htp-3(vc79). As expected, there are high levels of
RAD-51 foci throughout the wild type gonads which seem to be abolished in the HTP-3 null
(Figure 4). The interesting finding is that these foci are not completely abolished in htp3(vc79)
but simply decreased (data not shown), which is consistent with an overall phenotype that is
less severe than htp-3(tm3655). It is yet to be determined whether these are programmed
meiotic DSBs or whether something in pre-meiotic S phase triggered the appearance of these
cuts in the DNA. Next, I wanted to investigate whether any residual HTP-3 protein product
that may potentially load on the axis but not be detected by stainings could be sufficient for
DSB formation as measured by RAD-51 foci appearance. In order to assess this, I injected
dsRNA against htp-3 into wild type and htp-3(vc79) worm gonads and stained them with
29
RAD-51. I observed that in both cases, HTP-3 could be detected neither on chromosomal
tracks nor in aggregates, but that DSBs were still made to a level equivalent to htp-3(vc79)
(Figure 4). This could be a result of a partial RNAi, indicating that a very low, cytologically
undetectable amount of HTP-3 protein is sufficient for DSB formation. HTP-3
RNAi was functioning in wild type gonads as evidenced by a clear increased number of
DAPI-stained bodies at diakinesis (data not shown). To determine whether these results are
true, particularly because observing RAD-51 foci in htp-3(RNAi) is not consistent with what
has been published previously (Goodyer, 2008), the experiments should be repeated.
However, there is evidence to suggest it is a bona fide result: high levels of RAD-51 foci in
wild type and inexistent foci in spo-11(ok79) suggest these foci are not stainings artefacts,
and the clear RNAi phenotype indicates at least a partial removal of HTP-3 from these worms.
My preliminary findings are consistent with the interpretation that HTP-3 axis localization
and a functional HORMA domain are not required for DSB formation, at least not for a
minimal level.
HTP-3 expression levels
I was also interested in determining whether the levels of HTP-3 in htp-3(vc79) were
different than in wild type or if the issue was elsewhere, such as a localization difference or
a difference in quantity or type of post translational modifications on the protein. To assess
this, I examined levels HTP-3 protein by Western blot. The HTP-3 band present in wild types
disappears in htp-3(tm3655), which is consistent with this band representing the correct
30
protein product. However, HTP-3 was not detectable by Western blot in htp-3(vc79), which
is not consistent with the cytological data (Figure 8). This is an unusual result since antibodies
are usually known to detect proteins more reliably on Western blots than
immunohistochemistry (Kurien, 2011). This could be due to a technical matter regarding the
solubility of the aggregates present in htp-3(vc79). Other proteins known to be present in the
same aggregates, such as SYP-1, should be tested in the same Western blot to determine
whether the issue is with HTP-3 itself or with the meiotic aggregate.
Suppressor screen
In order to pursue my inquiries into the requirements for loading of HTP-3 onto the
chromosomal axis, I decided to perform a forward genetic screen in hopes of identifying
suppressors of htp-3(vc79). I was looking for phenotypic rescue of the high embryonic
lethality and incidence of males in these worms. This would have allowed me to obtain a list
of compensatory mutations that would have opened new lines of inquiry for this project to
be continued. I initially performed a pilot ethyl methanesulfonate (EMS) screen at 33.5 mM
on 500 homozygous htp-3(vc79) worms that yielded no suppressors. The worms with the
lowest embryonic lethality and brood size had values that did not statistically differ from the
values for htp-3(vc79) (data not shown). I decided to design a larger screen with a much
larger P0 generation made up of 2000 homozygous htp-3(vc79) at a higher dose of EMS
(50mM) to increase the chances of finding suppressors. This screen gave rise to no
suppressors although there were 3 revertants; in which the htp3-(vc79) allele was reverted
back to wild type sequence. The binomial probability formula revealed a low (6%) chance of
this screen yielding 3 revertants. From the screens, we could have retrieved revertants,
31
intragenic suppressors and/or extragenic suppressors. The fact that neither screen yielded any
suppressors could indicate that any additional mutation in htp-3 is very lethal (intragenic
suppressor), or that the mutation causes a drastic change at the protein level that is not easily
compensated by change in another protein such as a binding partner (extragenic suppressor).
Since the interaction between REC-8 and HTP-3 seems to be abrogated in htp-3(vc79) based
on cytological evidence, I hypothesized that a compensatory mutation in REC-8 might come
up as an extragenic suppressor but this was not the case. Since htp-3(vc79) was isolated from
an EMS screen, it implies that this locus is not entirely refractory to mutations via this
chemical yet my screens did not yield any suppressors; this could be explained if the
htp3(vc79) allele cannot easily be compensated for.
32
Chapter 3: Materials and Methods
Worm culture:
Worms are kept on solid NGM plates made with 20g agar, 0.55g Tris HCl, 0.24g Tris
base, 3.1g bacto-peptone, 2g NaCl and 0.008g cholesterol with distilled H2O up to 1L which
is autoclaved to sterilize it before being poured and streaked with OP50 bacteria.
Staining:
L4 worms of the desired genotype were isolated on plates at 20 degrees one day
before staining. The slides were coated twice at 95 degrees with the following solution:
100µL polylysine, 100µL of gelatin 2%, 10µL of chrome alum and 790µL of distilled water.
The worms were dissected in 25µL of PBS x1 after which as much of the liquid as possible
was removed and replaced with 11µL of 1% PFA (in PBST). A cover slip was added, the
extra liquid was removed and the slide was left at room temperature for 5mn before
undergoing freeze cracking in liquid nitrogen. After which the cover slip was removed and a
series of washes were started: first methanol for 1mn, then 4 washes of PBST for 5mn each.
After the washes, the samples were blocked with BSA in PBST for 2 hours, after which 50µL
of primary antibodies were applied and the slides were stored at 4 degrees overnight. The
next day, after 4 washes of PBST BSA for 10mn, 50µL of secondary antibodies were applied
on the slide for 2h at room temperature. Following that waiting period, 4 washes of PBST
for 10mn were done then 13uL of DAPI in vectashield was put on the slide and another cover
slip was applied and sealed with nail polish after 1 hour. For antibodies, HTP-3 guinea pig
was used at 1:500 dilution, REC-8 commercial mouse (Abcam ab38372) was used at 1:100
dilution, SYP-1 goat was used at 1:100 dilution, RAD-51 rabbit was used at 1:1125.
33
Microinjections:
The master mix for injection was prepared at a concentration of between 0.5 and 5
ug/uL of dsRNA. A Leica DM-IRB inverted fluorescence microscope was used to perform
microinjections. Each day previous to a microinjection section, a plate with L4 worms was
prepared (larval stage 4, the last stage before adult molt – recognizable via their white
crescent shaped vulva unique to this larval stage). These young adult worms were
microinjected the next day in both distal gonadal arms, releasing an amount of mix large
enough to swell the gonad every time. The worms were left to recover at room temperature
for an hour before singling on fresh plates. They would then grow and lay their progeny at
20 degrees. At 70h post-injection, the P0 (HTP-3 RNAi injections) were dissected, fixed, and
stained as described above.
RNAi:
scc-3(RNAi) feeding clone is from Ahringer RNAi library, it is plated on IPTG-Amp
plates (concentration is 1mM and 100ug/mL respectively). Worms of the desired genotypes
are put on as L4 stage larvae, allowed to lay eggs and the progeny is dissected 24h after the
L4 stage. L4440 plasmid is used as a control. RNAi experiments are performed at 20° C.
HTP-3 RNAi is PCR amplified from the Ahringer RNAi library clone templates using
T7 universal primers, the DNA is then purified (using a Bio Basic Column PCR Purification
Kit) before an in vitro transcription (using an Ambion MEGAScript T7 Kit) generates the
RNA which is purified again using 3 rounds.
34
Western Blot
Plates with >100 worms of the desired genotypes were washed off, and rinsed with
PBS 3 times to collect the animals. Sample protein buffer (31mL H2O, 4mL glycerol, 2.4 mL
Tris-Cl 1M pH 6.8, 0.8g SDS, 4mg bromophenol blue, 0.5 mL β-mercaptoethanol) was added
in a 1:1 ratio before 3 cycles of 5mn at 95° and 5mn at -20°. Proteins and 7uL of ladder were
loaded in the stacking gel (6%) which consists of 5.2 mL dH2O, 2 mL 30% PAA, 2.5 mL
Tris pH 6.8 (1M), 100 uL SDS 10% as well as 100uL APS and 10uL TEMED added right
before pouring. This gel is located above the resolving gel (10%) which consists of 6.3 mL
dH2O, 5.33 mL 30% PAA, 4 mL Tris pH 8.8 (1.5M), 160uL SDS 10% as well 160uL APS
and 16uL TEMED added right before pouring. The combined gel, once loaded with the
proteins, is first run at 80V for 30mn (or until the dye reaches the end of the stacking gel),
then at 120V for 90mn (or until the dye just about runs out of the gel) in 1x running buffer.
It is then transferred to a membrane by being run at 100V for 80mn in transfer buffer (150mL
of 10x running buffer, 300mL of methanol, 1.5mL of 10% SDS, water up to 1.5L) in the
following order: negative pole - sponge - filter papers – gel – membrane - filter papers –
sponge – positive. The membrane is then blocked overnight with blocking buffer at 4° while
shaking. Primary antibodies are added diluted in the same blocking buffer, and the membrane
rests at 4° overnight while shaking again. After 3 washes of 10mn in blocking buffer, the
secondary antibodies are added for 2h at room temperature before additional 5 washes of
10mn in blocking buffer. Development using an ECL solution is then performed to visualize
the protein bands and the ladder.
35
Chapter 4: Future direction, discussion and conclusion
Future direction
There are a number of experiments that could be done to further our understanding
of synaptonemal complex protein loading, pairing and cohesion in C. elegans meiosis, as
well as to supplement this analysis of htp-3(vc79). First, the inconsistent nature of the REC8
aggregates in pachytene nuclei of htp-3(vc79) would require a Western blot for REC-8 to
determine protein expression levels. Since REC-8 is present in mitotic nuclei, it is easy to
wonder about the fate of the protein since it cannot be detected in stainings in the gonadal
zones following the first one. It can either be targeted for destruction or become extremely
diffuse throughout the nuclei, or even be post translationally modified such that the antibody
does not recognize the protein product anymore. A Western blot should determine whether
REC-8 is targeted for degradation or if another scenario should be considered.
The fundamental explanation for the lack of HTP-3 loading on the chromosomal axis
is still elusive. Is HTP-3 unable to undergo a conformational change which prevents it from
polymerizing along the SC, or does the mutation abolish binding, or perhaps does it enhance
some binding strength and change the dynamics of HTP-3 binding partners? An experiment
useful in determining the landscape of HTP-3 partners in htp-3(vc79) is a
coimmunoprecipitation. This will determine which proteins differentially bind HTP-3 in wild
type vs. htp-3(tm3655) vs. htp-3(vc79) and will be a first step in exploring the consequences
of the amino acid change with regards to binding of the HORMA domain to other domains
or proteins. Of course, feasibility of this experiment is yet to be determined since HTP-3 was
not detected in Western blots of htp-3(vc79) adult worms.
36
A number of findings emerge from the distribution of DAPI-stained bodies, including
that there is a minimal amount of DNA fragmentation as evidenced by the number of nuclei
in which there are more than 12 univalents. To determine whether this DNA fragmentation
is due to unrepaired DSB, spo-11(RNAi) could be introduced in gonads of htp-3(79) animals.
Since the number of nuclei with more than 12 univalents is fairly small, it will be hard to
detect a statistically significant difference after spo-11(RNAi) but it is worth a try.
htp-3 mutants are clearly defective in pairing but only HIM-8 stainings (for
preliminary X chromosome specific data) and FISH analysis as was done in Goodyer et al.
in 2011 can establish with certainty the extent of the deficiency; it is expected that htp3(vc79)
will have different pairing efficiency than htp-3(tm3655) that should be consistent with the
statistical differences between their DAPI-stained body counts (Goodyer, 2011).
Lastly, an additional experiment would be useful to better understand why there are
remaining RAD-51 foci in gonads of wild type gonads injected with htp-3(RNAi) despite
numerous lines of evidence pointing towards a successful RNAi: injecting htp-3(RNAi) in
htp-3(tm3655). It is clear that since there aren’t be any RAD-51 foci in htp-3(tm3655), there
shouldn’t be any either after injection of htp-3(RNAi). If a different result is obtained, this
might explain the presence of RAD-51 foci in the previously reported experiment when none
were expected. Since the RAD-51 antibody is different from the one previously published
(Goodyer, 2008), it is possible that it may behave differently and this experiment would test
that.
Of course, it would still be valuable to perform the suppressor screen again since it
has not given rise to any successful candidates so far, but the validity of the experiment
remains. Perhaps it would lead to a more successful outcome to increase the concentration of
37
EMS in order to increase the chances of hitting the locus of interest, or have a larger starting
pool of worms to mutate to increase chances of obtaining a suppressor.
Discussion
It is vital for the survival of sexually reproducing organisms that the number of
mistakes in meiosis be minimized or inexistent. Errors in chromosome segregation lead to
abnormal amount of DNA in daughter cells which in turn lead to serious disorders or even
halts in the embryonic development as soon as a vital gene is discovered to be missing
(Hassold, 2001). Thus we establish the importance of studying this cellular process, by which
haploid gametes are generated in a significant percentage of living organisms. Numerous
details of this pathway are still to be uncovered, including the mechanism and regulation
involved in HTP-3 loading on the synaptonemal complex from the chromatin where it is
located in pre-meiotically dividing nuclei. Additionally, the particular reason for the
requirement of HTP-3 in double strand break formation is still unknown, as well as the exact
significance of its predicted interaction with the C. elegans meiotic kleisin REC-8. It is with
these specific purposes in mind together with the aim to understand pairing and cohesion
better, that I delved into the characterization of htp-3(vc79).
First, I discovered that although the htp-3(vc79) exhibited embryonic lethality that
was consistent with its overall meiotic phenotype, its incidence of males (16%) was
particularly low compared to some other meiotic mutants. For example, syp-1 mutants
(transverse element of the synaptonemal complex) as well as him-8 mutants (X chromosome
pairing center) exhibit 38% males (MacQueen, 2002; Phillips, 2005); while zhp-3 mutants
38
(component of the complex localizing at recombination events) show 28% males (Bhalla,
2008). However, another class of meiotic mutants seem to have incidence of males in the
same range of numbers as does htp-3(vc79): plk-2 mutants (kinase with a clear role in
chromosome pairing and movement) display around 7% males (Harper, 2011; Labella, 2011)
while him-3 mutants (lateral element of the synaptonemal complex) and rec-8(RNAi)
(meiotic kleisin, element of the cohesin complex) exhibit 11% males (Hodgkin, 1979;
Pasierbek et al., 2001). Therefore, it seems that amongst all proteins known to have roles in
meiosis, some affect non-disjunction of the X chromosome more than others. It would appear
that sex chromosome pairing in C. elegans is particularly resistant to lack of HTP-3 at the
axis, perhaps because a different mechanism of control is in place for this particular set of
chromosomes.
Next was revealed that htp-3(vc79) had a DAPI stain body distribution statistically
different than wild type (6 bivalents) as well as htp-3(tm3655) in that it had no clear peak
around 12 univalents as could have been expected. Since REC-8 requires HTP-3 loading on
the axis to load itself, one can expect a mutant in which HTP-3 is unable to load to show a
phenotype similar to a REC-8 null mutant. There are two ways to describe what is observed
when REC-8 is depleted or mutated; it has been reported that there are usually 24 DAPI
stained bodies, loosely paired throughout the nuclei as was reported by Pasierbek et al (2001)
in an rec-8(RNAi) background, or that there are 12 DAPI stained bodies with a cohesion
defect leading to a slight separation between the sister chromatids, as was reported more
recently by Severson et al. (2009) and again in 2014 when looking at rec-8(ok978) (Paseribek,
2001; Severson, 2009; Severson, 2014). Data shown in this study support the evidence that
39
rec-8(ok978) shows a clear peak of 12 DAPI stained body count. There are two other meiotic
kleisins that can also mediate cohesion (COH-3/4) so we don’t expect the full separation of
all 24 sister chromatids in diakinesis nuclei of rec-8(ok978), in fact, the consensus is that
there are only about 12 univalents in this mutant and they all exhibit the typical partial sister
chromatid separation: a clear line demarcating the two lobes on each univalents while these
are still held together.
As expected because of the loading co-dependence of HTP-3 and REC-8, a peak of
11 to 12 univalents can be found in htp-3(tm3655) diakinesis nuclei similar to the situation
in rec-8 mutants. However, instead of a predictably similar phenotype in htp-3(vc79), it
exhibits a much larger variability of DAPI stain body count. Two main differences between
htp-3(tm3655) and htp-3(vc79) is that HTP-3 is most likely completely absent throughout the
gonads of the null allele but loads appropriately in mitotic nuclei and is present in aggregates
in pachytene nuclei in the gonads of the other allele. Therefore, the lack of phenotypic
similitude with rec-8 mutants might be due to an interaction between REC-8 and HTP-3 in
mitotic nuclei lacking in htp-3(tm3655) but remaining in htp-3(vc79), which is able to
mitigate the lack of REC-8 on the chromosomal axis later on in pachytene. Alternatively, this
could be due to unexpected repercussions in meiotic nuclei containing an aggregate since this
is a very unnatural situation for a cell that can easily cause unanticipated consequences.
We know that the meiotic aggregates of HTP-3 in htp-3(vc79) are able to colocalize
with the meiotic aggregates of SYP-1 while not being able to colocalize with REC-8 (when
observed). Of note here is that although it is unclear why REC-8 cannot be consistently
observed in gonads of htp-3(vc79), it seems that if HTP-3 is completely absent so is REC-8,
40
and if HTP-3 is present in aggregates during pachytene, REC-8 has the potential to be in
aggregates as well. Staining data presented here is consistent with the model put forth by
Kim et al (2014) which proposes that the N-terminal of HTP-3 (with the HORMA domain)
mediates interaction with REC-8 leading to cohesion and loading on the axis while its
Cterminal mediates synapsis via interaction with SYP-1 (Kim, 2014). Since the mutation in
htp-3(vc79) is located in the HORMA domain of HTP-3, it seems to prevent interaction with
REC-8 but the C-terminal of the protein does not seem to be affected.
Conclusion
In conclusion, this project was undertaken in an attempt to further understand meiotic
chromosome pairing and cohesion, as well as protein loading at the synaptonemal complex,
by performing a thorough analysis of the novel mutant htp-3(vc79). Results obtained bring
strength to the idea that the HORMA domain of HTP-3 needs to be properly folded and not
contain any mutations, at least not at the most conserved amino acid sites, for HTP-3 to load
onto the chromosomal axis and perform its pairing functions. However, neither an intact
HORMA domain nor HTP-3 localization to the chromosomal axis seems necessary for
meiotic double strand break formation, at least to a minimal level. Further experiments such
as a co-immunoprecipitation or a large scale suppressor screen would help focus the direction
this project may take in the future.
41
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