Cover Letter

To: Research Fellowship Program Committee

From: Fenmiao Zhong

Date: November 2, 2023

Subject: Investigating how aging affects meiotic progression via double-strand breaks in

Caenorhabditis elegans

My research proposal involves the investigation of connections between meiotic double-strand

breaks (DSBs) and aging using the model organism Caenorhabditis elegans (C.elegans).

C.elegans is a suitable model for meiosis and aging study since it is simple, easily genetically

manipulated, has whole genome sequenced, contains conserved meiotic genes, and has a

transparent gonad for visualization of meiotic progression. In the proposal, I discuss 3 specific

aims of the research and experimental design to approach the aims. I will study the aging process

of C.elegans by carefully controlling worms’ chronological age and study the meiotic

progression by visualizing meiotic protein localization and chromosome morphology. Besides, I

will construct genetically manipulated worm strains that have elevated or reduced expression of

specific meiotic genes to study their regulatory roles in meiosis. Overall my research will help to

reveal the relationship between aging and DSB formation and repair processes in C.elegans

germline, which will provide insights into understanding the interplay between genome integrity,

aging, and fertility in not only C.elegans but also other eukaryotes undergoing sexual

reproduction.
 Investigating how aging affects meiotic progression via double-strand breaks in
Caenorhabditis elegans

Background:
Meiosis is a critical biological process in the life cycles of sexually reproducing organisms to generate
gametes. It consists of one round of DNA replication followed by two rounds of cell division, where
homologous chromosomes and sister chromatids segregate respectively during each cell division (Yu et
al., 2016). For accurate chromosome segregation, homologous chromosomes undergo pairing, synapsis,
and crossover recombination, leading to the formation of chiasmata and chromosome bivalents. Meiotic
recombination events start with programmed DNA double-strand breaks (DSBs) during the first cycle of
meiotic cell division. DSBs are generated by SPO-11, which is an evolutionarily conserved endonuclease.
A subset of DSBs are repaired via homologous recombination and are resolved to form crossovers (COs)
between homolog pairs (Rosu et al., 2013). DSBs are essential to promote crossovers on every
chromosome pair, but excessive DSBs can result in genome instability by introducing mutations into the
genome (Stamper et al., 2013). Therefore, DSB formation and repair need to be highly regulated to
balance the requirement for CO formation and genome integrity. Aging negatively impacts fertility and
gamete quality in many organisms and genome integrity is suggested to be important for maintaining
gamete quality during aging (Toraason et al., 2022). However, how aging affects meiotic progression
through genome integrity is still unclear.

Methods:
To study meiosis and aging, Caenorhabditis elegans (C.elegans) is a good model organism due to its
simplicity and amenableness for genetic manipulation and phenotypic analyses. Genetic manipulation
approaches such as crossing, CRISPR gene editing, and RNA interference will be utilized to obtain
worms of different genotypes. Viability, fertility, and male progeny frequency will be assessed in young
and old worms to evaluate gamete quality and genome stability. Immunostaining of DSB-related proteins
and visualizing via high-resolution microscopy will be performed in young and old worms to examine
chromosome morphologies, meiotic protein localization and function, and DSB levels.

Aims:
The research focuses on understanding the mechanisms of aging regulating meiotic DSB formation and
repair to affect genome integrity and meiosis progression.
Aim 1: Investigate how aging affects meiotic DSB levels via regulation of DSB initiation and repair
The programmed DSB level is an important aspect in genome integrity. To understand the mechanism of
aging effect, I first want to investigate DSB distribution in worms upon aging. To evaluate aging effects, I
will collect worms 1 days post L4 to 7 days post L4 for experimentation. To examine DSB levels, I will
perform immunostaining and quantification of RAD-51 foci in C.elegans germline, which marks meiotic
DSB sites. Appearance of RAD-51 indicates formation of DSBs and disappearance of RAD-51 indicates
repair of DSBs. RAD-51 staining in wildtype N2 worms of different ages will be conducted to provide a
baseline level of DSBs over time. DSB levels upon aging will also be investigated in spo-11 mutants and
dsb-2 mutants which are defective in DSB initiation and rad-54 mutants which are defective in DSB
repair, which will provide insight into whether aging changes DSB levels via affecting the initiation
process or repair process.
Aim 2: Investigate how aging affects gamete quality via regulating DSB levels
To understand how changes in DSB levels upon aging affect gamete quality and fertility, I will assess
gamete production and viability of progeny by counting the number of hatched and unhatched eggs
produced by C.elegans N2 wildtype hermaphrodites, where the percentage of viable progeny is a marker
for gamete quality. I will also assess male progeny frequency in N2, where the presence of males is an
indicator of chromosome missegregation and defects in meiosis. Similar experiments will be performed in
spo-11, dsb-2, and rad-54 mutants to assess gamete quality and meiotic progression in mutants with
known defects in the DSB process. This will help illuminate the correlation between meiotic DSB levels
and fertility.
Aim 3: Investigating potential regulators of meiotic DSB levels in the aging process
DSB levels are mediated by CHK-2 activity, a master regulator of meiotic progression in C.elegans (Yu et
al., 2016). If CHK-2 activity exhibits an age-dependent change, it is likely that aging affects DSB levels
via regulating CHK-2 activity. I will assess potential changes in CHK-2 activity by measuring the length
of the transition zone designated by phospho-HIM/ZIM staining over time in wildtype and mutant worms
to examine if there is a correlation between DSB levels and CHK-2 activity, and if changes in CHK-2 can
explain age-dependent changes of DSB levels. Then I will upregulate or downregulate CHK-2 activity in
the young and old worms via genetic approaches and compare their phenotype to examine if changing
CHK-2 activity can mimic aging effects on DSB levels in the germline.

Impact:
C.elegans have many revolutionarily conserved genes, so knowledge of molecular mechanisms and
cellular processes of C.elegans can facilitate understanding of essential biological processes in other
eukaryotes. The research will shed light on how aging mediate DSB levels and potential regulators in the
age-dependent pathway. Understanding the connections between genome integrity, fertility, and aging will
help to investigate age-related infertility or reproductive problems as well as other diseases associated
with aging processes.

References:
1. Raices M, Bowman R, S695333molikove S, Yanowitz JL. Aging Negatively Impacts DNA Repair
and Bivalent Formation in the C. elegans Germ Line. Front Cell Dev Biol. 2021;9:695333.
doi:10.3389/fcell.2021.
2. Rosu S, Zawadzki KA, Stamper EL, et al. The C. elegans DSB-2 protein reveals a regulatory network
that controls competence for meiotic DSB formation and promotes crossover assurance. PLoS Genet.
2013;9(8):e1003674. doi:10.1371/journal.pgen.1003674
3. Stamper EL, Rodenbusch SE, Rosu S, Ahringer J, Villeneuve AM, et al. (2013) Identification of
DSB-1, a Protein Required for Initiation of Meiotic Recombination in Caenorhabditis elegans,
Illuminates a Crossover Assurance Checkpoint. PLOS Genetics 9(8): e1003679. doi:
10.1371/journal.pgen.1003679
4. Toraason E, Adler VL, Libuda DE. Aging and sperm signals alter DNA break formation and repair in
the C. elegans germline. PLoS Genet. 2022;18(11):e1010282. doi:10.1371/journal.pgen.1010282
5. Yu Z, Kim Y, Dernburg AF. Meiotic recombination and the crossover assurance checkpoint in
Caenorhabditis elegans. Semin Cell Dev Biol. 2016;54:106-116. doi:10.1016/j.semcdb.2016.03.014