Human milk variation is shaped by maternal genetics and impacts the infant gut microbiome.

Human milk is a complex mix of nutritional and bioactive components that provide complete nourishment for the infant. However, we lack a systematic knowledge of the factors shaping milk composition and how milk variation influences infant health. Here, we characterize relationships between maternal genetics, milk gene expression, milk composition, and the infant fecal microbiome in up to 310 exclusively breastfeeding mother-infant pairs. We identified 482 genetic loci associated with milk gene expression unique to the lactating mammary gland and link these loci to breast cancer risk and human milk oligosaccharide concentration. Integrative analyses uncovered connections between milk gene expression and infant gut microbiome, including an association between the expression of inflammation-related genes with milk interleukin-6 (IL-6) concentration and the abundance of Bifidobacterium and Escherichia in the infant gut. Our results show how an improved understanding of the genetics and genomics of human milk connects lactation biology with maternal and infant health.

Chondroitin sulfate proteoglycan 4 (CSPG4) increases invasion of recessive dystrophic epidermolysis bullosa-associated cutaneous squamous cell carcinoma by modifying TGFß signaling.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic skin-blistering disorder often progressing to metastatic cutaneous squamous cell carcinoma (cSCC) at chronic wound sites. Chondroitin sulfate proteoglycan 4 (CSPG4) is a cell-surface proteoglycan that is an oncoantigen in multiple malignancies, where it modulates oncogenic signaling, drives epithelial-to-mesenchymal transition (EMT), and enables cell motility. OBJECTIVES: To evaluate CSPG4 expression and function in RDEB-cSCC. METHODS: RDEB-cSCC cell lines were used to assess CSPG4-dependent changes in invasive potential, TGFß1-stimulated signal activation, and clinically relevant cytopathology metrics in an in vitro full-thickness tumor model. CSPG4 expression in RDEB-cSCC and non-RDEB cSCC tumors was analyzed via immunohistochemistry and single-cell RNA sequencing (scRNA-seq), respectively. RESULTS: Inhibiting CSPG4 expression reduced invasive potential in multiple RDEB-cSCC cell lines and altered membrane-proximal TGFß signal activation through changes in SMAD3 phosphorylation. CSPG4 expression was uniformly localized to basal-layer keratinocytes in fibrotic RDEB skin and tumor cells at the tumor/stroma interface at the invasive front in RDEB-cSCC tumors in vivo. Analysis of published scRNA-seq data revealed that CSPG4 expression was correlated with an enhanced EMT transcriptomic signature in cells at the tumor/stroma interface of non-RDEB cSCC tumors. Cytopathological metrics, like nucleus:cell area ratio, were influenced by CSPG4 expression in in vitro tumor models. CONCLUSIONS: We determined that CSPG4 expression in RDEB-cSCC cell lines enhanced invasive potential. Mechanistically, CSPG4 was found to enhance membrane-proximal TGFß-stimulated signaling through SMAD3, which is a key mediator of EMT in RDEB-cSCC. The implication of these studies is that CSPG4 may represent a therapeutic target that can be leveraged for clinical management in patients with RDEB-cSCC.

Human cytomegalovirus in breast milk is associated with milk composition and the infant gut microbiome and growth.

Human cytomegalovirus (CMV) is a highly prevalent herpesvirus that is often transmitted to the neonate via breast milk. Postnatal CMV transmission can have negative health consequences for preterm and immunocompromised infants, but any effects on healthy term infants are thought to be benign. Furthermore, the impact of CMV on the composition of the hundreds of bioactive factors in human milk has not been tested. Here, we utilize a cohort of exclusively breastfeeding full-term mother-infant pairs to test for differences in the milk transcriptome and metabolome associated with CMV, and the impact of CMV in breast milk on the infant gut microbiome and infant growth. We find upregulation of the indoleamine 2,3-dioxygenase (IDO) tryptophan-to-kynurenine metabolic pathway in CMV+ milk samples, and that CMV+ milk is associated with decreased Bifidobacterium in the infant gut. Our data indicate two opposing CMV-associated effects on infant growth; with kynurenine positively correlated, and CMV viral load negatively correlated, with infant weight-for-length at 1 month of age. These results suggest CMV transmission, CMV-related changes in milk composition, or both may be modulators of full-term infant development.

Assembly, stability, and dynamics of the infant gut microbiome are linked to bacterial strains and functions in mother's milk.

The establishment of the gut microbiome in early life is critical for healthy infant development. Although human milk is recommended as the sole source of nutrition for the human infant, little is known about how variation in milk composition, and especially the milk microbiome, shapes the microbial communities in the infant gut. Here, we quantified the similarity between the maternal milk and the infant gut microbiome using 507 metagenomic samples collected from 195 mother-infant pairs at one, three, and six months postpartum. We found that the microbial taxonomic overlap between milk and the infant gut was driven by bifidobacteria, in particular by B. longum. Infant stool samples dominated by B. longum also showed higher temporal stability compared to samples dominated by other species. We identified two instances of strain sharing between maternal milk and the infant gut, one involving a commensal (B. longum) and one a pathobiont (K. pneumoniae). In addition, strain sharing between unrelated infants was higher among infants born at the same hospital compared to infants born in different hospitals, suggesting a potential role of the hospital environment in shaping the infant gut microbiome composition. The infant gut microbiome at one month compared to six months of age was enriched in metabolic pathways associated with de-novo molecule biosynthesis, suggesting that early colonisers might be more versatile and metabolically independent compared to later colonizers. Lastly, we found a significant overlap in antimicrobial resistance genes carriage between the mother's milk and their infant's gut microbiome. Taken together, our results suggest that the human milk microbiome has an important role in the assembly, composition, and stability of the infant gut microbiome.

Trans-eQTL hotspots shape complex traits by modulating cellular states.

Regulatory genetic variation shapes gene expression, providing an important mechanism connecting DNA variation and complex traits. The causal relationships between gene expression and complex traits remain poorly understood. Here, we integrated transcriptomes and 46 genetically complex growth traits in a large cross between two strains of the yeast Saccharomyces cerevisiae. We discovered thousands of genetic correlations between gene expression and growth, suggesting functional connections. Local regulatory variation was a minor source of these genetic correlations. Instead, genetic correlations tended to arise from multiple independent trans-acting regulatory loci. Trans-acting hotspots that affect the expression of numerous genes accounted for particularly large fractions of genetic growth variation and of genetic correlations between gene expression and growth. Genes with genetic correlations were enriched for similar biological processes across traits, but with heterogeneous direction of effect. Our results reveal how trans-acting regulatory hotspots shape complex traits by altering cellular states.

Integration of 168,000 samples reveals global patterns of the human gut microbiome.

Understanding the factors that shape variation in the human microbiome is a major goal of research in biology. While other genomics fields have used large, pre-compiled compendia to extract systematic insights requiring otherwise impractical sample sizes, there has been no comparable resource for the 16S rRNA sequencing data commonly used to quantify microbiome composition. To help close this gap, we have assembled a set of 168,484 publicly available human gut microbiome samples, processed with a single pipeline and combined into the largest unified microbiome dataset to date. We use this resource, which is freely available at microbiomap.org, to shed light on global variation in the human gut microbiome. We find that Firmicutes, particularly Bacilli and Clostridia, are almost universally present in the human gut. At the same time, the relative abundance of the 65 most common microbial genera differ between at least two world regions. We also show that gut microbiomes in undersampled world regions, such as Central and Southern Asia, differ significantly from the more thoroughly characterized microbiomes of Europe and Northern America. Moreover, humans in these overlooked regions likely harbor hundreds of taxa that have not yet been discovered due to this undersampling, highlighting the need for diversity in microbiome studies. We anticipate that this new compendium can serve the community and enable advanced applied and methodological research.

Human Cytomegalovirus in breast milk is associated with milk composition, the infant gut microbiome, and infant growth.

Human cytomegalovirus (CMV) is a highly prevalent herpesvirus that is often transmitted to the neonate via breast milk. Postnatal CMV transmission can have negative health consequences for preterm and immunocompromised infants, but any effects on healthy term infants are thought to be benign. Furthermore, the impact of CMV on the composition of the hundreds of bioactive factors in human milk has not been tested. Here, we utilize a cohort of exclusively breastfeeding full term mother-infant pairs to test for differences in the milk transcriptome and metabolome associated with CMV, and the impact of CMV in breast milk on the infant gut microbiome and infant growth. We find upregulation of the indoleamine 2,3- dioxygenase (IDO) tryptophan-to-kynurenine metabolic pathway in CMV+ milk samples, and that CMV+ milk is associated with decreased Bifidobacterium in the infant gut. Our data indicate a complex relationship between milk CMV, milk kynurenine, and infant growth; with kynurenine positively correlated, and CMV viral load negatively correlated, with infant weight-for-length at 1 month of age. These results suggest CMV transmission, CMV-related changes in milk composition, or both may be modulators of full term infant development.

Substrate-specific effects of natural genetic variation on proteasome activity.

Protein degradation is an essential biological process that regulates protein abundance and removes misfolded and damaged proteins from cells. In eukaryotes, most protein degradation occurs through the stepwise actions of two functionally distinct entities, the ubiquitin system and the proteasome. Ubiquitin system enzymes attach ubiquitin to cellular proteins, targeting them for degradation. The proteasome then selectively binds and degrades ubiquitinated substrate proteins. Genetic variation in ubiquitin system genes creates heritable differences in the degradation of their substrates. However, the challenges of measuring the degradative activity of the proteasome independently of the ubiquitin system in large samples have limited our understanding of genetic influences on the proteasome. Here, using the yeast Saccharomyces cerevisiae, we built and characterized reporters that provide high-throughput, ubiquitin system-independent measurements of proteasome activity. Using single-cell measurements of proteasome activity from millions of genetically diverse yeast cells, we mapped 15 loci across the genome that influence proteasomal protein degradation. Twelve of these 15 loci exerted specific effects on the degradation of two distinct proteasome substrates, revealing a high degree of substrate-specificity in the genetics of proteasome activity. Using CRISPR-Cas9-based allelic engineering, we resolved a locus to a causal variant in the promoter of RPT6, a gene that encodes a subunit of the proteasome's 19S regulatory particle. The variant increases RPT6 expression, which we show results in increased proteasome activity. Our results reveal the complex genetic architecture of proteasome activity and suggest that genetic influences on the proteasome may be an important source of variation in the many cellular and organismal traits shaped by protein degradation.

Human milk variation is shaped by maternal genetics and impacts the infant gut microbiome.

Human milk is a complex mix of nutritional and bioactive components that provide complete nutrition for the infant. However, we lack a systematic knowledge of the factors shaping milk composition and how milk variation influences infant health. Here, we used multi-omic profiling to characterize interactions between maternal genetics, milk gene expression, milk composition, and the infant fecal microbiome in 242 exclusively breastfeeding mother-infant pairs. We identified 487 genetic loci associated with milk gene expression unique to the lactating mammary gland, including loci that impacted breast cancer risk and human milk oligosaccharide concentration. Integrative analyses uncovered connections between milk gene expression and infant gut microbiome, including an association between the expression of inflammation-related genes with IL-6 concentration in milk and the abundance of Bifidobacteria in the infant gut. Our results show how an improved understanding of the genetics and genomics of human milk connects lactation biology with maternal and infant health.

Genes and compounds that increase type VII collagen expression as potential treatments for dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a skin-blistering disease caused by mutations in COL7A1, which encodes type VII collagen (C7). There is no cure for DEB, but previous work has shown potential therapeutic benefit of increased production of even partially functional C7. Genome-wide screens using CRISPR-Cas9 have enabled the identification of genes involved in cancer development, drug resistance and other genetic diseases, suggesting that they could be used to identify drivers of C7 production. A keratinocyte C7 reporter cell line was created and used in a genome-wide CRISPR activation (CRISPRa) screen to identify genes and pathways that increase C7 expression. The CRISPRa screen results were used to develop a targeted drug screen to identify compounds that upregulate C7 expression. The C7_tdTomato cell line was validated as an effective reporter for detection of C7 upregulation. The CRISPRa screen identified DENND4B and TYROBP as top gene hits plus pathways related to calcium uptake and immune signalling in C7 regulation. The targeted drug screen identified several compounds that increase C7 expression in keratinocytes, of which kaempferol, a plant flavonoid, also significantly increased C7 mRNA and protein in DEB patient cells.

Multiple epistatic DNA variants in a single gene affect gene expression in trans.

DNA variants that alter gene expression in trans are important sources of phenotypic variation. Nevertheless, the identity of trans-acting variants remains poorly understood. Single causal variants in several genes have been reported to affect the expression of numerous distant genes in trans. Whether these simple molecular architectures are representative of trans-acting variation is unknown. Here, we studied the large RAS signaling regulator gene IRA2, which contains variants with extensive trans-acting effects on gene expression in the yeast Saccharomyces cerevisiae. We used systematic CRISPR-based genome engineering and a sensitive phenotyping strategy to dissect causal variants to the nucleotide level. In contrast to the simple molecular architectures known so far, IRA2 contained at least seven causal nonsynonymous variants. The effects of these variants were modulated by nonadditive, epistatic interactions. Two variants at the 5'-end affected gene expression and growth only when combined with a third variant that also had no effect in isolation. Our findings indicate that the molecular basis of trans-acting genetic variation may be considerably more complex than previously appreciated.

Trans-acting genetic variation affects the expression of adjacent genes.

Gene expression differences among individuals are shaped by trans-acting expression quantitative trait loci (eQTLs). Most trans-eQTLs map to hotspot locations that influence many genes. The molecular mechanisms perturbed by hotspots are often assumed to involve "vertical" cascades of effects in pathways that can ultimately affect the expression of thousands of genes. Here, we report that trans-eQTLs can affect the expression of adjacent genes via "horizontal" mechanisms that extend along a chromosome. Genes affected by trans-eQTL hotspots in the yeast Saccharomyces cerevisiae were more likely to be located next to each other than expected by chance. These paired hotspot effects tended to occur at adjacent genes that also show coexpression in response to genetic and environmental perturbations, suggesting shared mechanisms. Physical proximity and shared chromatin state, in addition to regulation of adjacent genes by similar transcription factors, were independently associated with paired hotspot effects among adjacent genes. Paired effects of trans-eQTLs can occur at neighboring genes even when these genes do not share a common function. This phenomenon could result in unexpected connections between regulatory genetic variation and phenotypes.

Systematic identification of cis-regulatory variants that cause gene expression differences in a yeast cross.

Sequence variation in regulatory DNA alters gene expression and shapes genetically complex traits. However, the identification of individual, causal regulatory variants is challenging. Here, we used a massively parallel reporter assay to measure the cis-regulatory consequences of 5832 natural DNA variants in the promoters of 2503 genes in the yeast Saccharomyces cerevisiae. We identified 451 causal variants, which underlie genetic loci known to affect gene expression. Several promoters harbored multiple causal variants. In five promoters, pairs of variants showed non-additive, epistatic interactions. Causal variants were enriched at conserved nucleotides, tended to have low derived allele frequency, and were depleted from promoters of essential genes, which is consistent with the action of negative selection. Causal variants were also enriched for alterations in transcription factor binding sites. Models integrating these features provided modest, but statistically significant, ability to predict causal variants. This work revealed a complex molecular basis for cis-acting regulatory variation.

DNA variants affecting the expression of numerous genes in trans have diverse mechanisms of action and evolutionary histories.

DNA variants that alter gene expression contribute to variation in many phenotypic traits. In particular, trans-acting variants, which are often located on different chromosomes from the genes they affect, are an important source of heritable gene expression variation. However, our knowledge about the identity and mechanism of causal trans-acting variants remains limited. Here, we developed a fine-mapping strategy called CRISPR-Swap and dissected three expression quantitative trait locus (eQTL) hotspots known to alter the expression of numerous genes in trans in the yeast Saccharomyces cerevisiae. Causal variants were identified by engineering recombinant alleles and quantifying the effects of these alleles on the expression of a green fluorescent protein-tagged gene affected by the given locus in trans. We validated the effect of each variant on the expression of multiple genes by RNA-sequencing. The three variants differed in their molecular mechanism, the type of genes they reside in, and their distribution in natural populations. While a missense leucine-to-serine variant at position 63 in the transcription factor Oaf1 (L63S) was almost exclusively present in the reference laboratory strain, the two other variants were frequent among S. cerevisiae isolates. A causal missense variant in the glucose receptor Rgt2 (V539I) occurred at a poorly conserved amino acid residue and its effect was strongly dependent on the concentration of glucose in the culture medium. A noncoding variant in the conserved fatty acid regulated (FAR) element of the OLE1 promoter influenced the expression of the fatty acid desaturase Ole1 in cis and, by modulating the level of this essential enzyme, other genes in trans. The OAF1 and OLE1 variants showed a non-additive genetic interaction, and affected cellular lipid metabolism. These results demonstrate that the molecular basis of trans-regulatory variation is diverse, highlighting the challenges in predicting which natural genetic variants affect gene expression.

Facial shape differences between rats selected for tame and aggressive behaviors.

Domestication has been consistently accompanied by a suite of traits called the domestication syndrome. These include increased docility, changes in coat coloration, prolonged juvenile behaviors, modified function of adrenal glands and reduced craniofacial dimensions. Wilkins et al recently proposed that the mechanistic factor underlying traits that encompass the domestication syndrome was altered neural crest cell (NCC) development. NCC form the precursors to a large number of tissue types including pigment cells, adrenal glands, teeth and the bones of the face. The hypothesis that deficits in NCC development can account for the domestication syndrome was partly based on the outcomes of Dmitri Belyaev's domestication experiments initially conducted on silver foxes. After generations of selecting for tameness, the foxes displayed phenotypes observed in domesticated species. Belyaev also had a colony of rats selected over 64 generations for either tameness or defensive aggression towards humans. Here we focus on the facial morphology of Belyaev's tame, 'domesticated' rats to test whether: 1) tameness in rats causes craniofacial changes similar to those observed in the foxes; 2) facial shape, i.e. NCC-derived region, is distinct in the tame and aggressive rats. We used computed-tomography scans of rat skulls and landmark-based geometric morphometrics to quantify and analyze the facial skeleton. We found facial shape differences between the tame and aggressive rats that were independent of size and which mirrored changes seen in domesticated animals compared to their wild counterparts. However, there was no evidence of reduced sexual dimorphism in the face of the tame rats. This indicates that not all morphological changes in NCC-derived regions in the rats follow the pattern of shape change reported in domesticated animals or the silver foxes. Thus, certain phenotypic trends that are part of the domestication syndrome might not be consistently present in all experimental animal models.

Genetic interactions contribute less than additive effects to quantitative trait variation in yeast.

Genetic mapping studies of quantitative traits typically focus on detecting loci that contribute additively to trait variation. Genetic interactions are often proposed as a contributing factor to trait variation, but the relative contribution of interactions to trait variation is a subject of debate. Here we use a very large cross between two yeast strains to accurately estimate the fraction of phenotypic variance due to pairwise QTL-QTL interactions for 20 quantitative traits. We find that this fraction is 9% on average, substantially less than the contribution of additive QTL (43%). Statistically significant QTL-QTL pairs typically have small individual effect sizes, but collectively explain 40% of the pairwise interaction variance. We show that pairwise interaction variance is largely explained by pairs of loci at least one of which has a significant additive effect. These results refine our understanding of the genetic architecture of quantitative traits and help guide future mapping studies.

The role of regulatory variation in complex traits and disease.

We are in a phase of unprecedented progress in identifying genetic loci that cause variation in traits ranging from growth and fitness in simple organisms to disease in humans. However, a mechanistic understanding of how these loci influence traits is lacking for the majority of loci. Studies of the genetics of gene expression have emerged as a key tool for linking DNA sequence variation to phenotypes. Here, we review recent insights into the molecular nature of regulatory variants and describe their influence on the transcriptome and the proteome. We discuss conceptual advances from studies in model organisms and present examples of complete chains of causality that link individual polymorphisms to changes in gene expression, which in turn result in physiological changes and, ultimately, disease risk.

Genetic mapping of MAPK-mediated complex traits Across S. cerevisiae.

Signaling pathways enable cells to sense and respond to their environment. Many cellular signaling strategies are conserved from fungi to humans, yet their activity and phenotypic consequences can vary extensively among individuals within a species. A systematic assessment of the impact of naturally occurring genetic variation on signaling pathways remains to be conducted. In S. cerevisiae, both response and resistance to stressors that activate signaling pathways differ between diverse isolates. Here, we present a quantitative trait locus (QTL) mapping approach that enables us to identify genetic variants underlying such phenotypic differences across the genetic and phenotypic diversity of S. cerevisiae. Using a Round-robin cross between twelve diverse strains, we identified QTL that influence phenotypes critically dependent on MAPK signaling cascades. Genetic variants under these QTL fall within MAPK signaling networks themselves as well as other interconnected signaling pathways. Finally, we demonstrate how the mapping results from multiple strain background can be leveraged to narrow the search space of causal genetic variants.

Genetic influences on translation in yeast.

Heritable differences in gene expression between individuals are an important source of phenotypic variation. The question of how closely the effects of genetic variation on protein levels mirror those on mRNA levels remains open. Here, we addressed this question by using ribosome profiling to examine how genetic differences between two strains of the yeast S. cerevisiae affect translation. Strain differences in translation were observed for hundreds of genes. Allele specific measurements in the diploid hybrid between the two strains revealed roughly half as many cis-acting effects on translation as were observed for mRNA levels. In both the parents and the hybrid, most effects on translation were of small magnitude, such that the direction of an mRNA difference was typically reflected in a concordant footprint difference. The relative importance of cis and trans acting variation on footprint levels was similar to that for mRNA levels. There was a tendency for translation to cause larger footprint differences than expected given the respective mRNA differences. This is in contrast to translational differences between yeast species that have been reported to more often oppose than reinforce mRNA differences. Finally, we catalogued instances of premature translation termination in the two yeast strains and also found several instances where erroneous reference gene annotations lead to apparent nonsense mutations that in fact reside outside of the translated gene body. Overall, genetic influences on translation subtly modulate gene expression differences, and translation does not create strong discrepancies between genetic influences on mRNA and protein levels.