Summary of Recent Research

Over the past 9 years, we have collected a unique set of human and non-human ape tissue samples that allows us to study differences in gene regulation and associated mechanisms within and between species. Our long-term goal is to identify the genetic basis for human specific traits, including genetic variation that underlies higher susceptibility to certain diseases in humans than in other primates. To do so, we use a combination of genomic approaches to characterize variation in gene expression levels and associated regulatory mechanisms within and between species. We then perform functional studies and/or genetic association studies, to identify causal links between genetic variation, changes in gene regulation, and ultimately – differences in complex phenotypes. Below are abstracts of a few recent papers, which demonstrate the scope of our work. • Khan Z, Ford MJ, Cusanovich DA, Mitrano A, Pritchard JK, and Gilad Y. Primate “Transcript and Protein Expression Levels Evolve under Compensatory Selection Pressures”, Science, 2013, Oct. 17. [Epub ahead of print]

Changes in gene regulation have likely played an important role in the evolution of primates. Differences in messenger RNA (mRNA) expression levels across primates have often been documented; however, it is not yet known to what extent measurements in divergence in mRNA levels reflect divergence in protein expression levels, which are probably more important in determining phenotypic differences. We used high-resolution, quantitative mass spectometry to collect protein expression measurements from human, chimpanzee, and rhesus macaque lymphoblastoid cell lines and compared them to transcript expression data from the same samples. We found dozens of genes with significant expression differences between species at the mRNA level yet little or no difference in protein expression. Overall, our data suggest that protein expression levels evolve under stronger evolutionary constraint than mRNA levels.

• Zhou X, Cain CE, Mythril M, Lewellen N, Michelini K, Davenport ER, Stephens M, Pritchard JK, and Gilad Y. “Epigenetic Modifications are Associated with Inter-species Gene Expression Variation in Primates”, Genome Biology, 2014, Dec 3; 15:547

Background

Changes in gene regulation have long been thought to play an important role in evolution and speciation, especially in primates. Over the past decade, comparative genomic studies have revealed extensive inter-species differences in gene expression levels, yet we know much less about the extent to which regulatory mechanisms differ between species.

Results

To begin addressing this gap, we perform a comparative epigenetic study in primate lymphoblastoid cell lines, to query the contribution of RNA polymerase II and four histone modifications, H3K4me1, H3K4me3, H3K27ac, and H3K27me3, to inter-species variation in gene expression levels. We find that inter-species differences in mark enrichment near transcription start sites are significantly more often associated with inter-species differences in the corresponding gene expression level than expected by chance alone. Interestingly, we also find that first-order interactions among the five marks, as well as chromatin states, do not markedly contribute to the degree of association between the marks and inter-species variation in gene expression levels, suggesting that the marginal effects of the five marks dominate the contribution.

Conclusions

Our observations suggest that epigenetic modifications are substantially associated with changes in gene expression levels among primates and may represent important molecular mechanisms in primate evolution.

• McVicker G, van de Gejin B, Degner DF, Cain CE, Banovich NE, Raj A, Lewellen N, Myrthil M, Gilad Y, and Pritchard JK. “Identification of genetic variants that affect histone modifications in human cells”, Science, 2013, Oct 17.

Histone modifications are important markers of function and chromatin state, yet the DNA sequence elements that direct them to specific genomic locations are poorly understood. Here, we identify hundreds of quantitative trait loci, genomic-wide, that affect histone modification or RNA polymerase II (Pol II) occupancy in Yoruba lymphoblastoid cell lines (LCLs). In many cases, the same variant is associated with quantitative changes in multiple histone marks and Pol II, as well as in deoxyribonuclease I sensitivity and nucleosome positioning. Transcription factor binding site polymorphisms are correlated overall with differences in local histone modification, and we identify specific transcription factors whose binding leads to histone modification in LCLs. Furthermore, variants that affect chromatin at distal regulatory sites frequently also direct changes in chromatin and gene expression at associated promoters.

• Tung J, Barreiro LB, Johnson ZP, Hansen KD, Michopoulos M, Toufexis T, Michelini K, Wilson ME, and Gilad Y. “Social environment is associated with gene regulatory variation in the rhesus macaque immune system”, PNAS, 2012, Apr 24; 109(17):6490-5

Variation in the social environment is a fundamental component of many vertebrate societies. In humans and other primates, adverse social environments often translate into lasting physiological costs. The biological mechanisms associated with these effects are therefore of great interest, both for understanding the evolutionary impacts of social behavior and in the context of human health. However, large gaps remain in our understanding of the mechanisms that mediate these effects at the molecular level. Here we addressed these questions by leveraging the power of an experimental system that consisted of 10 social groups of female macaques, in which each individual’s social status (i.e., dominance rank) could be experimentally controlled. Using this paradigm, we show that dominance rank results in a widespread, yet plastic, imprint on gene regulation, such that peripheral blood mononuclear cell gene expression data alone predict social status with 80% accuracy. We investigated the mechanistic basis of these effects using cell-type specific gene expression profiling and glucocorticoid resistance assays, which together contributed to rank effects on gene expression levels for 694 (70%) of the 987 rank-related genes. We also explored the possible contribution of DNA methylation levels to these effects, and identified global associations between dominance rank and methylation profiles that suggest epigenetic flexibility in response to status-related behavioral cues. Together, these results illuminate the importance of the molecular response to social conditions, particularly in the immune system, and demonstrate a key role for gene regulation in linking the social environment to individual physiology.

• Degner F.D., Pai, A.P., Pique-Regi, R*., Veyrieras, J.P., Gaffney, J.D., Pickrell, J.K., De Leon, S., Michelini, K., Lewellen, N., Crawford, G.E., Stephens, M., Gilad, Y., and Pritchard, J.K. “DNasal sensitivity QTLs are a major determinant of human expression variation”, Nature 2012, Feb 5. [Epub ahead of print]

The mapping of expression quantitative trait loci (eQTLs) has emerged as an important tool for linking genetic variation to changes in gene regulation. However, it remains difficult to identify the causal variants underlying eQTLs, and little is known about the regulatory mechanisms by which they act. Here we show that genetic variants that modify chromatin accessibility and transcription factor binding are a major mechanism through which genetic variation leads to gene expression differences among humans. We used DNase I sequencing to measure chromatin accessibility in 70 Yoruba lymphoblastoid cell lines, for which genome-wide genotypes and estimates of gene expression levels are also available. We obtained a total of 2.7 billion uniquely mapped DNase I-sequencing (DNase-seq) reads, which allowed us to produce genome-wide maps of chromatin accessibility for each individual. We identified 8,902 locations at which the DNase-seq read depth correlated significantly with genotype at a nearby single nucleotide polymorphism or insertion/deletion (false discovery rate = 10%). We call such variants ‘DNase I sensitivity quantitative trait loci’ (dsQTLs). We found that dsQTLs are strongly enriched with inferred transcription factor binding sites and are frequently associated with allele-specific changes in transcription factor binding. A substantial fraction (16%) of dsQTLs are also associated with variation in the expression levels of nearby genes (that is, these loci are also classified as eQTLs). Conversely, we estimate that as many as 55% of eQTL single nucleotide polymorphisms are also dsQTLs. Our observations indicate that dsQTLs are highly abundant in the human genome and are likely to be important contributors to phenotypic variation.