Metabolic changes in human brain evolution.

Because the human brain is considerably larger than those of other primates, it is not surprising that its energy requirements would far exceed that of any of the species within the order. Recently, the development of stem cell technologies and single-cell transcriptomics provides novel ways to address the question of what specific genomic changes underlie the human brain's unique phenotype. In this review, we consider what is currently known about human brain metabolism using a variety of methods from brain imaging and stereology to transcriptomics. Next, we examine novel opportunities that stem cell technologies and single-cell transcriptomics provide to further our knowledge of human brain energetics. These new experimental approaches provide the ability to elucidate the functional effects of changes in genetic sequence and expression levels that potentially had a profound impact on the evolution of the human brain.

Immune System Promiscuity in Human and Nonhuman Primate Evolution.

Many genes that respond to infection have functions outside of immunity and have been found to be under natural selection. Pathogens may therefore incidentally alter nonimmune physiology through engagement with immune system genes. This raises a logical question of how genetically promiscuous the immune system is, here defined as how heavily cross-referenced the immune system is into other physiological systems. This work examined immune gene promiscuity across physiological systems in primates by assessing the baseline (unperturbed) expression of key tissue and cell types for differences, and primate genomes for signatures of selection. These efforts revealed "immune" gene expression to be cross-referenced extensively in other physiological systems in primates. When immune and nonimmune tissues diverge in expression, the differentially expressed genes at baseline are enriched for cell biological activities not immediately identifiable as immune function based. Individual comparisons of immune and nonimmune tissues in primates revealed low divergence in gene expression between tissues, with the exception of whole blood. Immune gene promiscuity increases over evolutionary time, with hominoids exhibiting the most cross-referencing of such genes among primates. An assessment of genetic sequences also found positive selection in the coding regions of differentially expressed genes between tissues functionally associated with immunity. This suggests that, with increasing promiscuity, divergent gene expression between the immune system and other physiological systems tends to be adaptive and enriched for immune functions in hominoids.

The predictive nature of transcript expression levels on protein expression in adult human brain.

BACKGROUND: Next generation sequencing methods are the gold standard for evaluating expression of the transcriptome. When determining the biological implications of such studies, the assumption is often made that transcript expression levels correspond to protein levels in a meaningful way. However, the strength of the overall correlation between transcript and protein expression is inconsistent, particularly in brain samples. RESULTS: Following high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses of adult human brain samples, we compared the correlation in the expression of transcripts and proteins that support various biological processes, molecular functions, and that are located in different areas of the cell. Although most categories of transcripts have extremely weak predictive value for the expression of their associated proteins (R2 values of < 10%), transcripts coding for protein kinases and membrane-associated proteins, including those that are part of receptors or ion transporters, are among those that are most predictive of downstream protein expression levels. CONCLUSIONS: The predictive value of transcript expression for corresponding proteins is variable in human brain samples, reflecting the complex regulation of protein expression. However, we found that transcriptomic analyses are appropriate for assessing the expression levels of certain classes of proteins, including those that modify proteins, such as kinases and phosphatases, regulate metabolic and synaptic activity, or are associated with a cellular membrane. These findings can be used to guide the interpretation of gene expression results from primate brain samples.

Gene expression and adaptive noncoding changes during human evolution.

BACKGROUND: Despite evidence for adaptive changes in both gene expression and non-protein-coding, putatively regulatory regions of the genome during human evolution, the relationship between gene expression and adaptive changes in cis-regulatory regions remains unclear. RESULTS: Here we present new measurements of gene expression in five tissues of humans and chimpanzees, and use them to assess this relationship. We then compare our results with previous studies of adaptive noncoding changes, analyzing correlations at the level of gene ontology groups, in order to gain statistical power to detect correlations. CONCLUSIONS: Consistent with previous studies, we find little correlation between gene expression and adaptive noncoding changes at the level of individual genes; however, we do find significant correlations at the level of biological function ontology groups. The types of function include processes regulated by specific transcription factors, responses to genetic or chemical perturbations, and differentiation of cell types within the immune system. Among functional categories co-enriched with both differential expression and noncoding adaptation, prominent themes include cancer, particularly epithelial cancers, and neural development and function.

The impact of gene expression variation on the robustness and evolvability of a developmental gene regulatory network.

Regulatory interactions buffer development against genetic and environmental perturbations, but adaptation requires phenotypes to change. We investigated the relationship between robustness and evolvability within the gene regulatory network underlying development of the larval skeleton in the sea urchin Strongylocentrotus purpuratus. We find extensive variation in gene expression in this network throughout development in a natural population, some of which has a heritable genetic basis. Switch-like regulatory interactions predominate during early development, buffer expression variation, and may promote the accumulation of cryptic genetic variation affecting early stages. Regulatory interactions during later development are typically more sensitive (linear), allowing variation in expression to affect downstream target genes. Variation in skeletal morphology is associated primarily with expression variation of a few, primarily structural, genes at terminal positions within the network. These results indicate that the position and properties of gene interactions within a network can have important evolutionary consequences independent of their immediate regulatory role.

Recent advances in functional assays of transcriptional enhancers.

In this special edition of Genomics, we present reviews of the current state of the field in identifying and functionally understanding transcriptional enhancers in cells and developing tissues. Typically several enhancers coordinate the expression of an individual target gene, each controlling that gene's expression in specific cell types at specific times. Until recently, identifying each gene's enhancers had been challenging because enhancers do not occupy prescribed locations relative to their target genes. Recently there have been powerful advances in DNA sequencing and other technologies that make it possible to identify the majority of enhancers in virtually any cell type of interest. The reviews in this edition of Genomics highlight some of these new and powerful approaches.

Convergent adaptation of human lactase persistence in Africa and Europe.

A SNP in the gene encoding lactase (LCT) (C/T-13910) is associated with the ability to digest milk as adults (lactase persistence) in Europeans, but the genetic basis of lactase persistence in Africans was previously unknown. We conducted a genotype-phenotype association study in 470 Tanzanians, Kenyans and Sudanese and identified three SNPs (G/C-14010, T/G-13915 and C/G-13907) that are associated with lactase persistence and that have derived alleles that significantly enhance transcription from the LCT promoter in vitro. These SNPs originated on different haplotype backgrounds from the European C/T-13910 SNP and from each other. Genotyping across a 3-Mb region demonstrated haplotype homozygosity extending >2.0 Mb on chromosomes carrying C-14010, consistent with a selective sweep over the past approximately 7,000 years. These data provide a marked example of convergent evolution due to strong selective pressure resulting from shared cultural traits-animal domestication and adult milk consumption.

Extensive evolutionary changes in regulatory element activity during human origins are associated with altered gene expression and positive selection.

Understanding the molecular basis for phenotypic differences between humans and other primates remains an outstanding challenge. Mutations in non-coding regulatory DNA that alter gene expression have been hypothesized as a key driver of these phenotypic differences. This has been supported by differential gene expression analyses in general, but not by the identification of specific regulatory elements responsible for changes in transcription and phenotype. To identify the genetic source of regulatory differences, we mapped DNaseI hypersensitive (DHS) sites, which mark all types of active gene regulatory elements, genome-wide in the same cell type isolated from human, chimpanzee, and macaque. Most DHS sites were conserved among all three species, as expected based on their central role in regulating transcription. However, we found evidence that several hundred DHS sites were gained or lost on the lineages leading to modern human and chimpanzee. Species-specific DHS site gains are enriched near differentially expressed genes, are positively correlated with increased transcription, show evidence of branch-specific positive selection, and overlap with active chromatin marks. Species-specific sequence differences in transcription factor motifs found within these DHS sites are linked with species-specific changes in chromatin accessibility. Together, these indicate that the regulatory elements identified here are genetic contributors to transcriptional and phenotypic differences among primate species.

The appropriation of glucose through primate neurodevelopment.

The human brain is considerably larger and more energetically costly than that of other primate species. As such, discovering how human ancestors were able to provide sufficient energy to their brains is a central theme in the study of hominin evolution. However, many discussions of metabolism frequently omit the different ways in which energy, primarily glucose, is used once made available to the brain. In this review, we discuss two glucose metabolic pathways, oxidative phosphorylation and aerobic glycolysis, and their respective contributions to the energetic and anabolic budgets of the brain. While oxidative phosphorylation is a more efficient producer of energy, aerobic glycolysis contributes essential molecules for the growth of the brain and maintaining the structure of its cells. Although both pathways occur in the brain throughout the lifetime, aerobic glycolysis is a critical pathway during development, and oxidative phosphorylation is highest during adulthood. We outline how elevated levels of aerobic glycolysis may support the protracted neurodevelopmental sequence of humans compared with other primates. Finally, we review the genetic evidence for differences in metabolic function in the brains of primates and explore genes that may provide insight into how glucose metabolism may differ across species.

Genetic comparisons yield insight into the evolution of enamel thickness during human evolution.

Enamel thickness varies substantially among extant hominoids and is a key trait with significance for interpreting dietary adaptation, life history trajectory, and phylogenetic relationships. There is a strong link in humans between enamel formation and mutations in the exons of the four genes that code for the enamel matrix proteins and the associated protease. The evolution of thick enamel in humans may have included changes in the regulation of these genes during tooth development. The cis-regulatory region in the 5' flank (upstream non-coding region) of MMP20, which codes for enamelysin, the predominant protease active during enamel secretion, has previously been shown to be under strong positive selection in the lineages leading to both humans and chimpanzees. Here we examine evidence for positive selection in the 5' flank and 3' flank of AMELX, AMBN, ENAM, and MMP20. We contrast the human sequence changes with other hominoids (chimpanzees, gorillas, orangutans, gibbons) and rhesus macaques (outgroup), a sample comprising a range of enamel thickness. We find no evidence for positive selection in the protein-coding regions of any of these genes. In contrast, we find strong evidence for positive selection in the 5' flank region of MMP20 and ENAM along the lineage leading to humans, and in both the 5' flank and 3' flank regions of MMP20 along the lineage leading to chimpanzees. We also identify putative transcription factor binding sites overlapping some of the species-specific nucleotide sites and we refine which sections of the up- and downstream putative regulatory regions are most likely to harbor important changes. These non-coding changes and their potential for differential regulation by transcription factors known to regulate tooth development may offer insight into the mechanisms that allow for rapid evolutionary changes in enamel thickness across closely-related species, and contribute to our understanding of the enamel phenotype in hominoids.

Astrocytes Drive Divergent Metabolic Gene Expression in Humans and Chimpanzees.

The human brain utilizes ∼20% of all of the body's metabolic resources, while chimpanzee brains use <10%. Although previous work shows significant differences in metabolic gene expression between the brains of primates, we have yet to fully resolve the contribution of distinct brain cell types. To investigate cell type-specific interspecies differences in brain gene expression, we conducted RNA-seq on neural progenitor cells, neurons, and astrocytes generated from induced pluripotent stem cells from humans and chimpanzees. Interspecies differential expression analyses revealed that twice as many genes exhibit differential expression in astrocytes (12.2% of all genes expressed) than neurons (5.8%). Pathway enrichment analyses determined that astrocytes, rather than neurons, diverged in expression of glucose and lactate transmembrane transport, as well as pyruvate processing and oxidative phosphorylation. These findings suggest that astrocytes may have contributed significantly to the evolution of greater brain glucose metabolism with proximity to humans.

Tempo and mode of gene expression evolution in the brain across primates.

Primate evolution has led to a remarkable diversity of behavioral specializations and pronounced brain size variation among species (Barton, 2012; DeCasien and Higham, 2019; Powell et al., 2017). Gene expression provides a promising opportunity for studying the molecular basis of brain evolution, but it has been explored in very few primate species to date (e.g. Khaitovich et al., 2005; Khrameeva et al., 2020; Ma et al., 2022; Somel et al., 2009). To understand the landscape of gene expression evolution across the primate lineage, we generated and analyzed RNA-seq data from four brain regions in an unprecedented eighteen species. Here, we show a remarkable level of variation in gene expression among hominid species, including humans and chimpanzees, despite their relatively recent divergence time from other primates. We found that individual genes display a wide range of expression dynamics across evolutionary time reflective of the diverse selection pressures acting on genes within primate brain tissue. Using our samples that represent a 190-fold difference in primate brain size, we identified genes with variation in expression most correlated with brain size. Our study extensively broadens the phylogenetic context of what is known about the molecular evolution of the brain across primates and identifies novel candidate genes for the study of genetic regulation of brain evolution.

Contrasts between adaptive coding and noncoding changes during human evolution.

Changes in non-protein-coding regulatory DNA sequences have been proposed to play distinctive roles in adaptive evolution. We analyzed correlations between gene functions and evidence for positive selection in a common statistical framework across several large surveys of coding and noncoding sequences throughout the human genome. Strong correlations with both classifications in gene ontologies and measurements of gene expression indicate that neural development and function have adapted mainly through noncoding changes. In contrast, adaptation via coding changes is dominated by immunity, olfaction, and male reproduction. Genes with highly tissue-specific expression have undergone more adaptive coding changes, suggesting that pleiotropic constraints inhibit such changes in broadly expressed genes. In contrast, adaptive noncoding changes do not exhibit this pattern. Our findings underscore the probable importance of noncoding changes in the evolution of human traits, particularly cognitive traits.

Genetics of gene expression responses to temperature stress in a sea urchin gene network.

Stress responses play an important role in shaping species distributions and robustness to climate change. We investigated how stress responses alter the contribution of additive genetic variation to gene expression during development of the purple sea urchin, Strongylocentrotus purpuratus, under increased temperatures that model realistic climate change scenarios. We first measured gene expression responses in the embryos by RNA-seq to characterize molecular signatures of mild, chronic temperature stress in an unbiased manner. We found that an increase from 12 to 18 °C caused widespread alterations in gene expression including in genes involved in protein folding, RNA processing and development. To understand the quantitative genetic architecture of this response, we then focused on a well-characterized gene network involved in endomesoderm and ectoderm specification. Using a breeding design with wild-caught individuals, we measured genetic and gene-environment interaction effects on 72 genes within this network. We found genetic or maternal effects in 33 of these genes and that the genetic effects were correlated in the network. Fourteen network genes also responded to higher temperatures, but we found no significant genotype-environment interactions in any of the genes. This absence may be owing to an effective buffering of the temperature perturbations within the network. In support of this hypothesis, perturbations to regulatory genes did not affect the expression of the genes that they regulate. Together, these results provide novel insights into the relationship between environmental change and developmental evolution and suggest that climate change may not expose large amounts of cryptic genetic variation to selection in this species.

Differentially Active and Conserved Neural Enhancers Define Two Forms of Adaptive Noncoding Evolution in Humans.

The human and chimpanzee genomes are strikingly similar, but our neural phenotypes are very different. Many of these differences are likely driven by changes in gene expression, and some of those changes may have been adaptive during human evolution. Yet, the relative contributions of positive selection on regulatory regions or other functional regulatory changes are unclear. Where are these changes located throughout the human genome? Are functional regulatory changes near genes or are they in distal enhancer regions? In this study, we experimentally combined both human and chimpanzee cis-regulatory elements (CREs) that showed either (1) signs of accelerated evolution in humans or (2) that have been shown to be active in the human brain. Using a massively parallel reporter assay, we tested the ability of orthologous human and chimpanzee CREs to activate transcription in induced pluripotent stem-cell-derived neural progenitor cells and neurons. With this assay, we identified 179 CREs with differential activity between human and chimpanzee; in contrast, we found 722 CREs with signs of positive selection in humans. Selection and differentially expressed CREs strikingly differ in level of expression, size, and genomic location. We found a subset of 69 CREs in loci with genetic variants associated with neuropsychiatric diseases, which underscores the consequence of regulatory activity in these loci for proper neural development and function. By combining CREs that either experienced recent selection in humans or CREs that are functional brain enhancers, presents a novel way of studying the evolution of noncoding elements that contribute to human neural phenotypes.

Multiple Functional Variants in cis Modulate PDYN Expression.

Understanding genetic variation and its functional consequences within cis-regulatory regions remains an important challenge in human genetics and evolution. Here, we present a fine-scale functional analysis of segregating variation within the cis-regulatory region of prodynorphin, a gene that encodes an endogenous opioid precursor with roles in cognition and disease. In order to characterize the functional consequences of segregating variation in cis in a region under balancing selection in different human populations, we examined associations between specific polymorphisms and gene expression in vivo and in vitro. We identified five polymorphisms within the 5' flanking region that affect transcript abundance: a 68-bp repeat recognized in prior studies, as well as two microsatellites and two single nucleotide polymorphisms not previously implicated as functional variants. The impact of these variants on transcription differs by brain region, sex, and cell type, implying interactions between cis genotype and the differentiated state of cells. The effects of individual variants on expression level are not additive in some combinations, implying epistatic interactions between nearby variants. These data reveal an unexpectedly complex relationship between segregating genetic variation and its expression-trait consequences and highlights the importance of close functional scrutiny of natural genetic variation within even relatively well-studied cis-regulatory regions.

Genomic signatures of diet-related shifts during human origins.

There are numerous anthropological analyses concerning the importance of diet during human evolution. Diet is thought to have had a profound influence on the human phenotype, and dietary differences have been hypothesized to contribute to the dramatic morphological changes seen in modern humans as compared with non-human primates. Here, we attempt to integrate the results of new genomic studies within this well-developed anthropological context. We then review the current evidence for adaptation related to diet, both at the level of sequence changes and gene expression. Finally, we propose some ways in which new technologies can help identify specific genomic adaptations that have resulted in metabolic and morphological differences between humans and non-human primates.

Comparative expression analysis of the phosphocreatine circuit in extant primates: Implications for human brain evolution.

While the hominid fossil record clearly shows that brain size has rapidly expanded over the last ~2.5 M.yr. the forces driving this change remain unclear. One popular hypothesis proposes that metabolic adaptations in response to dietary shifts supported greater encephalization in humans. An increase in meat consumption distinguishes the human diet from that of other great apes. Creatine, an essential metabolite for energy homeostasis in muscle and brain tissue, is abundant in meat and was likely ingested in higher quantities during human origins. Five phosphocreatine circuit proteins help regulate creatine utilization within energy demanding cells. We compared the expression of all five phosphocreatine circuit genes in cerebral cortex, cerebellum, and skeletal muscle tissue for humans, chimpanzees, and rhesus macaques. Strikingly, SLC6A8 and CKB transcript levels are higher in the human brain, which should increase energy availability and turnover compared to non-human primates. Combined with other well-documented differences between humans and non-human primates, this allocation of energy to the cerebral cortex and cerebellum may be important in supporting the increased metabolic demands of the human brain.

A potential role for glucose transporters in the evolution of human brain size.

Differences in cognitive abilities and the relatively large brain are among the most striking differences between humans and their closest primate relatives. The energy trade-off hypothesis predicts that a major shift in energy allocation among tissues occurred during human origins in order to support the remarkable expansion of a metabolically expensive brain. However, the molecular basis of this adaptive scenario is unknown. Two glucose transporters (SLC2A1 and SLC2A4) are promising candidates and present intriguing mutations in humans, resulting, respectively, in microcephaly and disruptions in whole-body glucose homeostasis. We compared SLC2A1 and SLC2A4 expression between humans, chimpanzees and macaques, and found compensatory and biologically significant expression changes on the human lineage within cerebral cortex and skeletal muscle, consistent with mediating an energy trade-off. We also show that these two genes are likely to have undergone adaptation and participated in the development and maintenance of a larger brain in the human lineage by modulating brain and skeletal muscle energy allocation. We found that these two genes show human-specific signatures of positive selection on known regulatory elements within their 5'-untranslated region, suggesting an adaptation of their regulation during human origins. This study represents the first case where adaptive, functional and genetic lines of evidence implicate specific genes in the evolution of human brain size.

Genetic Mutations Associated with Hormone-Positive Breast Cancer in a Small Cohort of Ethiopian Women.

In Ethiopia, a breast cancer diagnosis is associated with a prognosis significantly worse than that of Europe and the US. Further, patients presenting with breast cancer in Ethiopia are far younger, on average, and patients are typically diagnosed at very late stages, relative to breast cancer patients of European descent. Emerging data suggest that a large proportion of Ethiopian patients have hormone-positive (ER+) breast cancer. This is surprising given (1) that patients have late-stage breast cancer at the time of diagnosis, (2) that African Americans with breast cancer frequently have triple negative breast cancer (TNBC), and (3) these patients typically receive chemotherapy, not hormone-targeting drugs. To further examine the similarity of Ethiopian breast tumors to those of African Americans or of those of European descent, we sequenced matched tumor and normal adjacent tissue from Ethiopian patients from a small pilot collection. We identified mutations in 615 genes across all three patients, unique to the tumor tissue. Across this analysis, we found far more mutations shared between Ethiopian patient tissue and that from white patients (103) than we did comparing to African Americans (3). Several mutations were found in extracellular matrix encoding genes with known roles in tumor cell growth and metastasis. We suggest future mechanistic studies on this disease focus on these genes first, toward finding new treatment strategies for breast cancer patients in Ethiopia.