Environment dominates over host genetics in shaping human gut microbiota.

Human gut microbiome composition is shaped by multiple factors but the relative contribution of host genetics remains elusive. Here we examine genotype and microbiome data from 1,046 healthy individuals with several distinct ancestral origins who share a relatively common environment, and demonstrate that the gut microbiome is not significantly associated with genetic ancestry, and that host genetics have a minor role in determining microbiome composition. We show that, by contrast, there are significant similarities in the compositions of the microbiomes of genetically unrelated individuals who share a household, and that over 20% of the inter-person microbiome variability is associated with factors related to diet, drugs and anthropometric measurements. We further demonstrate that microbiome data significantly improve the prediction accuracy for many human traits, such as glucose and obesity measures, compared to models that use only host genetic and environmental data. These results suggest that microbiome alterations aimed at improving clinical outcomes may be carried out across diverse genetic backgrounds.

Bread Affects Clinical Parameters and Induces Gut Microbiome-Associated Personal Glycemic Responses.

Bread is consumed daily by billions of people, yet evidence regarding its clinical effects is contradicting. Here, we performed a randomized crossover trial of two 1-week-long dietary interventions comprising consumption of either traditionally made sourdough-leavened whole-grain bread or industrially made white bread. We found no significant differential effects of bread type on multiple clinical parameters. The gut microbiota composition remained person specific throughout this trial and was generally resilient to the intervention. We demonstrate statistically significant interpersonal variability in the glycemic response to different bread types, suggesting that the lack of phenotypic difference between the bread types stems from a person-specific effect. We further show that the type of bread that induces the lower glycemic response in each person can be predicted based solely on microbiome data prior to the intervention. Together, we present marked personalization in both bread metabolism and the gut microbiome, suggesting that understanding dietary effects requires integration of person-specific factors.

Involvement of a gut-retina axis in protection against dietary glycemia-induced age-related macular degeneration.

Age-related macular degeneration (AMD) is the major cause of blindness in developed nations. AMD is characterized by retinal pigmented epithelial (RPE) cell dysfunction and loss of photoreceptor cells. Epidemiologic studies indicate important contributions of dietary patterns to the risk for AMD, but the mechanisms relating diet to disease remain unclear. Here we investigate the effect on AMD of isocaloric diets that differ only in the type of dietary carbohydrate in a wild-type aged-mouse model. The consumption of a high-glycemia (HG) diet resulted in many AMD features (AMDf), including RPE hypopigmentation and atrophy, lipofuscin accumulation, and photoreceptor degeneration, whereas consumption of the lower-glycemia (LG) diet did not. Critically, switching from the HG to the LG diet late in life arrested or reversed AMDf. LG diets limited the accumulation of advanced glycation end products, long-chain polyunsaturated lipids, and their peroxidation end-products and increased C3-carnitine in retina, plasma, or urine. Untargeted metabolomics revealed microbial cometabolites, particularly serotonin, as protective against AMDf. Gut microbiota were responsive to diet, and we identified microbiota in the Clostridiales order as being associated with AMDf and the HG diet, whereas protection from AMDf was associated with the Bacteroidales order and the LG diet. Network analysis revealed a nexus of metabolites and microbiota that appear to act within a gut-retina axis to protect against diet- and age-induced AMDf. The findings indicate a functional interaction between dietary carbohydrates, the metabolome, including microbial cometabolites, and AMDf. Our studies suggest a simple dietary intervention that may be useful in patients to arrest AMD.

Systematic Investigation of Transcription Factor Activity in the Context of Chromatin Using Massively Parallel Binding and Expression Assays.

Precise gene expression patterns are established by transcription factor (TFs) binding to regulatory sequences. While these events occur in the context of chromatin, our understanding of how TF-nucleosome interplay affects gene expression is highly limited. Here, we present an assay for high-resolution measurements of both DNA occupancy and gene expression on large-scale libraries of systematically designed regulatory sequences. Our assay reveals occupancy patterns at the single-cell level. It provides an accurate quantification of the fraction of the population bound by a nucleosome and captures distinct, even adjacent, TF binding events. By applying this assay to over 1,500 promoter variants in yeast, we reveal pronounced differences in the dependency of TF activity on chromatin and classify TFs by their differential capacity to alter chromatin and promote expression. We further demonstrate how different regulatory sequences give rise to nucleosome-mediated TF collaborations that quantitatively account for the resulting expression.

Personalized Nutrition by Prediction of Glycemic Responses.

Elevated postprandial blood glucose levels constitute a global epidemic and a major risk factor for prediabetes and type II diabetes, but existing dietary methods for controlling them have limited efficacy. Here, we continuously monitored week-long glucose levels in an 800-person cohort, measured responses to 46,898 meals, and found high variability in the response to identical meals, suggesting that universal dietary recommendations may have limited utility. We devised a machine-learning algorithm that integrates blood parameters, dietary habits, anthropometrics, physical activity, and gut microbiota measured in this cohort and showed that it accurately predicts personalized postprandial glycemic response to real-life meals. We validated these predictions in an independent 100-person cohort. Finally, a blinded randomized controlled dietary intervention based on this algorithm resulted in significantly lower postprandial responses and consistent alterations to gut microbiota configuration. Together, our results suggest that personalized diets may successfully modify elevated postprandial blood glucose and its metabolic consequences. VIDEO ABSTRACT.

Growth dynamics of gut microbiota in health and disease inferred from single metagenomic samples.

Metagenomic sequencing increased our understanding of the role of the microbiome in health and disease, yet it only provides a snapshot of a highly dynamic ecosystem. Here, we show that the pattern of metagenomic sequencing read coverage for different microbial genomes contains a single trough and a single peak, the latter coinciding with the bacterial origin of replication. Furthermore, the ratio of sequencing coverage between the peak and trough provides a quantitative measure of a species' growth rate. We demonstrate this in vitro and in vivo, under different growth conditions, and in complex bacterial communities. For several bacterial species, peak-to-trough coverage ratios, but not relative abundances, correlated with the manifestation of inflammatory bowel disease and type II diabetes.

Disparity between microRNA levels and promoter strength is associated with initiation rate and Pol II pausing.

MicroRNAs are transcribed by RNA polymerase II but the transcriptional features influencing their synthesis are poorly defined. Here we report that a TATA box in microRNA and protein-coding genes is associated with increased sensitivity to slow RNA polymerase II. Promoters driven by TATA box or NF-κB elicit high re-initiation rates, but paradoxically lower microRNA levels. MicroRNA synthesis becomes more productive by decreasing the initiation rate, but less productive when the re-initiation rate increases. This phenomenon is associated with a delay in miR-146a induction by NF-κB. Finally, we demonstrate that microRNAs are remarkably strong pause sites. Our findings suggest that lower efficiency of microRNA synthesis directed by TATA box or NF-κB is a consequence of frequent transcription initiations that lead to RNA polymerase II crowding at pause sites, thereby increasing the chance of collision and premature termination. These findings highlight the importance of the transcription initiation mechanism for microRNA synthesis, and have implications for TATA-box promoters in general.

Transcriptional mechanisms controlling miR-375 gene expression in the pancreas.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that play an important role in mediating a broad and expanding range of biological activities. miR-375 is expressed selectively in the pancreas. We have previously shown that selective expression of miR-375 in pancreatic beta cells is controlled by transcriptional mechanisms operating through a TATA box-containing promoter. Expression of miR-375 has been reported in non-beta cells within the endocrine pancreas, and indeed inactivation of miR-375 leads to perturbation in cell mass and number of both alpha and beta cells. Consistent with its expression throughout the endocrine pancreas, we now show that the promoter of the miR-375 gene shows selective activity in pancreatic endocrine alpha cells, comparable to that observed in beta cells. We previously identified a novel negative regulatory element located downstream of the miR-375 gene transcription start site. By generating luciferase reporter genes, we now show that the sequence is functional also when positioned upstream of a heterologous promoter, thus proving that the repressor effect is mediated at least in part at the level of transcription. Further characterization of the transcriptional control mechanism regulating expression of miR-375 and other pancreatic miRNAs will contribute to a better understanding of pancreas development and function.

Epithelial microRNAs regulate gut mucosal immunity via epithelium-T cell crosstalk.

Colonic homeostasis entails epithelium-lymphocyte cooperation, yet many participants in this process are unknown. We show here that epithelial microRNAs mediate the mucosa-immune system crosstalk necessary for mounting protective T helper type 2 (T(H)2) responses. Abolishing the induction of microRNA by gut-specific deletion of Dicer1 (Dicer1(Δgut)), which encodes an enzyme involved in microRNA biogenesis, deprived goblet cells of RELMß, a key T(H)2 antiparasitic cytokine; this predisposed the host to parasite infection. Infection of Dicer1(Δgut) mice with helminths favored a futile T(H)1 response with hallmarks of inflammatory bowel disease. Interleukin 13 (IL-13) induced the microRNA miR-375, which regulates the expression of TSLP, a T(H)2-facilitating epithelial cytokine; this indicated a T(H)2-amplification loop. We found that miR-375 was required for RELMß expression in vivo; miR-375-deficient mice had significantly less intestinal RELMß, which possibly explains the greater susceptibility of Dicer1(Δgut) mice to parasites. Our findings indicate that epithelial microRNAs are key regulators of gut homeostasis and mucosal immunity.

The promoter of the pri-miR-375 gene directs expression selectively to the endocrine pancreas.

microRNAs (miRNAs) are known to play an essential role in controlling a broad range of biological processes including animal development. Accordingly, many miRNAs are expressed preferentially in one or a small number of cell types. Yet the mechanisms responsible for this selectivity are not well understood. The aim of this study was to elucidate the molecular basis of cell-specific expression of the pri-miR-375 gene, which is selectively expressed in pancreatic islets, and has been implicated both in the development of islets, and the function of mature pancreatic beta cells. An evolutionarily conserved 768 bp region of DNA upstream of the pri-miR-375 gene was linked to GFP and luciferase reporter genes, and expression monitored in transgenic mice and transfected cultured cells. Deletion and targeted mutagenesis analysis was used to evaluate the functional significance of sequence blocks within the upstream fragment. 5'-RACE analysis was used for mapping the pri-miR-375 gene transcription start site. The conserved 768 bp region was able to direct preferential expression of a GFP reporter gene to pancreatic islets in transgenic mice. Deletion analysis using a luciferase reporter gene in transfected cultured cell lines confirmed the cell specificity of the putative promoter region, and identified several key cis-elements essential for optimal activity, including E-boxes and a TATA sequence. Consistent with this, 5'-RACE analysis identified a transcription start site within this DNA region, 24 bp downstream of the TATA sequence. These studies define the promoter of the pri-miR-375 gene, and show that islet-specific expression of the pri-miR-375 gene is controlled at the transcriptional level. Detailed analysis of the transcriptional mechanisms controlling expression of miRNA genes will be essential to permit a comprehensive understanding of the complex role of miRNAs such as miR-375 in developmental processes.

Regulation of the GPR40 locus: towards a molecular understanding.

GPR40 {FFAR1 [non-esterified ('free') fatty acid receptor 1]} is a G-protein-coupled receptor expressed preferentially in pancreatic beta-cells. GPR40 functions as a receptor for medium and long-chain fatty acids, and has been implicated in mediating both physiological and pathological effects of fatty acids on beta-cells. The GPR40 gene is encoded at an interesting chromosomal locus that contains several genes: at the 5'-end of the locus, located approximately 4 kb upstream of GPR40, is CD22, a gene encoding a receptor expressed selectively in lymphocytes and involved in B-lymphocyte maturation and function. At the 3'-end of the locus are the GPR41 (FFAR3) and GPR43 (FFAR2) genes encoding receptors activated by short-chain fatty acids. The intergenic region between CD22 and GPR40 contains several evolutionarily conserved sequence blocks, among them HR2 and HR3. beta-Cell-specific expression of GPR40 is controlled at the transcriptional level through HR2, a potent beta-cell-specific enhancer. The mechanisms controlling cell-specific expression of the remaining genes in the cluster are unknown. Given the divergent modes of expression of the genes within the locus and their demonstrated physiological significance, it is important to analyse further the locus with a view to fully understanding the basis for transcriptional regulation of the encoded genes.