A major goal in genetics is to directly measure the functional effects of DNA sequence variants throughout a genome. In the Kruglyak lab, I developed a method that uses CRISPR/Cas9 to engineer thousands of specific variants of interest in pools of the budding yeast Saccharomyces cerevisiae, and to screen them for functional effects. With the method, I examined the effects of premature termination codons in essential genes. Most premature termination codons were highly deleterious unless they occurred close to the C-terminal end and did not interrupt an annotated protein domain. Looking closely at the essential genes that were more PTC-tolerant, I found several that were only conditionally essential, as well as several that had large dispensable C-terminal ends. A manuscript describing these results was published in Nature Genetics.

Recombination is at the heart of mapping methods such as linkage analysis and GWAS, which use meiotic recombination events. Mitotic recombination could play the same role, but without the ability to target its occurrence, it normally occurs too rarely to be useful. In the Kruglyak lab, I developed a method to generate mitotic recombination at will at precise genomic loci. CRISPR-directed mitotic recombination has two major features. First, targeting recombination to the most desired loci allows us to identify causative polymorphisms much faster than using traditional methods. Second, since the method can be applied to diploid cells that can’t undergo meiosis, mapping could be possible in previously unapproachable scenarios, such as in sterile interspecies hybrids, or cell culture. I demonstrated that CRISPR can be used to target mitotic recombination in S. cerevisiae with high accuracy, and demonstrated its power in rapid fine-mapping by identifying the major polymorphism underlying manganese sensitivity in lab yeast. This study was published in Science.

One of the quantitative genetics interests of the Kruglyak lab is to quantify the importance of genetic interactions, or epistasis. These are cases where a combination of genetic variants has a different effect on a trait than predicted by summing the individual effects of the variants. I contributed to a study led by Josh Bloom showing that genetic interactions consistently play a small, but statistically significant, role in complex trait variation, published in Nature Communications. I was also involved in the follow-up to this study by Simon Forsberg, published in Nature Genetics, which showed that genetic interactions are particularly important in predicting traits of individuals with rare genotypes.

The Rine lab is interested in chromatin regulation as well as the effects of nutritional deficiencies, and so naturally is interested in how the two may interact, especially if understanding these connections can elucidate or alleviate the effects of nutritional deficiency. In my graduate work, I examined how histone methylation is affected by folate deficiency, as histone methylation requires the cofactor SAM, whose synthesis requires folate. I found that methylation on lysine 4 of histone H3 was decreased by folate deficiency, in both S. cerevisiae and Schizosaccharomyces pombe, two widely diverged species. I further demonstrated that this chromatin change had downstream effects on gene expression. This was published as a featured article in Genetics.

Yeast synthesize methionine, cysteine, and SAM from a single intermediate metabolite, homocysteine. The genes involved in these pathways are known to be transcriptionally regulated by cysteine availability, but much of the mechanics of this regulation remains to be understood. I showed that lack of SAM also induces these pathways by blocking cysteine synthesis. How cysteine is sensed is unknown. We used mass spectrometry to show that a key regulatory protein, Met30, is oxidized on highly conserved cysteine residues specifically in response to cysteine deficiency. Cysteine oxidation has only recently been recognized as a regulatory signal. This study was published in Molecular Biology of the Cell.

One curious fact of yeast chromatin regulation is that the genes involved in transcriptional silencing show signatures of rapid evolution. SIR4, in particular, is one of the most rapidly evolving genes in the yeast genome. I was involved in a study led by Oliver Zill to understand whether there are functional consequences to this rapid evolution, published in Genetics. We observed that Sir4 from S. cerevisiae is not functional in S. bayanus. We hypothesized this diversification may result from a role for Sir4 in genome protection through directing transposon integration into heterochromatin.