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Research Projects
Background
Meiosis,
a specialized cell cycle with a unique mode of chromosome segregation
Projects
The
control of DNA double-strand break formation
The
mechanism of meiotic recombination
The
distribution of meiotic recombination
Meiosis:
a specialized cell cycle with a unique mode of chromosome segregation
The conversion of diploid into haploid cells takes place during the meiotic
cell cycle: this cycle involves a single S phase followed by two successive
divisions. At the first division (reductional) homologous chromosomes
segregate from each other, at the second (equational) sister-chromatids
segregate. In terms of chromosome behavior, the second meiotic division is
similar to a mitotic division, whereas the reductional one is unique to meiotic
cells. The reductional segregation requires three events unique to meiotic
chromosomes: a specific regulation of sister-chromatid cohesion, the
establishment of at least one connection per chromosome arm and a monopolar
orientation of sister kinetochores at metaphase. These connections,
visualized as chiasmata, are established by recombination events between
homologs that lead to crossing-over. In the absence of crossing-over,
chromosomes cannot position properly on the metaphase plate, and this induces
either an arrest of the meiotic cycle or a random segregation of homologs,
and thus gametes with abnormal chromosome contents. More subtle deficiencies
in crossing-over, such as reduced frequencies and/or changes in distribution
are correlated with defects in chromosome segregation as well.
The establishment of crossing-over at the
right time, with proper frequencies and distribution, requires a complex
series of events from the pre-meiotic S phase, through the various stages of
prophase I (leptotene, zygotene, pachytene, diplotene, diakinesis) . These
involve structural changes: alignment of homologous chromosomes (leptotene), nuclear
re-organisation of chromosomes, in particular illustrated by the clustering
of telomeres at the onset of zygotene (the bouquet stage), chromosome
compaction and intimate chromosome pairing (pachytene), along with the
establishment of exchanges at the DNA level that are initiated at the
leptotene stage by the formation of localized DNA double-strand breaks
(DSBs). These events can be visualized at the cytological level by electron
microscopy and immunofluorescence (see Fig.1,
some examples on mouse spermatocytes and oocytes) in most organisms and at
the DNA level in the yeasts S. cerevisiae and S. pombe. Studies
mostly developed in yeast have allowed to define the main lines of the
mechanism of meiotic recombination (see below) and to correlate events taking
place at the DNA and at the chromosomal levels (Fig. 2).
Our lab is interested
in several aspects of the mechanism and regulation of meiotic recombination
in mice in particular those related with early events, involved in the
control of DSB formation, and in the distribution of recombination events in
the genome. We are using the mouse, Mus
musculus, as a model system.
Recent reviews:
Petronczki
M, Siomos M. F., Nasmyth K. (2003) Un ménage à quatre: the molecular
biology of chromosome segregation in meiosis. Cell, 112: 423-440
Cohen
PE, Pollack SE, Pollard JW (2006) Genetic analysis of chromosome pairing,
recombination and cell cycle control during first meiotic prophase in
mammals. Endocr Rev 27: 398-426.
Buard
and de Massy. (2007) Playing hide and seek with mammalian meiotic
crossover hotspots. Trends in genetics. 23: 301- 309
Hunter
N (2007) Meiotic recombination. In: Aguilera A, Rothstein R, editors.
Molecular genetics of recombination. Berlin Heidelberg:
Springer-Verlag. pp. 381-442.
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Projects
1) The control of DNA double-strand breaks (DSBs) formation, analysis
of the mouse Spo11 function.
The initiation step
of meiotic recombination, defined by the formation of DSBs, is particularly
fascinating as it potentially defines the distribution and frequencies of recombination
events, and clearly has to be highly controlled to achieve the various levels
of regulation that have been so far observed:
- The large number of DSB formed (about 200
estimated in yeast, 250 to 400 predicted in mouse) have obviously to be
faithfully repaired, without exception.
- DSB repair takes place preferentially by
interaction with the homolog, not the sister chromatid
- The repair of a DSB can lead to crossover and
non crossover, this is a major level of regulation that is thought to be
defined at or soon after initiation to ensure a least one crossover per
chromosome.
In 1997, it was shown
in S. cerevisiae, that DSB formation occurs by the predicted
transesterase activity of the Spo11 protein which is homologous to the
catalytic activity of a family of type II DNA topoisomerase (Bergerat A, de
Massy B, Gadelle D, Varoutas PC, Nicolas A, Forterre P. 1997. An atypical
topoisomerase II from Archaea with implications for meiotic recombination.
Nature 386: 414; Keeney, S., Giroux, C.N. & Kleckner, N. 1997
Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of
a widely conserved protein family. Cell 88, 375-84). DSB formation thus goes
through a protein-DNA covalent intermediate. One of the very interesting
features of this reaction is that Spo11 is not thought to have topoII
activity, but the DSB generated are channeled towards repair by interaction
with the homologous chromosome, implying a dissociation step of the Spo11-DNA
intermediate and according to an unknown mechanism. The recent
characterization of Spo11-oligonucleotide complexes in yeast and mouse shows
that Spo11 is dissociated from DSB ends by an endonucleolytic mechanism
(Neale et al., 2005, Nature, 436, 1053).
Ten other proteins have been shown to be
required for DSB formation in S. cerevisiae, and although their
activities have not been defined yet, they probably act in the context of one
or several protein complexes (Fig.3).
With respect to
initiation, many features remain to be understood in yeast and our knowledge
in other eukaryotes is very limited still. Based on the conservation of one
protein, Spo11, the mechanism of initiation is predicted to be conserved
among eukaryotes. However, besides Spo11, the other yeast genes required for
this process do not have recognizable homologs in multicellular eukaryotes.
We have identified the mouse Spo11 homolog and develop molecular and cellular
approaches to analyze its mechanism of action and its regulation.
2) The mechanism of meiotic recombination, a direct molecular approach
in the mouse.
To overcome the
limitations of genetic analysis for studying meiotic recombination events and
their controls in mice, we have developed a direct approach to detect,
measure and map recombination events, directly in the mouse germ line (male
and female). This approach is essentially based on allele-specific PCR
amplification similar to the strategy initially developed by A. Jeffreys in
humans.
We have
thus analyzed in details the properties of the Psmb9 crossover hotspot
on chr.17 (the Psmb9 hot-spot in the MHC was first identified by the
group of T. Shiroishi and previously named Lmp2) and shown that:
- This
site is an initiation site from meiotic recombination
- Most
initiation is predicted to take place in a 210bp interval
- Crossover
(CO) and noncrossover (NCO) events can be detected at this hotspot
- Gene conversion tracts
associated with CO are in average 500bp long, whereas those associated with NCO are much shorter (average less
than 150bp)
- Both CO and NCO are Spo11 dependent
- 90% of CO are MLH1 and
MLH3 dependent, whereas NCOs occur at normal levels in MLH1 and in MLH3 KO
mice.
- These results show the
presence of two distinct pathways for NCO and CO formation, and an MLH1/3
independent pathway for CO formation
Further investigations of the recombination mechanism can be addressed
through the analysis of other mouse mutants.
3) The distribution of meiotic recombination events along chromosomes
Recombination events are not randomly distributed in the genome, as shown by
the distribution of DSBs in yeasts and by the comparison of genetic and
physical maps in the human genome for instance. What defines a target or a
substrate for the recombination initiation machinery is still unknown. The
primary DNA sequence might play a role, but the activity of a recombination
site is probably defined by a combination of several additional factors
including chromatin and chromosome structural properties.
We are developing approaches to understand
what determines a recombination initiation site in the mouse. For this
purpose we have begun to characterize in detail the Psmb9 site (see
above). We are interested to understand several regulatory aspect of Psmb9
activity: cis/trans control elements, epigenetic control, male/female
regulations, influence of polymorphism, timing of events, comparison
between various hot-spots, and relation with recombination activities in
adjacent regions. These projects are developed both by molecular and
cytological approaches.
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