Adina Weinberger

Artificial sweeteners induce glucose intolerance by altering the gut microbiota

Non-caloric artificial sweeteners (NAS) are among the most widely used food additives worldwide, regularly consumed by lean and obese individuals alike. NAS consumption is considered safe and beneficial owing to their low caloric content, yet supporting scientific data remain sparse and controversial. Here we demonstrate that consumption of commonly used NAS formulations drives the development of glucose intolerance through induction of compositional and functional alterations to the intestinal microbiota. These NAS-mediated deleterious metabolic effects are abrogated by antibiotic treatment, and are fully transferrable to germ-free mice upon faecal transplantation of microbiota configurations from NAS-consuming mice, or of microbiota anaerobically incubated in the presence of NAS. We identify NAS-altered microbial metabolic pathways that are linked to host susceptibility to metabolic disease, and demonstrate similar NAS-induced dysbiosis and glucose intolerance in healthy human subjects. Collectively, our results link NAS consumption, dysbiosis and metabolic abnormalities, thereby calling for a reassessment of massive NAS usage.

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.

A genetic signature of interspecies variations in gene expression

Phenotypic diversity is generated through changes in gene structure or gene regulation. The availability of full genomic sequences allows for the analysis of gene sequence evolution. In contrast, little is known about the principles driving the evolution of gene expression. Here we describe the differential transcriptional response of four closely related yeast species to a variety of environmental stresses. Genes containing a TATA box in their promoters show an increased interspecies variability in expression, independent of their functional association. Examining additional data sets, we find that this enhanced expression divergence of TATA-containing genes is consistent across all eukaryotes studied to date, including nematodes, fruit flies, plants and mammals. TATA-dependent regulation may enhance the sensitivity of gene expression to genetic perturbations, thus facilitating expression divergence at particular genetic loci.

Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters

Despite extensive research, our understanding of the rules according to which cis-regulatory sequences are converted into gene expression is limited. We devised a method for obtaining parallel, highly accurate gene expression measurements from thousands of designed promoters and applied it to measure the effect of systematic changes in the location, number, orientation, affinity and organization of transcription-factor binding sites and nucleosome-disfavoring sequences. Our analyses reveal a clear relationship between expression and binding-site multiplicity, as well as dependencies of expression on the distance between transcription-factor binding sites and gene starts which are transcription-factor specific, including a striking ∼10-bp periodic relationship between gene expression and binding-site location. We show how this approach can measure transcription-factor sequence specificities and the sensitivity of transcription-factor sites to the surrounding sequence context, and compare the activity of 75 yeast transcription factors. Our method can be used to study both cis and trans effects of genotype on transcriptional, post-transcriptional and translational control.

Manipulating nucleosome disfavoring sequences allows fine-tune regulation of gene expression in yeast

Understanding how precise control of gene expression is specified within regulatory DNA sequences is a key challenge with far-reaching implications. Many studies have focused on the regulatory role of transcription factor–binding sites. Here, we explore the transcriptional effects of different elements, nucleosome-disfavoring sequences and, specifically, poly(dA:dT) tracts that are highly prevalent in eukaryotic promoters. By measuring promoter activity for a large-scale promoter library, designed with systematic manipulations to the properties and spatial arrangement of poly(dA:dT) tracts, we show that these tracts significantly and causally affect transcription. We show that manipulating these elements offers a general genetic mechanism, applicable to promoters regulated by different transcription factors, for tuning expression in a predictable manner, with resolution that can be even finer than that attained by altering transcription factor sites. Overall, our results advance the understanding of the regulatory code and suggest a potential mechanism by which promoters yielding prespecified expression patterns can be designed.

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

Estimating bacterial growth dynamics The pattern of sequencing read coverage of bacteria in metagenomic samples reflects the growth rate. This pattern is predictive of growth because bacterial genomes are circular, with a single origin of replication. So during growth, copies of the genome accumulate at the origin. Korem et al. use the ratio of copy number at the origin to the copy number at the terminus to detect the actively growing species in a microbiome (see the Perspective by Segre). They could spot the difference between virulent and avirulent strains, population diurnal oscillations, species that are growing in irritable bowel disease, and what happens when a hosts diet changes. Results were consistent in chemostats, in mice, and in human fecal samples. Science, this issue p. 1101; see also p. 1058 A new method provides a quantitative measure of the growth rate of multiple gut microbes in one go. [Also see Perspective by Segre] 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.

Deciphering the rules by which 5′-UTR sequences affect protein expression in yeast

Significance This study quantifies how protein levels are determined by the underlying 5′-UTR sequence of an mRNA. We accurately measured protein abundance in 2,041 5′-UTR sequence variants, differing only in positions −10 to −1. We show that a few nucleotide substitutions can significantly alter protein expression. We also developed a predictive model that explains two-thirds of the expression variation. We provide convincing evidence that key regulatory elements, including AUG sequence context, mRNA secondary structure, and out-of-frame upstream AUGs conjointly modulate protein levels. Our study can aid in synthetic biology applications, by suggesting sequence manipulations for fine-tuning protein expression in a predictable manner. The 5′-untranslated region (5′-UTR) of mRNAs contains elements that affect expression, yet the rules by which these regions exert their effect are poorly understood. Here, we studied the impact of 5′-UTR sequences on protein levels in yeast, by constructing a large-scale library of mutants that differ only in the 10 bp preceding the translational start site of a fluorescent reporter. Using a high-throughput sequencing strategy, we obtained highly accurate measurements of protein abundance for over 2,000 unique sequence variants. The resulting pool spanned an approximately sevenfold range of protein levels, demonstrating the powerful consequences of sequence manipulations of even 1-10 nucleotides immediately upstream of the start codon. We devised computational models that predicted over 70% of the measured expression variability in held-out sequence variants. Notably, a combined model of the most prominent features successfully explained protein abundance in an additional, independently constructed library, whose nucleotide composition differed greatly from the library used to parameterize the model. Our analysis reveals the dominant contribution of the start codon context at positions −3 to −1, mRNA secondary structure, and out-of-frame upstream AUGs (uAUGs) to phenotypic diversity, thereby advancing our understanding of how protein levels are modulated by 5′-UTR sequences, and paving the way toward predictably tuning protein expression through manipulations of 5′-UTRs.

Promoters maintain their relative activity levels under different growth conditions

Most genes change expression levels across conditions, but it is unclear which of these changes represents specific regulation and what determines their quantitative degree. Here, we accurately measured activities of ∼900 S. cerevisiae and ∼1800 E. coli promoters using fluorescent reporters. We show that in both organisms 60–90% of promoters change their expression between conditions by a constant global scaling factor that depends only on the conditions and not on the promoters identity. Quantifying such global effects allows precise characterization of specific regulation—promoters deviating from the global scale line. These are organized into few functionally related groups that also adhere to scale lines and preserve their relative activities across conditions. Thus, only several scaling factors suffice to accurately describe genome‐wide expression profiles across conditions. We present a parameter‐free passive resource allocation model that quantitatively accounts for the global scaling factors. It suggests that many changes in expression across conditions result from global effects and not specific regulation, and provides means for quantitative interpretation of expression profiles.

On the relation between promoter divergence and gene expression evolution

Recent studies have characterized significant differences in the cis‐regulatory sequences of related organisms, but the impact of these differences on gene expression remains largely unexplored. Here, we show that most previously identified differences in transcription factor (TF)‐binding sequences of yeasts and mammals have no detectable effect on gene expression, suggesting that compensatory mechanisms allow promoters to rapidly evolve while maintaining a stabilized expression pattern. To examine the impact of changes in cis‐regulatory elements in a more controlled setting, we compared the genes induced during mating of three yeast species. This response is governed by a single TF (STE12), and variations in its predicted binding sites can indeed account for about half of the observed expression differences. The remaining unexplained differences are correlated with the increased divergence of the sequences that flank the binding sites and an apparent modulation of chromatin structure. Our analysis emphasizes the flexibility of promoter structure, and highlights the interplay between specific binding sites and general chromatin structure in the control of gene expression.

Comparative genetics. Systematic discovery of cap-independent translation sequences in human and viral genomes.

Identifying the IRESs of humans and viruses Most proteins result from the translation of 5′ capped RNA transcripts. In viruses and a subset of human genes, RNA transcripts with internal ribosome entry sites (IRESs) are uncapped. Weingarten-Gabbay et al. systematically surveyed the presence of IRESs in human protein-coding transcripts, as well those of viruses (see the Perspective by Gebauer and Hentze). Large-scale mutagenesis profiling identified two classes of IRESs: those having a functional element localized to one small region of the IRES and those with important elements distributed across the entire region. An unbiased screen across human genes suggests that IRESs are more frequent than previously supposed in 3′ untranslated regions. Science, this issue p.10.1126/science.aad4939; see also p. 228 Ribosomal translation of both human and viral RNAs does not always require scanning from the 5′ end. [Also see Perspective by Gebauer and Hentze] INTRODUCTION The recruitment of the ribosome to a specific mRNA is a critical step in the production of proteins in cells. In addition to a general recognition of the “cap” structure at the beginning of eukaryotic mRNAs, ribosomes can also initiate translation from a regulatory RNA element termed internal ribosome entry site (IRES) in a cap-independent manner. IRESs are essential for the synthesis of many human and viral proteins and take part in a variety of biological functions, such as viral infections, the response of cells to stress, and organismal development. Despite their importance, we lack systematic methods for discovering and characterizing IRESs, and thus, little is known about their position in the human and viral genomes and the mechanisms by which they recruit the ribosome. RATIONALE Our method enables accurate measurement of thousands of fully designed sequences for cap-independent translation activity. By using a synthetic oligonucleotide library, we can determine the exact composition of the sequences tested and can profile sequences from hundreds of different viruses, as well as the human genome, in a single experiment. In addition, synthetic design enables the construction of oligos in which we carefully and systematically mutate native IRESs and measure the effect of these mutations on expression. This reverse-genetics approach enables the characterization of the regulatory elements that recruit the ribosome and provide specificity in translation. RESULTS We uncover thousands of human and viral sequences with cap-independent translation activity, which provide a 50-fold increase in the number of sequences known to date. Unbiased screening of cap-independent activity across human transcripts demonstrates enrichment of regulatory elements in the untranslated region in the beginning of transcripts (5′UTR). However, we also find enrichment in the untranslated region located downstream of the coding sequence (3′UTR), which suggests a mechanism by which ribosomes are recruited to the 3′UTR to enhance the translation of an upstream sequence. A genome-wide profiling of positive-strand RNA viruses ([+]ssRNA) reveals the existence of translational elements along their coding regions. This finding suggests that [+]ssRNA viruses can translate only part of their genome, in addition to the synthesis and cleavage of a premature polyprotein. Our analysis reveals two classes of functional elements that drive cap-independent translation: (i) highly structured elements and (ii) unstructured elements that act through a short sequence motif. We show that many 5′UTRs can attract the ribosome by Watson-Crick base pairing with the 18S ribosomal RNA, a structural RNA component of the small ribosomal subunit (40S). In addition, we systematically investigate the functional regions of the 18S rRNA involved in these interactions that enhance cap-independent translation. CONCLUSIONS These results reveal the wide existence of cap-independent translation sequences in both humans and viruses. They provide insights on the landscape of translational regulation and uncover the regulatory elements underlying cap-independent translation activity. High-throughput bicistronic assay provides insights on translational regulation in human and viruses. (A) A library of thousands designed oligonucleotides as synthesized and cloned into a bicistronic reporter. Measurements of eGFP production, representing cap-independent translation activity, were performed with fluorescence-activated cell sorting and deep sequencing (FACS-seq). (B) The landscape of cap-independent translation sequences in human and viruses and the identified cis-regulatory elements driving their activity. To investigate gene specificity at the level of translation in both the human genome and viruses, we devised a high-throughput bicistronic assay to quantify cap-independent translation. We uncovered thousands of novel cap-independent translation sequences, and we provide insights on the landscape of translational regulation in both humans and viruses. We find extensive translational elements in the 3′ untranslated region of human transcripts and the polyprotein region of uncapped RNA viruses. Through the characterization of regulatory elements underlying cap-independent translation activity, we identify potential mechanisms of secondary structure, short sequence motif, and base pairing with the 18S ribosomal RNA (rRNA). Furthermore, we systematically map the 18S rRNA regions for which reverse complementarity enhances translation. Thus, we make available insights into the mechanisms of translational control in humans and viruses.