A synthetic biology approach to probing nucleosome symmetry.

The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell.

Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden.

BACKGROUND: High tumor mutational burden (TMB) is an emerging biomarker of sensitivity to immune checkpoint inhibitors and has been shown to be more significantly associated with response to PD-1 and PD-L1 blockade immunotherapy than PD-1 or PD-L1 expression, as measured by immunohistochemistry (IHC). The distribution of TMB and the subset of patients with high TMB has not been well characterized in the majority of cancer types. METHODS: In this study, we compare TMB measured by a targeted comprehensive genomic profiling (CGP) assay to TMB measured by exome sequencing and simulate the expected variance in TMB when sequencing less than the whole exome. We then describe the distribution of TMB across a diverse cohort of 100,000 cancer cases and test for association between somatic alterations and TMB in over 100 tumor types. RESULTS: We demonstrate that measurements of TMB from comprehensive genomic profiling are strongly reflective of measurements from whole exome sequencing and model that below 0.5 Mb the variance in measurement increases significantly. We find that a subset of patients exhibits high TMB across almost all types of cancer, including many rare tumor types, and characterize the relationship between high TMB and microsatellite instability status. We find that TMB increases significantly with age, showing a 2.4-fold difference between age 10 and age 90 years. Finally, we investigate the molecular basis of TMB and identify genes and mutations associated with TMB level. We identify a cluster of somatic mutations in the promoter of the gene PMS2, which occur in 10% of skin cancers and are highly associated with increased TMB. CONCLUSIONS: These results show that a CGP assay targeting ~1.1 Mb of coding genome can accurately assess TMB compared with sequencing the whole exome. Using this method, we find that many disease types have a substantial portion of patients with high TMB who might benefit from immunotherapy. Finally, we identify novel, recurrent promoter mutations in PMS2, which may be another example of regulatory mutations contributing to tumorigenesis.

Evolution and genetic architecture of chromatin accessibility and function in yeast.

Chromatin accessibility is an important functional genomics phenotype that influences transcription factor binding and gene expression. Genome-scale technologies allow chromatin accessibility to be mapped with high-resolution, facilitating detailed analyses into the genetic architecture and evolution of chromatin structure within and between species. We performed Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-Seq) to map chromatin accessibility in two parental haploid yeast species, Saccharomyces cerevisiae and Saccharomyces paradoxus and their diploid hybrid. We show that although broad-scale characteristics of the chromatin landscape are well conserved between these species, accessibility is significantly different for 947 regions upstream of genes that are enriched for GO terms such as intracellular transport and protein localization exhibit. We also develop new statistical methods to investigate the genetic architecture of variation in chromatin accessibility between species, and find that cis effects are more common and of greater magnitude than trans effects. Interestingly, we find that cis and trans effects at individual genes are often negatively correlated, suggesting widespread compensatory evolution to stabilize levels of chromatin accessibility. Finally, we demonstrate that the relationship between chromatin accessibility and gene expression levels is complex, and a significant proportion of differences in chromatin accessibility might be functionally benign.

Integrative phenomics reveals insight into the structure of phenotypic diversity in budding yeast.

To better understand the quantitative characteristics and structure of phenotypic diversity, we measured over 14,000 transcript, protein, metabolite, and morphological traits in 22 genetically diverse strains of Saccharomyces cerevisiae. More than 50% of all measured traits varied significantly across strains [false discovery rate (FDR) = 5%]. The structure of phenotypic correlations is complex, with 85% of all traits significantly correlated with at least one other phenotype (median = 6, maximum = 328). We show how high-dimensional molecular phenomics data sets can be leveraged to accurately predict phenotypic variation between strains, often with greater precision than afforded by DNA sequence information alone. These results provide new insights into the spectrum and structure of phenotypic diversity and the characteristics influencing the ability to accurately predict phenotypes.

Population genomics and transcriptional consequences of regulatory motif variation in globally diverse Saccharomyces cerevisiae strains.

Noncoding genetic variation is known to significantly influence gene expression levels in a growing number of specific cases; however, the patterns of genome-wide noncoding variation present within populations, the evolutionary forces acting on noncoding variants, and the relative effects of regulatory polymorphisms on transcript abundance are not well characterized. Here, we address these questions by analyzing patterns of regulatory variation in motifs for 177 DNA binding proteins in 37 strains of Saccharomyces cerevisiae. Between S. cerevisiae strains, we found considerable polymorphism in regulatory motifs across strains (mean π = 0.005) as well as diversity in regulatory motifs (mean 0.91 motifs differences per regulatory region). Population genetics analyses reveal that motifs are under purifying selection, and there is considerable heterogeneity in the magnitude of selection across different motifs. Finally, we obtained RNA-Seq data in 22 strains and identified 49 polymorphic DNA sequence motifs in 30 distinct genes that are significantly associated with transcriptional differences between strains. In 22 of these genes, there was a single polymorphic motif associated with expression in the upstream region. Our results provide comprehensive insights into the evolutionary trajectory of regulatory variation in yeast and the characteristics of a compendium of regulatory alleles.

On the prospects of whole-genome association mapping in Saccharomyces cerevisiae.

Advances in sequencing technology have enabled whole-genome sequences to be obtained from multiple individuals within species, particularly in model organisms with compact genomes. For example, 36 genome sequences of Saccharomyces cerevisiae are now publicly available, and SNP data are available for even larger collections of strains. One potential use of these resources is mapping the genetic basis of phenotypic variation through genome-wide association (GWA) studies, with the benefit that associated variants can be studied experimentally with greater ease than in outbred populations such as humans. Here, we evaluate the prospects of GWA studies in S. cerevisiae strains through extensive simulations and a GWA study of mitochondrial copy number. We demonstrate that the complex and heterogeneous patterns of population structure present in yeast populations can lead to a high type I error rate in GWA studies of quantitative traits, and that methods typically used to control for population stratification do not provide adequate control of the type I error rate. Moreover, we show that while GWA studies of quantitative traits in S. cerevisiae may be difficult depending on the particular set of strains studied, association studies to map cis-acting quantitative trait loci (QTL) and Mendelian phenotypes are more feasible. We also discuss sampling strategies that could enable GWA studies in yeast and illustrate the utility of this approach in Saccharomyces paradoxus. Thus, our results provide important practical insights into the design and interpretation of GWA studies in yeast, and other model organisms that possess complex patterns of population structure.

Clusters of adaptive evolution in the human genome.

Considerable work has been devoted to identifying regions of the human genome that have been subjected to recent positive selection. Although detailed follow-up studies of putatively selected regions are critical for a deeper understanding of human evolutionary history, such studies have received comparably less attention. Recently, we have shown that ALMS1 has been the target of recent positive selection acting on standing variation in Eurasian populations. Here, we describe a careful follow-up analysis of genetic variation across the ALMS1 region, which unexpectedly revealed a cluster of substrates of positive selection. Specifically, through the analysis of SNP data from the HapMap and Human Genome Diversity Project-Centre d'Etude du Polymorphisme Humain samples as well sequence data from the region, we find compelling evidence for three independent and distinct signals of recent positive selection across this 3 Mb region surrounding ALMS1. Moreover, we analyzed the HapMap data to identify other putative clusters of independent selective events and conservatively discovered 19 additional clusters of adaptive evolution. This work has important implications for the interpretation of genome-scans for positive selection in humans and more broadly contributes to a better understanding of how recent positive selection has shaped genetic variation across the human genome.

Tracking footprints of artificial selection in the dog genome.

The size, shape, and behavior of the modern domesticated dog has been sculpted by artificial selection for at least 14,000 years. The genetic substrates of selective breeding, however, remain largely unknown. Here, we describe a genome-wide scan for selection in 275 dogs from 10 phenotypically diverse breeds that were genotyped for over 21,000 autosomal SNPs. We identified 155 genomic regions that possess strong signatures of recent selection and contain candidate genes for phenotypes that vary most conspicuously among breeds, including size, coat color and texture, behavior, skeletal morphology, and physiology. In addition, we demonstrate a significant association between HAS2 and skin wrinkling in the Shar-Pei, and provide evidence that regulatory evolution has played a prominent role in the phenotypic diversification of modern dog breeds. Our results provide a first-generation map of selection in the dog, illustrate how such maps can rapidly inform the genetic basis of canine phenotypic variation, and provide a framework for delineating the mechanistic basis of how artificial selection promotes rapid and pronounced phenotypic evolution.

Highly punctuated patterns of population structure on the X chromosome and implications for African evolutionary history.

It is well known that average levels of population structure are higher on the X chromosome compared to autosomes in humans. However, there have been surprisingly few analyses on the spatial distribution of population structure along the X chromosome. With publicly available data from the HapMap Project and Perlegen Sciences, we show a strikingly punctuated pattern of X chromosome population structure. Specifically, 87% of X-linked HapMap SNPs within the top 1% of F(ST) values cluster into five distinct loci. The largest of these regions spans 5.4 Mb and contains 66% of the most highly differentiated HapMap SNPs on the X chromosome. We demonstrate that the extreme clustering of highly differentiated SNPs on the X chromosome is not an artifact of ascertainment bias, nor is it specific to the populations genotyped in the HapMap Project. Rather, additional analyses and resequencing data suggest that these five regions have been substrates of recent and strong adaptive evolution. Finally, we discuss the implications that patterns of X-linked population structure have on the evolutionary history of African populations.

An ortholog of MIXTA-like2 controls epidermal cell shape in flowers of Thalictrum.

Here, we investigated the genetic underpinnings of pollination-related floral phenotypes in Thalictrum, a ranunculid with apetalous flowers. The variable presence of petaloid features in other floral organs correlates with distinct adaptations to insect vs. wind pollination. Conical cells are present in sepals or stamens of insect-pollinated species, and in stigmas. We characterized a Thalictrum ortholog of the Antirrhinum majus transcription factor MIXTA-like2, responsible for conical cells, from three species with distinct floral morphologies, representing two pollination syndromes. Genes were cloned by PCR and analysed phylogenetically. Expression analyses were conducted by quantitative PCR and in situ hybridization, followed by functional studies in transgenic tobacco. The cloned genes encode R2R3 MYB proteins closely related to Antirrhinum AmMYBML2 and Petunia hybrida PhMYB1. Spatial expression by in situ hybridization overlaps areas of conical cells. Overexpression in tobacco induces cell outgrowths in carpel epidermis and significantly increases the height of petal conical cells. We have described the first orthologs of AmMIXTA-like2 outside the core eudicots, likely ancestral to the MIXTA/MIXTA-like1 duplication. The conserved role in epidermal cell elongation results in conical cells, micromorphological markers for petaloidy. This adaptation to attract insect pollinators was apparently lost after the evolution of wind pollination in Thalictrum.

Population genomic analysis of ALMS1 in humans reveals a surprisingly complex evolutionary history.

Mutations in the human gene ALMS1 result in Alström Syndrome, which presents with early childhood obesity and insulin resistance leading to Type 2 diabetes. Previous genomewide scans for selection in the HapMap data based on linkage disequilibrium and population structure suggest that ALMS1 was subject to recent positive selection. Through a detailed population genomic analysis of existing genomewide data sets and new resequencing data obtained in geographically diverse populations, we find that the signature of selection at ALMS1 is considerably more complex than what would be expected for an idealized model of a selective sweep acting on a newly arisen advantageous mutation. Specifically, we observed three highly divergent and globally dispersed haplogroups, two of which carry a set of seven derived nonsynonymous single nucleotide polymorphisms that are nearly fixed in Asian populations. Our data suggest that the interaction of human demographic history and positive selection on standing variation in Eurasian populations approximately 15 thousand years ago parsimoniously explains the spectrum of extant ALMS1 variation. These results provide new insights into the evolutionary history of ALMS1 in humans and suggest that selective events identified in genomewide scans may be more complex than currently appreciated.

Population genomics of intron splicing in 38 Saccharomyces cerevisiae genome sequences.

Introns are a ubiquitous feature of eukaryotic genomes, and the dynamics of intron evolution between species has been extensively studied. However, comparatively few analyses have focused on the evolutionary forces shaping patterns of intron variation within species. To better understand the population genetic characteristics of introns, we performed an extensive population genetics analysis on key intron splice sequences obtained from 38 strains of Saccharomyces cerevisiae. As expected, we found that purifying selection is the dominant force governing intron splice sequence evolution in yeast, formally confirming that intron-containing alleles are a mutational liability. In addition, through extensive coalescent simulations, we obtain quantitative estimates of the strength of purifying selection (2N(e)s approximately 19) and use diffusion approximations to provide insights into the evolutionary dynamics and sojourn times of newly arising splice sequence mutations in natural yeast populations. In contrast to previous functional studies, evolutionary analyses comparing the prevalence of introns in essential and nonessential genes suggest that introns in nonribosomal protein genes are functionally important and tend to be actively maintained in natural populations of S. cerevisiae. Finally, we demonstrate that heritable variation in splicing efficiency is common in intron-containing genes with splice sequence polymorphisms. More generally, our study highlights the advantages of population genomics analyses for exploring the forces that have generated extant patterns of genome variation and for illuminating basic biological processes.