Biology subjects

Wittkopp, P. J.

Publications and source records attributed to Wittkopp, P. J..

16 recordsLinked to original sources

bioRxiv2026

Evolution of regulatory networks controlling plasticity in gene expression between Saccharomyces cerevisiae and Saccharomyces paradoxus

Organisms cope with environmental changes by modifying gene expression. To understand how regulatory networks controlling expression plasticity evolve, we analyzed RNAseq data from Saccharomyces cerevisiae, Saccharomyces paradoxus, and their F1 hybrids at multiple timepoints after transferring cells from standard laboratory conditions to five environments (low phosphorus, low nitrogen, hydroxyurea shock, heat stress, and cold stress) and during the diauxic shift. In each of the six datasets, we identified genes that changed expression following the transition to the new environment and used hierarchical clustering to identify genes that increased or decreased in expression. We then compared these classifications between orthologs to identify genes with divergent plasticity. For some genes, plasticity was more extreme in one species than the other, and for others, expression of orthologs changed in opposite directions when acclimating to the same environment. Most cases of plasticity divergence were seen only in one environment and were attributable primarily to trans-regulatory divergence. Using environment-specific regulatory networks inferred from data in Yeastract, we found that divergent plasticity of environment-specific transcription factors generally did not predict divergent plasticity of their target genes. We also found that, as a group, genes with conserved plasticity tended to have more regulatory interactions than genes with divergent plasticity. Interesting patterns of expression divergence were also observed for five transcription factors in the pleiotropic drug resistance network and their target genes that might contribute to phenotypic divergence. Together, these findings show how environment-specific trans-regulatory divergence and combinatorial gene regulation shape the evolution of expression plasticity.

evolutionary biology↗

bioRxiv2026

microRNAs affecting development of body pigmentation in adult Drosophila melanogaster

Phenotypic development is regulated by multiple mechanisms that ensure tight control of gene expression. Post-transcriptional regulation, including the silencing or degradation of messenger RNAs by microRNAs (miRNAs), is an important component of this process. Here, we use gain-of-function and loss-of-function screens to examine the effects of miRNAs on cuticular pigmentation in adult Drosophila melanogaster. We found that 48 of 166 miRNAs ectopically expressed in a stripe along the dorsal side of developing flies were each sufficient to affect pigmentation. We also found that 22 of 41 miRNAs competitively inhibited in the same tissue visibly altered pigmentation, showing that they were necessary for adult pigmentation development. For each of the 15 miRNAs with opposing effects in the gain- and loss-of-function screens, computational tools identified possible targets among 93 genes previously reported to affect adult pigmentation. Using cell culture, we found that one of these miRNAs (miR-8) was able to regulate gene expression through 3 UTR sequences from at least three pigmentation genes: ebony, bric-a-brac 1, and bric-a-brac 2. All three of these genes reduce development of black pigments, suggesting that miR-8 coordinately regulates expression of multiple genes with similar effects on pigmentation. These data show that miRNAs are developmental regulators of body pigmentation, which could also allow them to contribute to pigmentation divergence, as has been shown for miR-193 in butterflies.

genetics↗

bioRxiv2025

Trans-acting mutations reveal non-nuclear modulators of both intrinsic and extrinsic gene expression noise in a eukaryote.

Gene expression regulation is a stochastic process that can be modified by mutations not only in a deterministic manner but also in a probabilistic way, for example by changing the extent of cell-to-cell variability in gene expression (also called "gene expression noise"). Although systematic studies have successfully analyzed the properties of cis-acting mutations that modulate expression noise at their own locus, less is known about the type of genetic changes that alter expression noise in trans (at loci distant from the mutation). Here, we applied genetic mapping on yeast strains (Saccharomyces cerevisiae) generated by random mutagenesis and identified three mutations (in chs1, yme2 and msh1 genes) that changed the expression noise of a reporter gene in the nuclear genome regulated by the yeast TDH3 promoter. These mutations affected either extrinsic noise (variability due to cell-specific factors), intrinsic noise (inherent variability within each cell) or both, and their effects were found to not be specific to the TDH3 promoter. Surprisingly, all three mutations targeted proteins located outside of the nucleus: Yme2 and Msh1 being involved in the maintenance of mitochondrial genome integrity, and Chs1 being necessary for the repair of cell-wall defects in freshly-born daughter cells. Our results reveal that mitochondrial state can modulate the extent of intrinsic expression noise of eukaryotic nuclear genes.

genetics↗

bioRxiv2025

Natural variation in pigmentation caused by cis-regulatory changes affecting expression of the ebony gene in Drosophila elegans

Repetitive evolution has offered valuable insights into how similar traits can arise across different populations and species. The dimorphic body color morphs (brown and black) of geographically isolated Drosophila elegans Bock & Wheeler, 1972, along with its closely related species Drosophila gunungcola Sultana et al., 1999, which exhibits black morph, raise intriguing questions about whether these traits have emerged through independent evolution, introgression, or shared ancestral variants. To investigate the genetic and evolutionary origins of these differences, we perform quantitative trait locus (QTL) mapping on recombinant hybrids generated from reciprocal crosses between brown and black morphs D. elegans. QTL mapping reveals significant contributions from the X chromosome and Muller element E to intraspecific pigmentation polymorphism, with ebony identified as one candidate gene. Analysis of ebony expression, including allele-specific expression in F1 hybrids, and sequence variation suggests that the intra- and interspecific pigmentation differences within D. elegans species subgroup may have evolved independently from the lineage-specific region upstream of ebony. By identifying a shared candidate regulatory region associated with both intra- and interspecific variation, our findings suggest that recurrent use of the same genetic region may contribute to the emergence of similar traits across populations and species.

evolutionary biology↗

bioRxiv2025

Deep homology and design of proteasome chaperone proteins in Candida auris

A central tenet of biology is that protein structure mediates the sequence-function relationship. Recently, there has been excitement about the promise of advances in protein structure modeling to generate hypotheses about sequence-structure-function relationships based on successes with controlled benchmarks. Here, we leverage structural similarity to identify rapidly evolving proteasome assembly chaperones and characterize their function in the emerging fungal pathogen Candida auris. Despite the large sequence divergence, we demonstrate conservation of structure and function across hundreds of millions of years of evolution, representing a case of rapid neutral evolution. Using the functional constraints on structure from these naturally evolved sequences, we prospectively designed de novo chaperones and demonstrate that these artificial proteins can rescue complex biological processes in the context of the whole cell.

evolutionary biology↗

bioRxiv2023

Active compensation for changes in TDH3 expression mediated by direct regulators of TDH3 in Saccharomyces cerevisiae

Genetic networks are surprisingly robust to perturbations caused by new mutations. This robustness is conferred in part by compensation for loss of a genes activity by genes with overlapping functions, such as paralogs. Compensation occurs passively when the normal activity of one paralog can compensate for the loss of the other, or actively when a change in one paralogs expression, localization, or activity is required to compensate for loss of the other. The mechanisms of active compensation remain poorly understood in most cases. Here we investigate active compensation for the loss or reduction in expression of the Saccharomyces cerevisiae gene TDH3 by its paralogs TDH1 and TDH2. TDH1 and TDH2 are upregulated in a dose-dependent manner in response to reductions in TDH3 by a mechanism requiring the shared transcriptional regulators Gcr1p and Rap1p. Other glycolytic genes regulated by Rap1p and Gcr1p show changes in expression similar to TDH2, suggesting that the active compensation by TDH3 paralogs is part of a broader homeostatic response mediated by shared transcriptional regulators.

cell biology↗

bioRxiv2021

Mutational sources of trans-regulatory variation affecting gene expression in Saccharomyces cerevisiae

Heritable variation in a genes expression arises from mutations impacting cis- and trans-acting components of its regulatory network, with expression variation often derived from trans-regulatory mutations within species. Here, we investigate how trans-regulatory mutations are distributed within the genome and within a gene regulatory network by identifying and characterizing 69 mutations with trans-regulatory effects on expression of the same focal gene in Saccharomyces cerevisiae. Relative to 1766 mutations without effects on expression of this focal gene, we found that these trans-regulatory mutations were enriched in coding sequences of transcription factors previously predicted to regulate expression of the focal gene. However, over 90% of the trans-regulatory mutations identified mapped to other types of genes involved in diverse biological processes including chromatin state, metabolism and signal transduction. Finally, we find that the genomic distribution of these new regulatory mutations significantly overlaps with the genomic distribution of trans-regulatory variants segregating within S. cerevisiae.

genetics↗

bioRxiv2020

ebony affects pigmentation divergence and cuticular hydrocarbons in Drosophila americana and D. novamexicana

1Drosophila pigmentation has been a fruitful model system for understanding the genetic and developmental mechanisms underlying phenotypic evolution. For example, prior work has shown that divergence of the tan gene contributes to pigmentation differences between two members of the virilis group: Drosophila novamexicana, which has a light yellow body color, and D. americana, which has a dark brown body color. Quantitative trait locus (QTL) mapping and expression analysis has suggested that divergence of the ebony gene might also contribute to pigmentation differences between these two species. Here, we directly test this hypothesis by using CRISPR/Cas9 genome editing to generate ebony null mutants in D. americana and D. novamexicana and then using reciprocal hemizygosity testing to compare the effects of each species ebony allele on pigmentation. We find that divergence of ebony does indeed contribute to the pigmentation divergence between species, with effects on both the overall body color as well as a difference in pigmentation along the dorsal abdominal midline. Motivated by recent work in D. melanogaster, we also used the ebony null mutants to test for effects of ebony on cuticular hydrocarbon (CHC) profiles. We found that ebony affects CHC abundance in both species, but does not contribute to qualitative differences in the CHC profiles between these two species. Additional transgenic resources for working with D. americana and D. novamexicana, such as white mutants of both species and yellow mutants in D. novamexicana, were generated in the course of this work and are also described. Taken together, this study advances our understanding of loci contributing to phenotypic divergence and illustrates how the latest genome editing tools can be used for functional testing in non-model species.

evolutionary biology↗

bioRxiv2019

The yellow gene influences Drosophila male mating success through sex comb melanization

Drosophila melanogaster males perform a series of courtship behaviors that, when successful, result in copulation with a female. For over a century, mutations in the yellow gene, named for its effects on pigmentation, have been known to reduce male mating success. Prior work has suggested that yellow influences mating behavior through effects on wing extension, song, and/or courtship vigor. Here, we rule out these explanations, as well as effects on the nervous system more generally, and find instead that the effects of yellow on male mating success are mediated by its effects on pigmentation of male-specific leg structures called sex combs. Loss of yellow expression in these modified bristles reduces their melanization, which changes their structure and causes difficulty grasping females prior to copulation. These data illustrate why the mechanical properties of anatomy, and not just neural circuitry, must be considered to fully understand the development and evolution of behavior.

animal behavior and cognition↗

bioRxiv2019

Complex polymorphic genetic architecture underlies trans-regulatory variation among strains of Saccharomyces cerevisiae

Heritable variation in gene expression is common within species. Much of this variation is due to genetic changes at loci other than the affected gene and is thus trans-acting. This trans-regulatory variation is often polygenic, with individual variants typically having small effects, making the genetic architecture of trans-regulatory variation challenging to study. Consequently, key questions about trans-regulatory variation remain, including how selection affects this variation and how trans-regulatory variants are distributed throughout the genome and within species. Here, we show that trans-regulatory variation affecting TDH3 promoter activity is common among strains of Saccharomyces cerevisiae. Comparing this variation to neutral models of trans-regulatory evolution based on empirical measures of mutational effects revealed that stabilizing selection has constrained this variation. Using a powerful quantitative trait locus (QTL) mapping method, we identified [~]100 loci altering expression between a reference strain and each of three genetically distinct strains. In all three cases, the non-reference strain alleles increased and decreased TDH3 promoter activity with similar frequencies, suggesting that stabilizing selection maintained many trans-acting variants with opposing effects. Loci altering expression were located throughout the genome, with many loci being strain specific and others being shared among multiple strains. These findings are consistent with theory showing stabilizing selection for quantitative traits can maintain many alleles with opposing effects, and the wide-spread distribution of QTL throughout the genome is consistent with the omnigenic model of complex trait variation. Furthermore, the prevalence of alleles with opposing effects might provide raw material for compensatory evolution and developmental systems drift.\n\nSignificance statementGene expression varies among individuals in a population due to genetic differences in regulatory components. To determine how this variation is distributed within genomes and species, we used a powerful genetic mapping approach to examine multiple strains of Saccharomyces cerevisiae. Despite evidence of stabilizing selection maintaining gene expression levels among strains, we find hundreds of loci that affect expression of a single gene. These loci vary among strains and include similar frequencies of alleles that increase and decrease expression. As a result, each strain contains a unique set of compensatory alleles that lead to similar levels of gene expression among strains. This regulatory variation might form the basis for large scale regulatory rewiring observed between distantly related species.

evolutionary biology↗

bioRxiv2019

Empirical measures of mutational effects define neutral models of regulatory evolution in Saccharomyces cerevisiae

Understanding how phenotypes evolve requires disentangling the effects of mutation generating new variation from the effects of selection filtering it. Evolutionary models frequently assume that mutation introduces phenotypic variation symmetrically around the population mean, yet few studies have tested this assumption by deeply sampling the distributions of mutational effects for particular traits. Here, we examine distributions of mutational effects for gene expression in the budding yeast Saccharomyces cerevisiae by measuring the effects of thousands of point mutations introduced randomly throughout the genome. We find that the distributions of mutational effects differ for the 10 genes surveyed and violate the assumption of normality. For example, all ten distributions of mutational effects included more mutations with large effects than expected for normally distributed phenotypes. In addition, some genes also showed asymmetries in their distribution of mutational effects, with new mutations more likely to increase than decrease the genes expression or vice versa. Neutral models of regulatory evolution that take these empirically determined distributions into account suggest that neutral processes may explain more expression variation within natural populations than currently appreciated. Significance statementNew mutations tend to arise randomly throughout the genome, but their phenotypic effects are often not random. This disconnect results from interactions among genes that define the genotype-phenotype map. The structure of this map is poorly known and different for each trait, making it challenging to predict the distribution of mutational effects for specific phenotypes. Empirical measures of the distribution of mutational effects are thus necessary to understand how traits can change in the absence of natural selection. In this work, we define such distributions for expression of ten genes in S. cerevisiae and show that they predict greater neutral expression divergence than commonly used models of phenotypic evolution.

evolutionary biology↗

bioRxiv2019

Pleiotropic effects of ebony and tan on pigmentation and cuticular hydrocarbon composition in Drosophila melanogaster

Pleiotropic genes are genes that affect more than one trait. For example, many genes required for pigmentation in the fruit fly Drosophila melanogaster also affect traits such as circadian rhythms, vision, and mating behavior. Here, we present evidence that two pigmentation genes, ebony and tan, which encode enzymes catalyzing reciprocal reactions in the melanin biosynthesis pathway, also affect cuticular hydrocarbon (CHC) composition in D. melanogaster females. More specifically, we report that ebony loss-of-function mutants have a CHC profile that is biased toward long (>25C) chain CHCs, whereas tan loss-of-function mutants have a CHC profile that is biased toward short (<25C) chain CHCs. Moreover, pharmacological inhibition of dopamine synthesis, a key step in the melanin synthesis pathway, reversed the changes in CHC composition seen in ebony mutants, making the CHC profiles similar to those seen in tan mutants. These observations suggest that genetic variation affecting ebony and/or tan activity might cause correlated changes in pigmentation and CHC composition in natural populations. We tested this possibility using the Drosophila Genetic Reference Panel (DGRP) and found that CHC composition covaried with pigmentation as well as levels of ebony and tan expression in newly eclosed adults in a manner consistent with the ebony and tan mutant phenotypes. These data suggest that the pleiotropic effects of ebony and tan might contribute to covariation of pigmentation and CHC profiles in Drosophila.

genetics↗

bioRxiv2018

Redundant and cryptic enhancer activities of the Drosophila yellow gene

cis-regulatory sequences known as enhancers play a key role in regulating gene expression. Evolutionary changes in these DNA sequences contribute to phenotypic evolution. The Drosophila yellow gene, which is required for pigmentation, has emerged as a model system for understanding how cis-regulatory sequences evolve, providing some of the most detailed insights available into how activities of orthologous enhancers have diverged between species. Here, we examine the evolution of yellow cis-regulatory sequences on a broader scale by comparing the distribution and function of yellow enhancer activities throughout the 5 intergenic and intronic sequences of Drosophila melanogaster, Drosophila pseudoobscura, and Drosophila willistoni. We find that cis-regulatory sequences driving expression in a particular tissue are not as modular as previously described, but rather have many redundant and cryptic enhancer activities distributed throughout the regions surveyed. Interestingly, cryptic enhancer activities of sequences from one species often drove patterns of expression observed in other species, suggesting that the frequent evolutionary changes in yellow expression observed among Drosophila species may be facilitated by gaining and losing repression of pre-existing cis-regulatory sequences.

evolutionary biology↗

bioRxiv2018

Fitness effects of altering gene expression noise in Saccharomyces cerevisiae

Gene expression noise is an evolvable property of biological systems that describes differences in gene expression among genetically identical cells in the same environment. Prior work has shown that expression noise is heritable and can be shaped by natural selection, but the impact of variation in expression noise on organismal fitness has proven difficult to measure. Here, we quantify the fitness effects of altering expression noise for the TDH3 gene in Saccharomyces cerevisiae. We show that increases in expression noise can be deleterious or beneficial depending on the difference between the average expression level of a genotype and the expression level maximizing fitness. We also show that a simple model relating single-cell expression levels to population growth produces patterns that are consistent with our empirical data. We use this model to explore a broad range of average expression levels and expression noise, providing additional insight into the fitness effects of variation in expression noise.

evolutionary biology↗

bioRxiv2017

Effects of mutation and selection on plasticity of TDH3 promoter activity in Saccharomyces cerevisiae

Phenotypic plasticity is an evolvable property of biological systems that can arise from environment-specific regulation of gene expression. To better understand the evolutionary and molecular mechanisms that give rise to plasticity in gene expression, we quantified the effects of 235 single nucleotide mutations in the Saccharomyces cerevisiae TDH3 promoter (PTDH3) on the activity of this promoter in media containing glucose, galactose, or glycerol as a carbon source. We found that the distributions of mutational effects differed among environments because many mutations altered the plastic response exhibited by the wild type allele. Comparing the effects of these mutations to the effects of 30 PTDH3 polymorphisms on expression plasticity in the same environments provided evidence of natural selection acting to prevent the plastic response in PTDH3 activity between glucose and galactose from becoming larger. The largest changes in expression plasticity were observed between fermentable (glucose or galactose) and nonfermentable (glycerol) carbon sources and were caused by mutations located in the RAP1 and GCR1 transcription factor binding sites. Mutations altered expression plasticity most frequently between the two fermentable environments, however, with mutations causing significant changes in plasticity between glucose and galactose distributed throughout the promoter, suggesting they might affect chromatin structure. Taken together, these results provide insight into the molecular mechanisms underlying gene-by-environment interactions affecting gene expression as well as the evolutionary dynamics affecting natural variation in plasticity of gene expression.\n\nSignificance StatementFrom seasonal variation in the color of butterfly wings to trees bending toward the light, organisms often change in response to their environment. These changes, known as phenotypic plasticity, can result from differences in how genes are expressed among environments. Mutations causing environment-specific changes in gene expression provide raw material for phenotypic plasticity, but their frequency, effect size and direction of effects among environments are not well understood. This study shows that mutations in the promoter of a yeast metabolic gene often display environment-dependent effects on gene expression and that these environment-dependent effects have been shaped by selection in natural populations.

genetics↗

bioRxiv2017

Fitness effects of cis-regulatory variants in the Saccharomyces cerevisiae TDH3 promoter

Variation in gene expression is widespread within and between species, but fitness consequences of this variation are generally unknown. Here we use mutations in the Saccharomyces cerevisiae TDH3 promoter to assess how changes in TDH3 expression affect cell growth. From these data, we predict the fitness consequences of de novo mutations and natural polymorphisms in the TDH3 promoter. Nearly all mutations and polymorphisms in the TDH3 promoter were found to have no significant effect on fitness in the environment assayed, suggesting that the wild type allele of this promoter is robust to the effects of most new cis-regulatory mutations.

genetics↗