Grant: Discovery Grants Program - Individual

The remarkable extent to which protein-coding genes are still functionally equivalent between humans and yeast emphasizes the power of distant organisms for studying human genes. In preliminary work, we've already humanized >400 strains. Remarkably, 47% of the human genes can complement a lethal growth defect conferred by loss of the corresponding yeast gene. Thus, the assays reveal which conserved processes are still interchangeable, creating direct assays of human gene function for the swappable subset.Aim 1. Humanize budding yeast. In Aim 1, We will focus on systematically humanizing approximately 1,000 yeast genes, one at a time or as systems in their entirety, at their native loci thus maintaining native expression, building reagents to study gene function and variation in a simplified organism. Specifically, by focusing on human genes that have acquired lineage specific duplication, we will test which of the co-orthologs' (paralogs') tends to retain function and why? Upon completion, this will be the most extensive, rigorous and systematic test of humanization ever performed resulting in many humanized yeast strains to be used in subsequent studies (as in Aim 2) while also being made freely available to the scientific community. Aim 2. Deep scanning mutagenesis to characterize the contribution of variation to human gene function.In Aim 2, I will humanize yeast with libraries of human gene variants and directly measure the impact of genetic variation on human gene function. We will focus on both replaceable and non-replaceable human genes while aiming to understand the contribution of genetic variation to functional replaceability. In Aim 2a, we will test whether variations in the non-replaceable human 20S core subunits can allow functional replaceability in yeast, thus identifying key residues/interactions that allow replaceability. The humanization paradigm allows us to test whether complete essential yeast and human systems are, to a first approximation, interchangeable. Thus, allowing investigations in the effects of local functional network on genetic variation. In Aim 2b, we will test the effect of genetic variation in the context of largely yeast and humanized local functional network on human genes belonging to sterol biosynthesis pathway.Aim 3. Humanize fission yeastFission yeast and budding yeast have diverged >400 million years ago and while many cellular processes are similar between them, many other processes like Telomere Shelterin complex, RNAi systems that are more alike in humans. Notably, many of these critical cell systems are non-replaceable in budding yeast. In Aim 3, we will perform systematic screen for human gene replaceability in fission yeast, thus, testing whether similar properties of orthologous genes predict replaceability (as in budding yeast) while also creating many new humanized yeast strains.