SUMMARY
Fertilization of aneuploid gametes often leads to pregnancy loss or disorders such as Down’s syndrome.
Aneuploidy occurs in 10-30% of human gametes and typically arises from chromosome segregation errors during
meiosis. Meiotic events such as homologous chromosome pairing, double strand breaks (DSBs), recombination
and the formation of crossovers, ensure proper chromosome segregation. Nevertheless, the mechanisms that
regulate these events are incompletely understood. Consequently, there are currently few to no strategies to
predict or prevent aneuploidy during gametogenesis. To overcome some limitations in the field, we established
a fluorescence reporter-operator system (FROS) based on the lac operator-lac repressor (lacO-lacR) paradigm,
which enables protein targeting to distinct genomic regions. Our long-term goal is to elucidate the mechanisms
that regulate chromosome segregation during meiosis and thus ensure the formation of gametes with a normal
karyotype. Aim 1 use FROS to dissect rapid chromosome motions in mouse spermatocytes at prophase I. We
will also combine FROS with long-term 3D measurements of chromosome motions in seminiferous tubules to
directly test whether homologous chromosome pairing arises from increasingly productive interactions (reeling
in), versus reiterative rounds of transient interactions (catch and release). Additionally, we will analyze mutant
spermatocytes by FROS to determine how key players, including components of the Linker of Nucleoskeleton
and Cytoskeleton (LINC) complex and the synaptonemal complex, affect homologous and non-homologous
interactions at specific loci. Dynein and microtubules interact with the LINC complex and are known to contribute
to RPMs. Aim 2 will identify additional motors and cytoskeleton components that support these movements,
building on our preliminary data uncovering kinesins as candidate molecular motors involved in generating
RPMs. We will extend these findings with unbiased proteomic approaches, functional assays and FROS to
decipher the dynamic forces that govern homolog pairing. Aim 3 will investigate the mechanism underlying the
association between ANKRD31 and pro-DSB factors at recombination hotspots. Using FROS, we will target
ANKRD31-GFP-lacI to the lacO region in spermatocytes, which we predict will recruit pro-DSB factors and
recombination proteins. We will also analyze the effect of ANKRD31-GFP-lacI on downstream recombination
intermediates at lacO sites. The proposed research will for the first time discern how chromosome context affects
the mechanisms underlying chromosome segregation and thus advance new knowledge of fundamental meiotic
processes and the causes of aneuploidy.
