Convergent expansions of keystone gene families drive metabolic innovation in a major eukaryotic clade.

Many remarkable innovations have repeatedly occurred across vast evolutionary distances. When convergent traits emerge on the tree of life, they are sometimes driven by the same underlying gene families, while other times many different gene families are involved. Conversely, a gene family may be repeatedly recruited for a single trait or many different traits. To understand the general rules governing convergence at both genomic and phenotypic levels, we systematically tested associations between 56 binary metabolic traits and gene count in 14,710 gene families from 993 species of Saccharomycotina yeasts. Using a recently developed phylogenetic approach that reduces spurious correlations, we discovered that gene family expansion and contraction was significantly linked to trait gain and loss in 45/56 (80%) of traits. While 601/746 (81%) of significant gene families were associated with only one trait, we also identified several 'keystone' gene families that were significantly associated with up to 13/56 (23%) of all traits. These results indicate that metabolic innovations in yeasts are governed by a narrow set of major genetic elements and mechanisms.

Specialization restricts the evolutionary paths available to yeast sugar transporters.

Functional innovation at the protein level is a key source of evolutionary novelties. The constraints on functional innovations are likely to be highly specific in different proteins, which are shaped by their unique histories and the extent of global epistasis that arises from their structures and biochemistries. These contextual nuances in the sequence-function relationship have implications both for a basic understanding of the evolutionary process and for engineering proteins with desirable properties. Here, we have investigated the molecular basis of novel function in a model member of an ancient, conserved, and biotechnologically relevant protein family. These Major Facilitator Superfamily sugar porters are a functionally diverse group of proteins that are thought to be highly plastic and evolvable. By dissecting a recent evolutionary innovation in an α-glucoside transporter from the yeast Saccharomyces eubayanus, we show that the ability to transport a novel substrate requires high-order interactions between many protein regions and numerous specific residues proximal to the transport channel. To reconcile the functional diversity of this family with the constrained evolution of this model protein, we generated new, state-of-the-art genome annotations for 332 Saccharomycotina yeast species spanning approximately 400 million years of evolution. By integrating phylogenetic and phenotypic analyses across these species, we show that the model yeast α-glucoside transporters likely evolved from a multifunctional ancestor and became subfunctionalized. The accumulation of additive and epistatic substitutions likely entrenched this subfunction, which made the simultaneous acquisition of multiple interacting substitutions the only reasonably accessible path to novelty.

Ploidy evolution in a wild yeast is linked to an interaction between cell type and metabolism.

Ploidy is an evolutionarily labile trait, and its variation across the tree of life has profound impacts on evolutionary trajectories and life histories. The immediate consequences and molecular causes of ploidy variation on organismal fitness are frequently less clear, although extreme mating type skews in some fungi hint at links between cell type and adaptive traits. Here, we report an unusual recurrent ploidy reduction in replicate populations of the budding yeast Saccharomyces eubayanus experimentally evolved for improvement of a key metabolic trait, the ability to use maltose as a carbon source. We find that haploids have a substantial, but conditional, fitness advantage in the absence of other genetic variation. Using engineered genotypes that decouple the effects of ploidy and cell type, we show that increased fitness is primarily due to the distinct transcriptional program deployed by haploid-like cell types, with a significant but smaller contribution from absolute ploidy. The link between cell-type specification and the carbon metabolism adaptation can be traced to the noncanonical regulation of a maltose transporter by a haploid-specific gene. This study provides novel mechanistic insight into the molecular basis of an environment-cell type fitness interaction and illustrates how selection on traits unexpectedly linked to ploidy states or cell types can drive karyotypic evolution in fungi.

Rapid and inexpensive preparation of genome-wide nucleosome footprints from model and non-model organisms.

MNase-seq (micrococcal nuclease sequencing) is used to map nucleosome positions in eukaryotic genomes to study the relationship between chromatin structure and DNA-dependent processes. Current protocols require at least two days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for S. cerevisiae and other fungi which takes only three hours. Modified protocols were developed for wild fungi and mammalian cells. This method for rapidly producing sequencing-ready nucleosome footprints from several organisms makes MNase-seq faster and easier, with less chemical waste.

Basis of specificity for a conserved and promiscuous chromatin remodeling protein.

Eukaryotic genomes are organized dynamically through the repositioning of nucleosomes. Isw2 is an enzyme that has been previously defined as a genome-wide, nonspecific nucleosome spacing factor. Here, we show that Isw2 instead acts as an obligately targeted nucleosome remodeler in vivo through physical interactions with sequence-specific factors. We demonstrate that Isw2-recruiting factors use small and previously uncharacterized epitopes, which direct Isw2 activity through highly conserved acidic residues in the Isw2 accessory protein Itc1. This interaction orients Isw2 on target nucleosomes, allowing for precise nucleosome positioning at targeted loci. Finally, we show that these critical acidic residues have been lost in the Drosophila lineage, potentially explaining the inconsistently characterized function of Isw2-like proteins. Altogether, these data suggest an 'interacting barrier model,' where Isw2 interacts with a sequence-specific factor to accurately and reproducibly position a single, targeted nucleosome to define the precise border of phased chromatin arrays.

DNA encodes the genetic instructions for life in a long, flexible molecular chain that is packaged up neatly to fit inside cells. Short sections of DNA are wound around proteins to form bundles called nucleosomes, and then spun into chromatin fibres, a more compact form of DNA. While nucleosomes are a fundamental part of this space-saving packaging process, they also play a key regulatory role in gene expression, which is where genes are decoded into working proteins. Placing nucleosomes at regular intervals along DNA invariably controls which parts of the DNA ­ and which genes ­ the cell's machinery can access and 'read' to make proteins. But the nucleosomes' positions are not fixed, and gene expression is a dynamic process. The cell often uncoils and repackages its DNA while molecular motors called chromatin remodelling proteins move nucleosomes up and down the DNA, exposing some genes and obstructing others. One group of chromatin remodelling proteins are called Imitation Switch (ISWI) complexes. It has long been thought that these complexes position nucleosomes with little regard to the underlying DNA sequence or the genes encoded, that is to say in a non-specific way. However, this theory has not been thoroughly tested. It is possible that ISWI complexes actually place nucleosomes on certain parts of DNA at particular times in an organism's development, or in response to other environmental factors. Except how such precision is achieved remains unknown. To test this alternative theory of nucleosome positioning, Donovan et al. studied ISWI proteins and nucleosomes in common baker's yeast. This involved systematically removing sections of ISWI proteins to see whether the complexes could still position nucleosomes, and which parts of the proteins where essential for the job. By doing so, Donovan et al. identified multiple 'targeting' proteins that bind to ISWI proteins and deliver the complexes to specific target sequences of DNA. From there, the complex remodels the nucleosome, positioning it at a specific distance from its landing site on DNA, as further experiments showed. This research provides a new model for explaining how nucleosomes are positioned to package DNA and control gene expression. Donovan et al. have identified a new mechanism of interaction between nucleosomes and chromatin remodelling proteins of the ISWI variety. It is possible that more interactions of this kind will be discovered with further research.

Engineered Chromatin Remodeling Proteins for Precise Nucleosome Positioning.

Regulation of chromatin structure is essential for controlling access of DNA to factors that require association with specific DNA sequences. Here we describe the development and validation of engineered chromatin remodeling proteins (E-ChRPs) for inducing programmable changes in nucleosome positioning by design. We demonstrate that E-ChRPs function both in vitro and in vivo to specifically reposition target nucleosomes and entire nucleosomal arrays. We show that induced, systematic positioning of nucleosomes over yeast Ume6 binding sites leads to Ume6 exclusion, hyperacetylation, and transcriptional induction at target genes. We also show that programmed global loss of nucleosome-free regions at Reb1 targets is generally inhibitory with mildly repressive transcriptional effects. E-ChRPs are compatible with multiple targeting modalities, including the SpyCatcher and dCas9 moieties, resulting in high versatility and enabling diverse future applications. Thus, engineered chromatin remodeling proteins represent a simple and robust means to probe and disrupt DNA-dependent processes in different chromatin contexts.

Selection, Linkage, and Population Structure Interact To Shape Genetic Variation Among Threespine Stickleback Genomes.

The outcome of selection on genetic variation depends on the geographic organization of individuals and populations as well as the organization of loci within the genome. Spatially variable selection between marine and freshwater habitats has had a significant and heterogeneous impact on patterns of genetic variation across the genome of threespine stickleback fish. When marine stickleback invade freshwater habitats, more than a quarter of the genome can respond to divergent selection, even in as little as 50 years. This process largely uses standing genetic variation that can be found ubiquitously at low frequency in marine populations, can be millions of years old, and is likely maintained by significant bidirectional gene flow. Here, we combine population genomic data of marine and freshwater stickleback from Cook Inlet, Alaska, with genetic maps of stickleback fish derived from those same populations to examine how linkage to loci under selection affects genetic variation across the stickleback genome. Divergent selection has had opposing effects on linked genetic variation on chromosomes from marine and freshwater stickleback populations: near loci under selection, marine chromosomes are depauperate of variation, while these same regions among freshwater genomes are the most genetically diverse. Forward genetic simulations recapitulate this pattern when different selective environments also differ in population structure. Lastly, dense genetic maps demonstrate that the interaction between selection and population structure may impact large stretches of the stickleback genome. These findings advance our understanding of how the structuring of populations across geography influences the outcomes of selection, and how the recombination landscape broadens the genomic reach of selection.