Browsing by Subject "Population sequencing"

Fluctuations in nutrient availability occur for nearly all species, and adaptation to endure starvation conditions is essential. Genetic pathways involved in regulating starvation resistance are implicated in aging and complex diseases such as cancer, diabetes, and obesity in humans. Consequences of experiencing starvation persist later in life and subsequent generations, suggesting epigenetic regulation. However, much is still unknown about how starvation resistance is regulated and the contributions of different types of regulation. The roundworm Caenorhabditis elegans reversibly arrests development in the absence of food and can endure starvation for several weeks. Here, we investigate how transcriptional, epigenetic, and genetic regulation impact starvation resistance during developmental arrest in C. elegans. Gene expression dynamics change quickly during the first few hours of starvation, and broadly conserved transcription factors are required for starvation survival. However, temporal- and tissue-specific requirements of transcription for supporting starvation survival and recovery are largely unknown. In chapter 2, we used mRNA-seq combined with temporal degradation of RNA Polymerase II in the soma and germline to better understand gene regulation throughout arrest. We find that transcription is required in the soma for survival early in starvation but is dispensable thereafter, and known transcriptional regulators primarily act early in arrest. In contrast, the germline is transcriptionally quiescent throughout starvation, but germline transcripts are relatively stable compared to somatic transcripts. This reveals alternative gene-regulatory strategies in the soma and germline during starvation-induced developmental arrest, with the soma relying on a robust early transcriptional response while the germline relies on mRNA stability to maintain integrity. Phenotypic plasticity is facilitated by epigenetic regulation, and remnants of such regulation may persist after plasticity-inducing cues are gone, even affecting germ cells to impact subsequent generations. However, the relationship between plasticity and transgenerational epigenetic memory is not understood. Dauer diapause provides an opportunity to determine how a plastic response to the early-life environment affects traits later in life and in subsequent generations. In chapter 3, we find that, after extended diapause, postdauer worms initially exhibit reduced reproductive success and greater interindividual variation. In contrast, F3 progeny of postdauers display increased starvation resistance and lifespan, revealing potentially adaptive transgenerational effects. Transgenerational effects are dependent on the duration of diapause, indicating an effect of extended starvation. In agreement, RNA-seq demonstrates a transgenerational effect on nutrient-responsive genes. This work reveals complex effects of nutrient stress over different time scales in an animal that evolved to thrive in feast and famine. Many conserved genes and pathways regulate starvation resistance, but most genetic analysis in C. elegans has been restricted to a single genetic background, potentially restricting identification of additional genes. Hundreds of genetically distinct wild strains of C. elegans have been whole-genome sequenced and can be used for GWAS. In chapters 4 and 5, we implemented two high-throughput sequencing approaches, RAD-seq and MIP-seq, to determine relative starvation resistance of over 100 wild strains over time. We used GWAS to identify QTL associated with starvation resistance, near-isogenic lines to validate QTL, and CRISPR gene editing to modify specific genes within QTL. We focused on genes in the insulin receptor-like domain (irld) family, as this family has been virtually uncharacterized, but the genes share homology with the sole known insulin-like receptor in C. elegans, DAF-2, which is a major regulator of starvation resistance. We found that specific variants in two members of the irld family confers increased starvation resistance in multiple genetic backgrounds, and this is dependent on the transcription factor downstream of insulin signaling, DAF-16/FOXO. Thus, this work shows that natural genetic variation in novel modifiers of insulin-signaling regulates starvation resistance.

The advent of high coverage and low cost sequencing technologies has allowed for

newer and more powerful approaches in molecular and population genetics. Transposon

sequencing, where genome-saturated mutant populations allele frequencies are

measured before and after selection, functionally characterizes each and every gene

in the genome in a single experiment. The approach has been successfully applied

to a variety of phenotypes in a variety of unicellular systems: growth and motility

in E. coli, synthetic genetic interactions in yeast, and in vitro pathogen-resistance in

mammalian cell lines. However, transposon insertion typically produces null alleles,

which can be valuable to identify gene function, but evolutionary insight relies on

identifcation of naturally occurring polymorphisms affecting the trait of interest.

Genome-wide association studies (GWAS) can be used to study the effect of natural

genetic variation on a trait, but they grow prohibitively expensive if the number of

individuals to genotype and phenotype becomes large.

Here I describe the application of transposon sequencing and pooled sequencing

GWAS in the whole metazoan model, Caenorhabditis elegans. Transposon sequencing

has not been previously implemented in an animal model. I have sequenced a control

library using our method, C. elegans transposon sequencing (CeTnSeq). We have

constructed a new Mos1 transposon mutator strain that is more convenient to use

than the existing strain and allows for extra-chromosomal insertions to be degraded

by restriction digest. My preliminary results show that our method is qualitatively

effective at identifying transposon insertion sites, but suffers from PCR duplication

error. I propose to optimize the number of PCR cycles in the library and to include

unique molecular identifiers (UMI) in the library adaptor. I also show that the

restriction digest is effective at removing extra-chromosomal array insertions from

the library.

I constructed simulation models to help design optimal Ce-TnSeq experiments

with respect to statistical power for a proposed starvation survival assay. I considered

many parameters affecting the design, including: culture size, number of generations,

expected effect size, sequencing coverage, and sample size. I show that the number

of homozygous mutant animals in the screen is a critical factor in the design of

experiments. I also saw diminishing returns with respect to increasing sample size

and sequencing depth. These simulations will be invaluable in designing future Ce-

TnSeq experiments and identifying critical aspects of the protocol to optimize.

We performed pooled sequencing (using restriction-site associated DNA sequencing)

on a population of 95 wild isolates subjected to starvation. I identified strains

that were resistant and sensitive to starvation, and we verified these results using

traditional methods. We used our population sequencing data to perform an association

study of starvation survival across the 95 strains, and identified two statistically

significant quantitative trait loci.