Characterization of caffeine response regulatory variants in vascular endothelial cells.

Genetic variants in gene regulatory sequences can modify gene expression and mediate the molecular response to environmental stimuli. In addition, genotype-environment interactions (GxE) contribute to complex traits such as cardiovascular disease. Caffeine is the most widely consumed stimulant and is known to produce a vascular response. To investigate GxE for caffeine, we treated vascular endothelial cells with caffeine and used a massively parallel reporter assay to measure allelic effects on gene regulation for over 43,000 genetic variants. We identified 665 variants with allelic effects on gene regulation and 6 variants that regulate the gene expression response to caffeine (GxE, false discovery rate [FDR] < 5%). When overlapping our GxE results with expression quantitative trait loci colocalized with coronary artery disease and hypertension, we dissected their regulatory mechanisms and showed a modulatory role for caffeine. Our results demonstrate that massively parallel reporter assay is a powerful approach to identify and molecularly characterize GxE in the specific context of caffeine consumption.

Single nucleus transcriptomics of ventral midbrain identifies glial activation associated with chronic opioid use disorder.

Dynamic interactions of neurons and glia in the ventral midbrain mediate reward and addiction behavior. We studied gene expression in 212,713 ventral midbrain single nuclei from 95 individuals with history of opioid misuse, and individuals without drug exposure. Chronic exposure to opioids was not associated with change in proportions of glial and neuronal subtypes, however glial transcriptomes were broadly altered, involving 9.5 - 6.2% of expressed genes within microglia, oligodendrocytes, and astrocytes. Genes associated with activation of the immune response including interferon, NFkB signaling, and cell motility pathways were upregulated, contrasting with down-regulated expression of synaptic signaling and plasticity genes in ventral midbrain non-dopaminergic neurons. Ventral midbrain transcriptomic reprogramming in the context of chronic opioid exposure included 325 genes that previous genome-wide studies had linked to risk of substance use traits in the broader population, thereby pointing to heritable risk architectures in the genomic organization of the brain's reward circuitry.

Genetic control of the dynamic transcriptional response to immune stimuli and glucocorticoids at single-cell resolution.

Synthetic glucocorticoids, such as dexamethasone, have been used as a treatment for many immune conditions, such as asthma and, more recently, severe COVID-19. Single-cell data can capture more fine-grained details on transcriptional variability and dynamics to gain a better understanding of the molecular underpinnings of inter-individual variation in drug response. Here, we used single-cell RNA-seq to study the dynamics of the transcriptional response to glucocorticoids in activated peripheral blood mononuclear cells from 96 African American children. We used novel statistical approaches to calculate a mean-independent measure of gene expression variability and a measure of transcriptional response pseudotime. Using these approaches, we showed that glucocorticoids reverse the effects of immune stimulation on both gene expression mean and variability. Our novel measure of gene expression response dynamics, based on the diagonal linear discriminant analysis, separated individual cells by response status on the basis of their transcriptional profiles and allowed us to identify different dynamic patterns of gene expression along the response pseudotime. We identified genetic variants regulating gene expression mean and variability, including treatment-specific effects, and showed widespread genetic regulation of the transcriptional dynamics of the gene expression response.

Single Nucleus Transcriptomics Reveals Pervasive Glial Activation in Opioid Overdose Cases.

Dynamic interactions of neurons and glia in the ventral midbrain (VM) mediate reward and addiction behavior. We studied gene expression in 212,713 VM single nuclei from 95 human opioid overdose cases and drug-free controls. Chronic exposure to opioids left numerical proportions of VM glial and neuronal subtypes unaltered, while broadly affecting glial transcriptomes, involving 9.5 - 6.2% of expressed genes within microglia, oligodendrocytes, and astrocytes, with prominent activation of the immune response including interferon, NFkB signaling, and cell motility pathways, sharply contrasting with down-regulated expression of synaptic signaling and plasticity genes in VM non-dopaminergic neurons. VM transcriptomic reprogramming in the context of opioid exposure and overdose included 325 genes with genetic variation linked to substance use traits in the broader population, thereby pointing to heritable risk architectures in the genomic organization of the brain's reward circuitry.

Analysis of transcriptional changes in the immune system associated with pubertal development in a longitudinal cohort of children with asthma.

Puberty is an important developmental period marked by hormonal, metabolic and immune changes. Puberty also marks a shift in sex differences in susceptibility to asthma. Yet, little is known about the gene expression changes in immune cells that occur during pubertal development. Here we assess pubertal development and leukocyte gene expression in a longitudinal cohort of 251 children with asthma. We identify substantial gene expression changes associated with age and pubertal development. Gene expression changes between pre- and post-menarcheal females suggest a shift from predominantly innate to adaptive immunity. We show that genetic effects on gene expression change dynamically during pubertal development. Gene expression changes during puberty are correlated with gene expression changes associated with asthma and may explain sex differences in prevalence. Our results show that molecular data used to study the genetics of early onset diseases should consider pubertal development as an important factor that modifies the transcriptome.

Genetic control of the dynamic transcriptional response to immune stimuli and glucocorticoids at single cell resolution.

Synthetic glucocorticoids, such as dexamethasone, have been used as treatment for many immune conditions, such as asthma and more recently severe COVID-19. Single cell data can capture more fine-grained details on transcriptional variability and dynamics to gain a better understanding of the molecular underpinnings of inter-individual variation in drug response. Here, we used single cell RNA-seq to study the dynamics of the transcriptional response to glucocorticoids in activated Peripheral Blood Mononuclear Cells from 96 African American children. We employed novel statistical approaches to calculate a mean-independent measure of gene expression variability and a measure of transcriptional response pseudotime. Using these approaches, we demonstrated that glucocorticoids reverse the effects of immune stimulation on both gene expression mean and variability. Our novel measure of gene expression response dynamics, based on the diagonal linear discriminant analysis, separated individual cells by response status on the basis of their transcriptional profiles and allowed us to identify different dynamic patterns of gene expression along the response pseudotime. We identified genetic variants regulating gene expression mean and variability, including treatment-specific effects, and demonstrated widespread genetic regulation of the transcriptional dynamics of the gene expression response.

Interspecies variation in hominid gut microbiota controls host gene regulation.

The gut microbiome exhibits extreme compositional variation between hominid hosts. However, it is unclear how this variation impacts host physiology across species and whether this effect can be mediated through microbial regulation of host gene expression in interacting epithelial cells. Here, we characterize the transcriptional response of human colonic epithelial cells in vitro to live microbial communities extracted from humans, chimpanzees, gorillas, and orangutans. We find that most host genes exhibit a conserved response, whereby they respond similarly to the four hominid microbiomes. However, hundreds of host genes exhibit a divergent response, whereby they respond only to microbiomes from specific host species. Such genes are associated with intestinal diseases in humans, including inflammatory bowel disease and Crohn's disease. Last, we find that inflammation-associated microbial species regulate the expression of host genes previously associated with inflammatory bowel disease, suggesting health-related consequences for species-specific host-microbiome interactions across hominids.

Psychosocial experiences modulate asthma-associated genes through gene-environment interactions.

Social interactions and the overall psychosocial environment have a demonstrated impact on health, particularly for people living in disadvantaged urban areas. Here, we investigated the effect of psychosocial experiences on gene expression in peripheral blood immune cells of children with asthma in Metro Detroit. Using RNA-sequencing and a new machine learning approach, we identified transcriptional signatures of 19 variables including psychosocial factors, blood cell composition, and asthma symptoms. Importantly, we found 169 genes associated with asthma or allergic disease that are regulated by psychosocial factors and 344 significant gene-environment interactions for gene expression levels. These results demonstrate that immune gene expression mediates the link between negative psychosocial experiences and asthma risk.

Functional dynamic genetic effects on gene regulation are specific to particular cell types and environmental conditions.

Genetic effects on gene expression and splicing can be modulated by cellular and environmental factors; yet interactions between genotypes, cell type, and treatment have not been comprehensively studied together. We used an induced pluripotent stem cell system to study multiple cell types derived from the same individuals and exposed them to a large panel of treatments. Cellular responses involved different genes and pathways for gene expression and splicing and were highly variable across contexts. For thousands of genes, we identified variable allelic expression across contexts and characterized different types of gene-environment interactions, many of which are associated with complex traits. Promoter functional and evolutionary features distinguished genes with elevated allelic imbalance mean and variance. On average, half of the genes with dynamic regulatory interactions were missed by large eQTL mapping studies, indicating the importance of exploring multiple treatments to reveal previously unrecognized regulatory loci that may be important for disease.

The activity of the genes in a cell depends on the type of cell they are in, the interactions with other genes, the environment and genetics. Active genes produce a greater number of mRNA molecules, which act as messenger molecules to instruct the cell to produce proteins. The amount of mRNA molecules in cells can be measured to assess the levels of gene activity. Genes produce mRNAs through a process called transcription, and the collection of all the mRNA molecules in a cell is called the transcriptome. Cells obtained from human samples can be grown in the lab under different conditions, and this can be used to transform them into different types of cells. These cells can then be exposed to different treatments ­ such as specific chemicals ­ to understand how the environment affects them. Cells derived from different people may respond differently to the same treatment based on their unique genetics. Exposing different types of cells from many people to different treatments can help explain how genetics, the environment and cell type affect gene activity. Findley et al. grew three different types of cells from six different people in the lab. The cells were exposed to 28 different treatments, which reflect different environmental changes. Studying all these different factors together allowed Findley et al. to understand how genetics, cell type and environment affect the activity of over 53,000 genes. Around half of the effects due to an interaction between genetics and the environment and had not been seen in other larger studies of the transcriptome. Many of these newly observed changes are in genes that have connections to different diseases, including heart disease. The results of Findley et al. provide evidence indicating to which extent lifestyle and the environment can interact with an individual's genetic makeup to impact gene activity and long-term health. The more researchers can understand these factors, the more useful they can be in helping to predict, detect and treat illnesses. The findings also show how genes and the environment interact, which may be relevant to understanding disease development. There is more work to be done to understand a wider range of environmental factors across more cell types. It will also be important to establish how this work on cells grown in the lab translates to human health.

Does the human placenta express the canonical cell entry mediators for SARS-CoV-2?

The pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected more than 10 million people, including pregnant women. To date, no consistent evidence for the vertical transmission of SARS-CoV-2 exists. The novel coronavirus canonically utilizes the angiotensin-converting enzyme 2 (ACE2) receptor and the serine protease TMPRSS2 for cell entry. Herein, building upon our previous single-cell study (Pique-Regi et al., 2019), another study, and new single-cell/nuclei RNA-sequencing data, we investigated the expression of ACE2 and TMPRSS2 throughout pregnancy in the placenta as well as in third-trimester chorioamniotic membranes. We report that co-transcription of ACE2 and TMPRSS2 is negligible in the placenta, thus not a likely path of vertical transmission for SARS-CoV-2. By contrast, receptors for Zika virus and cytomegalovirus, which cause congenital infections, are highly expressed by placental cell types. These data show that the placenta minimally expresses the canonical cell-entry mediators for SARS-CoV-2.

Interpreting Coronary Artery Disease Risk Through Gene-Environment Interactions in Gene Regulation.

GWAS and eQTL studies identified thousands of genetic variants associated with complex traits and gene expression. Despite the important role of environmental exposures in complex traits, only a limited number of environmental factors were measured in these studies. Measuring molecular phenotypes in tightly controlled cellular environments provides a more tractable setting to study gene-environment interactions in the absence of other confounding variables. We performed RNA-seq and ATAC-seq in endothelial cells exposed to retinoic acid, dexamethasone, caffeine, and selenium to model genetic and environmental effects on gene regulation in the vascular endothelium-a common site of pathology in cardiovascular disease. We found that genes near regions of differentially accessible chromatin were more likely to be differentially expressed [OR = (3.41, 6.52), [Formula: see text]]. Furthermore, we confirmed that environment-specific changes in transcription factor binding are a key mechanism for cellular response to environmental stimuli. Single nucleotide polymorphisms (SNPs) in these transcription response factor footprints for dexamethasone, caffeine, and retinoic acid were enriched in GTEx eQTLs from artery tissues, indicating that these environmental conditions are latently present in GTEx samples. Additionally, SNPs in footprints for response factors in caffeine are enriched in colocalized eQTLs for coronary artery disease (CAD), suggesting a role for caffeine in CAD risk. By combining GWAS, eQTLs, and response genes, we annotated environmental components that can increase or decrease disease risk through changes in gene expression in 43 genes. Interestingly, each treatment may amplify or buffer genetic risk for CAD, depending on the particular SNP or gene considered.

Gut Microbiota Has a Widespread and Modifiable Effect on Host Gene Regulation.

Variation in gut microbiome is associated with wellness and disease in humans, and yet the molecular mechanisms by which this variation affects the host are not well understood. A likely mechanism is that of changing gene regulation in interfacing host epithelial cells. Here, we treated colonic epithelial cells with live microbiota from five healthy individuals and quantified induced changes in transcriptional regulation and chromatin accessibility in host cells. We identified over 5,000 host genes that change expression, including 588 distinct associations between specific taxa and host genes. The taxa with the strongest influence on gene expression alter the response of genes associated with complex traits. Using ATAC-seq, we showed that a subset of these changes in gene expression are associated with changes in host chromatin accessibility and transcription factor binding induced by exposure to gut microbiota. We then created a manipulated microbial community with titrated doses of Collinsella, demonstrating that manipulation of the composition of the microbiome under both natural and controlled conditions leads to distinct and predictable gene expression profiles in host cells. Taken together, our results suggest that specific microbes play an important role in regulating expression of individual host genes involved in human complex traits. The ability to fine-tune the expression of host genes by manipulating the microbiome suggests future therapeutic routes.IMPORTANCE The composition of the gut microbiome has been associated with various aspects of human health, but the mechanism of this interaction is still unclear. We utilized a cellular system to characterize the effect of the microbiome on human gene expression. We showed that some of these changes in expression may be mediated by changes in chromatin accessibility. Furthermore, we validate the role of a specific microbe and show that changes in its abundance can modify the host gene expression response. These results show an important role of gut microbiota in regulating host gene expression and suggest that manipulation of microbiome composition could be useful in future therapies.

Differentially expressed gene networks, biomarkers, long noncoding RNAs, and shared responses with cocaine identified in the midbrains of human opioid abusers.

Opioid abuse is now the most common cause of accidental death in the US. Although opioids and most other drugs of abuse acutely increase signaling mediated by midbrain dopamine (DA)-synthesizing neurons, little is known about long-lasting changes in DA cells that may contribute to continued opioid abuse, craving, and relapse. A better understanding of the molecular and cellular bases of opioid abuse could lead to advancements in therapeutics. This study comprises, to our knowledge, the first unbiased examination of genome-wide changes in midbrain gene expression associated with human opioid abuse. Our analyses identified differentially expressed genes and distinct gene networks associated with opioid abuse, specific genes with predictive capability for subject assignment to the opioid abuse cohort, and genes most similarly affected in chronic opioid and cocaine abusers. We also identified differentially expressed long noncoding RNAs capable of regulating known drug-responsive protein-coding genes. Opioid-regulated genes identified in this study warrant further investigation as potential biomarkers and/or therapeutic targets for human substance abuse.

A hypermorphic antioxidant response element is associated with increased MS4A6A expression and Alzheimer's disease.

Late onset Alzheimer's disease (AD) is a multifactorial disorder, with AD risk influenced by both environmental and genetic factors. Recent genome-wide association studies (GWAS) have identified genetic loci associated with increased risk of developing AD. The MS4A (membrane-spanning 4-domains subfamily A) gene cluster is one of the most significant loci associated with AD risk, and MS4A6A expression is correlated with AD pathology. We identified a single nucleotide polymorphism, rs667897, at the MS4A locus that creates an antioxidant response element and links MS4A6A expression to the stress responsive Cap-n-Collar (CNC) transcription factors NRF1 (encoded by NFE2L1) and NRF2 (encoded by NFE2L2). The risk allele of rs667897 generates a strong CNC binding sequence that is activated by proteostatic stress in an NRF1-dependent manner, and is associated with increased expression of the gene MS4A6A. Together, these findings suggest that the cytoprotective CNC regulatory network aberrantly activates MS4A6A expression and increases AD risk in a subset of the population.

Environmental perturbations lead to extensive directional shifts in RNA processing.

Environmental perturbations have large effects on both organismal and cellular traits, including gene expression, but the extent to which the environment affects RNA processing remains largely uncharacterized. Recent studies have identified a large number of genetic variants associated with variation in RNA processing that also have an important role in complex traits; yet we do not know in which contexts the different underlying isoforms are used. Here, we comprehensively characterized changes in RNA processing events across 89 environments in five human cell types and identified 15,300 event shifts (FDR = 15%) comprised of eight event types in over 4,000 genes. Many of these changes occur consistently in the same direction across conditions, indicative of global regulation by trans factors. Accordingly, we demonstrate that environmental modulation of splicing factor binding predicts shifts in intron retention, and that binding of transcription factors predicts shifts in alternative first exon (AFE) usage in response to specific treatments. We validated the mechanism hypothesized for AFE in two independent datasets. Using ATAC-seq, we found altered binding of 64 factors in response to selenium at sites of AFE shift, including ELF2 and other factors in the ETS family. We also performed AFE QTL mapping in 373 individuals and found an enrichment for SNPs predicted to disrupt binding of the ELF2 factor. Together, these results demonstrate that RNA processing is dramatically changed in response to environmental perturbations through specific mechanisms regulated by trans factors.

RNA-sequencing Identifies Novel Pathways in Sarcoidosis Monocytes.

Sarcoidosis is a complex systemic granulomatous disorder of unknown etiology. Genome-wide association studies have not been able to explain a causative role for nucleotide variation in its pathogenesis. The goal of the present study was to identify the gene expression profile and the cellular pathways altered in sarcoidosis monocytes via RNA-sequencing. Peripheral blood monocytes play a role in sarcoidosis inflammation. Therefore, we determined and compared the transcriptional signature of monocytes from peripheral blood from sarcoidosis patients and healthy controls via RNA-sequencing. We found 2,446 differentially expressed (DE) genes between sarcoidosis and healthy control monocytes. Analysis of these DE genes showed enrichment for ribosome, phagocytosis, lysosome, proteasome, oxidative phosphorylation and metabolic pathways. RNA-sequencing identified upregulation of genes involved in phagocytosis and lysosomal pathway in sarcoidosis monocytes, whereas genes involved in proteasome degradation and ribosomal pathways were downregulated. Further studies are needed to investigate the role of specific genes involved in the identified pathways and their possible interaction leading to sarcoidosis pathology.

High-throughput allele-specific expression across 250 environmental conditions.

Gene-by-environment (GxE) interactions determine common disease risk factors and biomedically relevant complex traits. However, quantifying how the environment modulates genetic effects on human quantitative phenotypes presents unique challenges. Environmental covariates are complex and difficult to measure and control at the organismal level, as found in GWAS and epidemiological studies. An alternative approach focuses on the cellular environment using in vitro treatments as a proxy for the organismal environment. These cellular environments simplify the organism-level environmental exposures to provide a tractable influence on subcellular phenotypes, such as gene expression. Expression quantitative trait loci (eQTL) mapping studies identified GxE interactions in response to drug treatment and pathogen exposure. However, eQTL mapping approaches are infeasible for large-scale analysis of multiple cellular environments. Recently, allele-specific expression (ASE) analysis emerged as a powerful tool to identify GxE interactions in gene expression patterns by exploiting naturally occurring environmental exposures. Here we characterized genetic effects on the transcriptional response to 50 treatments in five cell types. We discovered 1455 genes with ASE (FDR < 10%) and 215 genes with GxE interactions. We demonstrated a major role for GxE interactions in complex traits. Genes with a transcriptional response to environmental perturbations showed sevenfold higher odds of being found in GWAS. Additionally, 105 genes that indicated GxE interactions (49%) were identified by GWAS as associated with complex traits. Examples include GIPR-caffeine interaction and obesity and include LAMP3-selenium interaction and Parkinson disease. Our results demonstrate that comprehensive catalogs of GxE interactions are indispensable to thoroughly annotate genes and bridge epidemiological and genome-wide association studies.

Genetic and transcriptional analysis of human host response to healthy gut microbiota.

Many studies have demonstrated the importance of the gut microbiota in healthy and disease states. However, establishing the causality of host-microbiota interactions in humans is still challenging. Here, we describe a novel experimental system to define the transcriptional response induced by the microbiota in human cells and to shed light on the molecular mechanisms underlying host-gut microbiota interactions. In primary human colonic epithelial cells, we identified over 6,000 genes that change expression at various time points following co-culturing with the gut microbiota of a healthy individual. Among the differentially expressed genes we found a 1.8-fold enrichment of genes associated with diseases that have been previously linked to the microbiome, such as obesity and colorectal cancer. In addition, our experimental system allowed us to identify 87 host SNPs that show allele-specific expression in 69 genes. Furthermore, for 12 SNPs in 12 different genes, allele-specific expression is conditional on the exposure to the microbiota. Of these 12 genes, eight have been associated with diseases linked to the gut microbiota, specifically colorectal cancer, obesity and type 2 diabetes. Our study demonstrates a scalable approach to study host-gut microbiota interactions and can be used to identify putative mechanisms for the interplay between host genetics and microbiota in health and disease.

Emerging lung cancer therapeutic targets based on the pathogenesis of bone metastases.

Lung cancer is the second most common cancer and the leading cause of cancer related mortality in both men and women. Each year, more people die of lung cancer than of colon, breast, and prostate cancers combined. It is widely accepted that tumor metastasis is a formidable barrier to effective treatment of lung cancer. The bone is one of the frequent metastatic sites for lung cancer occurring in a large number of patients. Bone metastases can cause a wide range of symptoms that could impair quality of life of lung cancer patients and shorten their survival. We strongly believe that molecular targets (tumor-related and bone microenvironment based) that have been implicated in lung cancer bone metastases hold great promise in lung cancer therapeutics. Thus, this paper discusses some of the emerging molecular targets that have provided insights into the cascade of metastases in lung cancer with the focus on bone invasion. It is anticipated that the information gathered might be useful in future efforts of optimizing lung cancer treatment strategies.

Screening and identification of novel compounds with potential anti-proliferative effects on gallium-resistant lung cancer through an AXL kinase pathway.

The clinical application of gallium compounds as anticancer agents is hampered by development of resistance. As a potential strategy to overcome the limitation, eight series of compounds were identified through virtual screening of AXL kinase homology model. Anti-proliferative studies were carried using gallium-sensitive (S) and gallium-resistant (R) human lung adenocarcinoma (A549) cells. Compounds 5476423 and 7919469 were identified as leads. The IC50 values from treating R-cells showed compounds 5476423 and 7919469 had 80 fold and 13 fold increased potency, respectively, compared to gallium acetylacetonate (GaAcAc). The efficacy of GaAcAc against R-cells was increased 2 fold and 1.2 fold when combined with compounds 5476423 and 7919469, respectively. Compared with S-cells, R-cells showed elevated expression of AXL protein, which was significantly suppressed through treatments with the lead compounds. It is anticipated that the lead compounds could be applied in virtual screening programs to identify novel scaffolds for new therapeutic agents as well as combinatorial therapy agents in gallium resistant lung cancer.