Biology subjects

Day, L.

Publications and source records attributed to Day, L..

5 recordsLinked to original sources

bioRxiv2022

The Mycobacterium tuberculosis protein O-phosphorylation landscape

Bacterial phosphosignaling has long been synonymous with the histidine kinases of the two component systems, but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignaling enzymes in eukaryotes, but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria remains unclear. Here, we explored the global signaling capacity of the STPKs in Mtb. We generated STPK loss-and gain-of-function strains and measured the resulting O-phosphorylation and transcriptional changes. This deep phosphoproteome shows that O-phosphorylation in Mtb is an underexplored protein modification that affects >70% of the proteome. The substrate-STPK interactions show an extensive interface with the transcriptional machinery, resulting in regulation of gene expression of [~]30% of Mtb genes. Mtb O-phosphorylation gives rise to an expansive, distributed, and cooperative network of a complexity that has previously only been associated with eukaryotic phosphosignaling networks.

microbiology↗

bioRxiv2020

Massively parallel identification of causal variants underlying gene expression differences in a yeast cross

Sequence variation in regulatory DNA alters gene expression and shapes genetically complex traits. However, the identification of individual, causal regulatory variants is challenging. Here, we used a massively parallel reporter assay to measure the cis-regulatory consequences of 5,832 natural DNA variants in the promoters of 2,503 genes in the yeast Saccharomyces cerevisiae. We identified 451 causal variants, which underlie genetic loci known to affect gene expression. Several promoters harbored multiple causal variants. In five promoters, pairs of variants showed non-additive, epistatic interactions. Causal variants were enriched at conserved nucleotides, tended to have low derived allele frequency, and were depleted from promoters of essential genes, which is consistent with the action of negative selection. Causal variants were also enriched for alterations in transcription factor binding sites. Models integrating these features provided modest, but statistically significant, ability to predict causal variants. This work revealed a complex molecular basis for cis-acting regulatory variation.

genetics↗

bioRxiv2019

Rare variants contribute disproportionately to quantitative trait variation in yeast

A detailed understanding of the sources of heritable variation is a central goal of modern genetics. Genome-wide association studies (GWAS) in humans1 have implicated tens of thousands of DNA sequence variants in disease risk and quantitative trait variation, but these variants fail to account for the entire heritability of diseases and traits. GWAS have by design focused on common DNA sequence variants; however, recent studies underscore the likely importance of the contribution of rare variants to heritable variation2. Further, finding the genes that underlie the GWAS signals remains a major challenge. Here, we use a unique model system to disentangle the contributions of common and rare variants to a large number of quantitative traits. We generated large crosses among 16 diverse yeast strains and identified thousands of quantitative trait loci (QTLs) that explain most of the heritable variation in 38 traits. We combined our results with sequencing data for 1,011 yeast isolates3 to decouple variant effect size estimation from allele frequency and showed that rare variants make a disproportionate contribution to trait variation as a consequence of their larger effect sizes. Evolutionary analyses revealed that this contribution is driven by rare variants that arose recently, that such variants are more likely to decrease fitness, and that negative selection has shaped the relationship between variant frequency and effect size. Finally, we leveraged the structure of the crosses to resolve hundreds of QTLs to single genes. These results refine our understanding of trait variation at the population level and suggest that studies of rare variants are a fertile ground for discovery of genetic effects.

genetics↗

bioRxiv2017

Genetics of trans-regulatory variation in gene expression

Heritable variation in gene expression provides a critical bridge between differences in genome sequence and the biology of many traits, including common human diseases. However, the sources of most regulatory genetic variation remain unknown. Here, we used transcriptome profiling in 1,012 yeast segregants to map the genetic basis of variation in gene expression with high statistical power. We identified expression quantitative trait loci (eQTL) that together account for over 70% of the total genetic contribution to variation in mRNA levels, allowing us to examine the sources of regulatory variation comprehensively. We found that variation in the expression of a typical gene has a complex genetic architecture involving multiple eQTL. We also detected hundreds of eQTL pairs with significant non-additive interactions in an unbiased genome-wide scan. Although most genes were influenced by a local eQTL located close to the gene, most expression variation arose from distant, trans-acting eQTL located far from their target genes. Nearly all distant eQTL clustered at 102 \"hotspot\" locations, some of which influenced the expression of thousands of genes. Hotspot regions were enriched for transcription factor genes and altered expression of their target genes though both direct and indirect mechanisms. Many local eQTL had no detectable effects on the expression of other genes in trans. These results reveal the complexity of genetic influences on transcriptome variation in unprecedented depth and detail.

genetics↗

bioRxiv2017

Highly parallel genome variant engineering with CRISPR/Cas9 in eukaryotic cells

Direct measurement of functional effects of DNA sequence variants throughout a genome is a major challenge. We developed a method that uses CRISPR/Cas9 to engineer many specific variants of interest in parallel in the budding yeast Saccharomyces cerevisiae, and to screen them for functional effects. We used the method to examine the functional consequences of premature termination codons (PTCs) at different locations within all annotated essential genes in yeast. We found that most PTCs were highly deleterious unless they occurred close to the C-terminal end and did not interrupt an annotated protein domain. Surprisingly, we discovered that some putatively essential genes are dispensable, while others have large dispensable regions. This approach can be used to profile the effects of large classes of variants in a high-throughput manner.

genomics↗