Transposable elements in mammalian chromatin organization.

Transposable elements (TEs) are mobile DNA elements that comprise almost 50% of mammalian genomic sequence. TEs are capable of making additional copies of themselves that integrate into new positions in host genomes. This unique property has had an important impact on mammalian genome evolution and on the regulation of gene expression because TE-derived sequences can function as cis-regulatory elements such as enhancers, promoters and silencers. Now, advances in our ability to identify and characterize TEs have revealed that TE-derived sequences also regulate gene expression by both maintaining and shaping 3D genome architecture. Studies are revealing how TEs contribute raw sequence that can give rise to the structures that shape chromatin organization, and thus gene expression, allowing for species-specific genome innovation and evolutionary novelty.

The Human Pangenome Project: a global resource to map genomic diversity.

The human reference genome is the most widely used resource in human genetics and is due for a major update. Its current structure is a linear composite of merged haplotypes from more than 20 people, with a single individual comprising most of the sequence. It contains biases and errors within a framework that does not represent global human genomic variation. A high-quality reference with global representation of common variants, including single-nucleotide variants, structural variants and functional elements, is needed. The Human Pangenome Reference Consortium aims to create a more sophisticated and complete human reference genome with a graph-based, telomere-to-telomere representation of global genomic diversity. Here we leverage innovations in technology, study design and global partnerships with the goal of constructing the highest-possible quality human pangenome reference. Our goal is to improve data representation and streamline analyses to enable routine assembly of complete diploid genomes. With attention to ethical frameworks, the human pangenome reference will contain a more accurate and diverse representation of global genomic variation, improve gene-disease association studies across populations, expand the scope of genomics research to the most repetitive and polymorphic regions of the genome, and serve as the ultimate genetic resource for future biomedical research and precision medicine.

Genetic, epigenetic, and environmental mechanisms govern allele-specific gene expression.

Allele-specific expression (ASE) is a phenomenon in which one allele is preferentially expressed over the other. Genetic and epigenetic factors cause ASE by altering the final composition of a gene's product, leading to expression imbalances that can have functional consequences on phenotypes. Environmental signals also impact allele-specific expression, but how they contribute to this cross talk remains understudied. Here, we explored how genotype, parent-of-origin, tissue, sex, and dietary fat simultaneously influence ASE biases. Male and female mice from a F1 reciprocal cross of the LG/J and SM/J strains were fed a high or low fat diet. We harnessed strain-specific variants to distinguish between two ASE classes: parent-of-origin-dependent (unequal expression based on parental origin) and sequence-dependent (unequal expression based on nucleotide identity). We present a comprehensive map of ASE patterns in 2853 genes across three tissues and nine environmental contexts. We found that both ASE classes are highly dependent on tissue and environmental context. They vary across metabolically relevant tissues, between males and females, and in response to dietary fat. We also found 45 genes with inconsistent ASE biases that switched direction across tissues and/or environments. Finally, we integrated ASE and QTL data from published intercrosses of the LG/J and SM/J strains. Our ASE genes are often enriched in QTLs for metabolic and musculoskeletal traits, highlighting how this orthogonal approach can prioritize candidate genes. Together, our results provide novel insights into how genetic, epigenetic, and environmental mechanisms govern allele-specific expression, which is an essential step toward deciphering the genotype-to-phenotype map.

Epigenomic analysis reveals prevalent contribution of transposable elements to cis-regulatory elements, tissue-specific expression, and alternative promoters in zebrafish.

Transposable elements (TEs) encode regulatory elements that impact gene expression in multiple species, yet a comprehensive analysis of zebrafish TEs in the context of gene regulation is lacking. Here, we systematically investigate the epigenomic and transcriptomic landscape of TEs across 11 adult zebrafish tissues using multidimensional sequencing data. We find that TEs contribute substantially to a diverse array of regulatory elements in the zebrafish genome and that 37% of TEs are positioned in active regulatory states in adult zebrafish tissues. We identify TE subfamilies enriched in highly specific regulatory elements among different tissues. We use transcript assembly to discover TE-derived transcriptional units expressed across tissues. Finally, we show that novel TE-derived promoters can initiate tissue-specific transcription of alternate gene isoforms. This work provides a comprehensive profile of TE activity across normal zebrafish tissues, shedding light on mechanisms underlying the regulation of gene expression in this widely used model organism.

Integrated transcriptomics contrasts fatty acid metabolism with hypoxia response in ß-cell subpopulations associated with glycemic control.

BACKGROUND: Understanding how heterogeneous ß-cell function impacts diabetes is imperative for therapy development. Standard single-cell RNA sequencing analysis illuminates some factors driving heterogeneity, but new strategies are required to enhance information capture. RESULTS: We integrate pancreatic islet single-cell and bulk RNA sequencing data to identify ß-cell subpopulations based on gene expression and characterize genetic networks associated with ß-cell function in obese SM/J mice. We identify ß-cell subpopulations associated with basal insulin secretion, hypoxia response, cell polarity, and stress response. Network analysis associates fatty acid metabolism and basal insulin secretion with hyperglycemic-obesity, while expression of Pdyn and hypoxia response is associated with normoglycemic-obesity. CONCLUSIONS: By integrating single-cell and bulk islet transcriptomes, our study explores ß-cell heterogeneity and identifies novel subpopulations and genetic pathways associated with ß-cell function in obesity.

Pancreatic ß-cell heterogeneity in health and diabetes: classes, sources, and subtypes.

Pancreatic ß-cells perform glucose-stimulated insulin secretion, a process at the center of type 2 diabetes etiology. Efforts to understand how ß-cells behave in healthy and stressful conditions have revealed a wide degree of morphological, functional, and transcriptional heterogeneity. Sources of heterogeneity include ß-cell topography, developmental origin, maturation state, and stress response. Advances in sequencing and imaging technologies have led to the identification of ß-cell subtypes, which play distinct roles in the islet niche. This review examines ß-cell heterogeneity from morphological, functional, and transcriptional perspectives, and considers the relevance of topography, maturation, development, and stress response. It also discusses how these factors have been used to identify ß-cell subtypes, and how heterogeneity is impacted by diabetes. We examine open questions in the field and discuss recent technological innovations that could advance understanding of ß-cell heterogeneity in health and disease.

Genetic background and diet affect brown adipose gene coexpression networks associated with metabolic phenotypes.

Adipose is a dynamic endocrine organ that is critical for regulating metabolism and is highly responsive to nutritional environment. Brown adipose tissue is an exciting potential therapeutic target; however, there are no systematic studies of gene-by-environment interactions affecting function of this organ. We leveraged a weighted gene coexpression network analysis to identify transcriptional networks in brown adipose tissue from LG/J and SM/J inbred mice fed high- or low-fat diets and correlate these networks with metabolic phenotypes. We identified eight primary gene network modules associated with variation in obesity and diabetes-related traits. Four modules were enriched for metabolically relevant processes such as immune and cytokine response, cell division, peroxisome functions, and organic molecule metabolic processes. The relative expression of genes in these modules is highly dependent on both genetic background and dietary environment. Genes in the immune/cytokine response and cell division modules are particularly highly expressed in high fat-fed SM/J mice, which show unique brown adipose-dependent remission of diabetes. The interconnectivity of genes in these modules is also heavily dependent on diet and strain, with most genes showing both higher expression and coexpression under the same context. We highlight several genes of interest, Col28a1, Cyp26b1, Bmp8b, and Ngef, that have distinct expression patterns among strain-by-diet contexts and fall under metabolic quantitative trait loci previously mapped in an F16 generation of an advanced intercross between LG/J and SM/J. Each of these genes have some connection to obesity and diabetes-related traits, but have not been studied in brown adipose tissue. Our results provide important insights into the relationship between brown adipose and systemic metabolism by being the first gene-by-environment study of brown adipose transcriptional networks.

Genomic imprinting and parent-of-origin effects on complex traits.

Parent-of-origin effects occur when the phenotypic effect of an allele depends on whether it is inherited from the mother or the father. Several phenomena can cause parent-of-origin effects, but the best characterized is parent-of-origin-dependent gene expression associated with genomic imprinting. The development of new mapping approaches applied to the growing abundance of genomic data has demonstrated that imprinted genes can be important contributors to complex trait variation. Therefore, to understand the genetic architecture and evolution of complex traits, including complex diseases and traits of agricultural importance, it is crucial to account for these parent-of-origin effects. Here, we discuss patterns of phenotypic variation associated with imprinting, evidence supporting its role in complex trait variation and approaches for identifying its molecular signatures.

Epigenetics of metabolic syndrome.

The dramatic increase in global prevalence of metabolic disease is inexplicable when considering only environmental or only genetic factors, leading to the need to explore the possible roles of epigenetic factors. A great deal of progress has been made in this interdisciplinary field in recent years, with many studies investigating various aspects of the metabolic syndrome and its associated epigenetic changes. Rodent models of metabolic diseases have been particularly illuminating because of the ability to leverage tools such as genetic and environmental modifications. The current review summarizes recent breakthroughs regarding epigenetic markers in studies of obesity, Type II diabetes, and cardiovascular disease, the three major disorders associated with metabolic syndrome. We also discuss open questions and future directions for integrating genomic, epigenomic, and phenotypic big biodata toward understanding metabolic syndrome etiology.

Using whole-genome sequences of the LG/J and SM/J inbred mouse strains to prioritize quantitative trait genes and nucleotides.

BACKGROUND: The laboratory mouse is the most commonly used model for studying variation in complex traits relevant to human disease. Here we present the whole-genome sequences of two inbred strains, LG/J and SM/J, which are frequently used to study variation in complex traits as diverse as aging, bone-growth, adiposity, maternal behavior, and methamphetamine sensitivity. RESULTS: We identified small nucleotide variants (SNVs) and structural variants (SVs) in the LG/J and SM/J strains relative to the reference genome and discovered novel variants in these two strains by comparing their sequences to other mouse genomes. We find that 39% of the LG/J and SM/J genomes are identical-by-descent (IBD). We characterized amino-acid changing mutations using three algorithms: LRT, PolyPhen-2 and SIFT. We also identified polymorphisms between LG/J and SM/J that fall in regulatory regions and highly informative transcription factor binding sites (TFBS). We intersected these functional predictions with quantitative trait loci (QTL) mapped in advanced intercrosses of these two strains. We find that QTL are both over-represented in non-IBD regions and highly enriched for variants predicted to have a functional impact. Variants in QTL associated with metabolic (231 QTL identified in an F16 generation) and developmental (41 QTL identified in an F34 generation) traits were interrogated and we highlight candidate quantitative trait genes (QTG) and nucleotides (QTN) in a QTL on chr13 associated with variation in basal glucose levels and in a QTL on chr6 associated with variation in tibia length. CONCLUSIONS: We show how integrating genomic sequence with QTL reduces the QTL search space and helps researchers prioritize candidate genes and nucleotides for experimental follow-up. Additionally, given the LG/J and SM/J phylogenetic context among inbred strains, these data contribute important information to the genomic landscape of the laboratory mouse.

Genetic effects at pleiotropic loci are context-dependent with consequences for the maintenance of genetic variation in populations.

Context-dependent genetic effects, including genotype-by-environment and genotype-by-sex interactions, are a potential mechanism by which genetic variation of complex traits is maintained in populations. Pleiotropic genetic effects are also thought to play an important role in evolution, reflecting functional and developmental relationships among traits. We examine context-dependent genetic effects at pleiotropic loci associated with normal variation in multiple metabolic syndrome (MetS) components (obesity, dyslipidemia, and diabetes-related traits). MetS prevalence is increasing in Western societies and, while environmental in origin, presents substantial variation in individual response. We identify 23 pleiotropic MetS quantitative trait loci (QTL) in an F(16) advanced intercross between the LG/J and SM/J inbred mouse strains (Wustl:LG,SM-G16; n = 1002). Half of each family was fed a high-fat diet and half fed a low-fat diet; and additive, dominance, and parent-of-origin imprinting genotypic effects were examined in animals partitioned into sex, diet, and sex-by-diet cohorts. We examine the context-dependency of the underlying additive, dominance, and imprinting genetic effects of the traits associated with these pleiotropic QTL. Further, we examine sequence polymorphisms (SNPs) between LG/J and SM/J as well as differential expression of positional candidate genes in these regions. We show that genetic associations are different in different sex, diet, and sex-by-diet settings. We also show that over- or underdominance and ecological cross-over interactions for single phenotypes may not be common, however multidimensional synthetic phenotypes at loci with pleiotropic effects can produce situations that favor the maintenance of genetic variation in populations. Our findings have important implications for evolution and the notion of personalized medicine.

Parent-of-origin effects propagate through networks to shape metabolic traits.

Parent-of-origin effects are unexpectedly common in complex traits, including metabolic and neurological traits. Parent-of-origin effects can be modified by the environment, but the architecture of these gene-by-environmental effects on phenotypes remains to be unraveled. Previously, quantitative trait loci (QTL) showing context-specific parent-of-origin effects on metabolic traits were mapped in the F16 generation of an advanced intercross between LG/J and SM/J inbred mice. However, these QTL were not enriched for known imprinted genes, suggesting another mechanism is needed to explain these parent-of-origin effects phenomena. We propose that non-imprinted genes can generate complex parent-of-origin effects on metabolic traits through interactions with imprinted genes. Here, we employ data from mouse populations at different levels of intercrossing (F0, F1, F2, F16) of the LG/J and SM/J inbred mouse lines to test this hypothesis. Using multiple populations and incorporating genetic, genomic, and physiological data, we leverage orthogonal evidence to identify networks of genes through which parent-of-origin effects propagate. We identify a network comprised of three imprinted and six non-imprinted genes that show parent-of-origin effects. This epistatic network forms a nutritional responsive pathway and the genes comprising it jointly serve cellular functions associated with growth. We focus on two genes, Nnat and F2r, whose interaction associates with serum glucose levels across generations in high-fat-fed females. Single-cell RNAseq reveals that Nnat expression increases and F2r expression decreases in pre-adipocytes along an adipogenic trajectory, a result that is consistent with our observations in bulk white adipose tissue.

Genetic factors and diet affect long-bone length in the F34 LG,SM advanced intercross.

Previous studies on the LG,SM advanced intercross line have identified approximately 40 quantitative trait loci (QTL) for long -bone (humerus, ulna, femur, and tibia) lengths. In this study, long-bone-length QTL were fine-mapped in the F(34) generation (n = 1424) of the LG,SM advanced intercross. Environmental effects were assessed by dividing the population by sex between high-fat and low-fat diets, producing eight sex/diet cohorts. We identified 145 individual bone-length QTL comprising 45 pleiotropic QTL; 69 replicated QTL from previous studies, 35 were new traits significant at previously identified loci, and 41 were novel QTL. Many QTL affected only a subset of the population based on sex and/or diet. Eight of ten known skeletal growth genes were upregulated in 3-week-old LG/J male proximal tibial growth plates relative to SM/J. The sequences of parental strains LG/J and SM/J indicated the presence of over half a million polymorphisms in the confidence intervals of these 45 QTL. We examined 526 polymorphisms and found that 97 represented radical changes to amino acid composition while 40 were predicted to be deleterious to protein function. Additional experimentation is required to understand how changes in gene regulation or protein function can alter the genetic architecture and interact with the environment to produce phenotypic variation.

The importance of context to the genetic architecture of diabetes-related traits is revealed in a genome-wide scan of a LG/J × SM/J murine model.

Variations in diabetic phenotypes are caused by complex interactions of genetic effects, environmental factors, and the interplay between the two. We tease apart these complex interactions by examining genome-wide genetic and epigenetic effects on diabetes-related traits among different sex, diet, and sex-by-diet cohorts in a Mus musculus model. We conducted a genome-wide scan for quantitative trait loci that affect serum glucose and insulin levels and response to glucose stress in an F(16) Advanced Intercross Line of the LG/J and SM/J intercross (Wustl:LG,SM-G16). Half of each sibship was fed a high-fat diet and half was fed a relatively low-fat diet. Context-dependent genetic (additive and dominance) and epigenetic (parent-of-origin imprinting) effects were characterized by partitioning animals into sex, diet, and sex-by-diet cohorts. We found that different cohorts often have unique genetic effects at the same loci, and that genetic signals can be masked or erroneously assigned to specific cohorts if they are not considered individually. Our data demonstrate that the effects of genes on complex trait variation are highly context-dependent and that the same genomic sequence can affect traits differently depending on an individual's sex and/or dietary environment. Our results have important implications for studies of complex traits in humans.

Genetic, epigenetic, and gene-by-diet interaction effects underlie variation in serum lipids in a LG/JxSM/J murine model.

Variation in serum cholesterol, free-fatty acids, and triglycerides is associated with cardiovascular disease (CVD) risk factors. There is great interest in characterizing the underlying genetic architecture of these risk factors, because they vary greatly within and among human populations and between the sexes. We present results of a genome-wide scan for quantitative trait loci (QTL) affecting serum cholesterol, free-fatty acids, and triglycerides in an F(16) advanced intercross line of LG/J and SM/J (Wustl:LG,SM-G16). Half of the population was fed a high-fat diet and half was fed a relatively low-fat diet. Context-dependent genetic (additive and dominance) and epigenetic (imprinting) effects were characterized by partitioning animals into sex, diet, and sex-by-diet cohorts. Here we examine genetic, environmental, and genetic-by-environmental interactions of QTL overlapping previously identified loci associated with CVD risk factors, and we add to the serum lipid QTL landscape by identifying new loci.

Brown Adipose Expansion and Remission of Glycemic Dysfunction in Obese SM/J Mice.

We leverage the SM/J mouse to understand glycemic control in obesity. High-fat-fed SM/J mice initially develop poor glucose homeostasis relative to controls. Strikingly, their glycemic dysfunction resolves by 30 weeks of age despite persistent obesity. The mice dramatically expand their brown adipose depots as they resolve glycemic dysfunction. This occurs naturally and spontaneously on a high-fat diet, with no temperature or genetic manipulation. Removal of the brown adipose depot impairs insulin sensitivity, indicating that the expanded tissue is functioning as an insulin-stimulated glucose sink. We describe morphological, physiological, and transcriptomic changes that occur during the brown adipose expansion and remission of glycemic dysfunction, and focus on Sfrp1 (secreted frizzled-related protein 1) as a compelling candidate that may underlie this phenomenon. Understanding how the expanded brown adipose contributes to glycemic control in SM/J mice will open the door for innovative therapies aimed at improving metabolic complications in obesity.

Spontaneous restoration of functional ß-cell mass in obese SM/J mice.

Maintenance of functional ß-cell mass is critical to preventing diabetes, but the physiological mechanisms that cause ß-cell populations to thrive or fail in the context of obesity are unknown. High fat-fed SM/J mice spontaneously transition from hyperglycemic-obese to normoglycemic-obese with age, providing a unique opportunity to study ß-cell adaptation. Here, we characterize insulin homeostasis, islet morphology, and ß-cell function during SM/J's diabetic remission. As they resolve hyperglycemia, obese SM/J mice dramatically increase circulating and pancreatic insulin levels while improving insulin sensitivity. Immunostaining of pancreatic sections reveals that obese SM/J mice selectively increase ß-cell mass but not α-cell mass. Obese SM/J mice do not show elevated ß-cell mitotic index, but rather elevated α-cell mitotic index. Functional assessment of isolated islets reveals that obese SM/J mice increase glucose-stimulated insulin secretion, decrease basal insulin secretion, and increase islet insulin content. These results establish that ß-cell mass expansion and improved ß-cell function underlie the resolution of hyperglycemia, indicating that obese SM/J mice are a valuable tool for exploring how functional ß-cell mass can be recovered in the context of obesity.

Dietary iron interacts with genetic background to influence glucose homeostasis.

BACKGROUND: Iron is a critical component of metabolic homeostasis, but consumption of dietary iron has increased dramatically in the last 30 years, corresponding with the rise of metabolic disease. While the link between iron metabolism and metabolic health is well established, the extent to which dietary iron contributes to metabolic disease risk is unexplored. Further, it is unknown how dietary iron interacts with genetic background to modify metabolic disease risk. METHODS: LG/J and SM/J inbred mouse strains were used to investigate the relationship between genetic background and metabolic function during an 8-week high iron diet. Glucose tolerance and adiposity were assessed, colorimetric assays determined levels of circulating metabolic markers, and hepatic iron content was measured. RNA sequencing was performed on white adipose tissue to identify genes differentially expressed across strain, diet, and strain X diet cohorts. Hepatic Hamp expression and circulating hepcidin was measured, and small nucleotide variants were identified in the Hamp genic region. RESULTS: LG/J mice experienced elevated fasting glucose and glucose intolerance during the high iron diet, corresponding with increased hepatic iron load, increased circulating ferritin, and signs of liver injury. Adipose function was also altered in high iron-fed LG/J mice, including decreased adiposity and leptin production and differential expression of genes involved in iron and glucose homeostasis. LG/J mice failed to upregulate hepatic Hamp expression during the high iron diet, resulting in low circulating hepcidin levels compared to SM/J mice. CONCLUSIONS: This study highlights the importance of accounting for genetic variation when assessing the effects of diet on metabolic health, and suggests dietary iron's impact on liver and adipose tissue is an underappreciated component of metabolic disease risk.

Ironing out the Details: Untangling Dietary Iron and Genetic Background in Diabetes.

The search for genetic risk factors in type-II diabetes has been hindered by a failure to consider dietary variables. Dietary nutrients impact metabolic disease risk and severity and are essential to maintaining metabolic health. Genetic variation between individuals confers differences in metabolism, which directly impacts response to diet. Most studies attempting to identify genetic risk factors in disease fail to incorporate dietary components, and thus are ill-equipped to capture the breadth of the genome's impact on metabolism. Understanding how genetic background interacts with nutrients holds the key to predicting and preventing metabolic diseases through the implementation of personalized nutrition. Dysregulation of iron homeostasis is associated with type-II diabetes, but the link between dietary iron and metabolic dysfunction is poorly defined. High iron burden in adipose tissue induces insulin resistance, but the mechanisms underlying adipose iron accumulation remain unknown. Hepcidin controls dietary iron absorption and distribution in metabolic tissues, but it is unknown whether genetic variation influencing hepcidin expression modifies susceptibility to dietary iron-induced insulin resistance. This review highlights discoveries concerning the axis of iron homeostasis and adipose function and suggests that genetic variation underlying dietary iron metabolism is an understudied component of metabolic disease.