Publications by authors named "Stanley Fields"

Article Synopsis
  • Human genetics has evolved significantly over the last 30 years, shifting focus from rare Mendelian diseases to the intricate genetic factors influencing common diseases.
  • Researchers highlight the critical role of genetic context—including variants, gene regulation, and environmental interactions—in understanding how these genetic variants impact health.
  • The article calls for unified methods to analyze the complex interplay of molecular and environmental factors, proposing that combining cellular, animal, and epidemiological data can enhance our interpretation of genetic variants and improve disease treatment strategies.
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Insulators are -regulatory elements that separate transcriptional units, whereas silencers are elements that repress transcription regardless of their position. In plants, these elements remain largely uncharacterized. Here, we use the massively parallel reporter assay Plant STARR-seq with short fragments of eight large insulators to identify more than 100 fragments that block enhancer activity.

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The 3' end of a gene, often called a terminator, modulates mRNA stability, localization, translation, and polyadenylation. Here, we adapted Plant STARR-seq, a massively parallel reporter assay, to measure the activity of over 50,000 terminators from the plants Arabidopsis thaliana and Zea mays. We characterize thousands of plant terminators, including many that outperform bacterial terminators commonly used in plants.

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Enhancers are cis-regulatory elements that shape gene expression in response to numerous developmental and environmental cues. In animals, several models have been proposed to explain how enhancers integrate the activity of multiple transcription factors. However, it remains largely unclear how plant enhancers integrate transcription factor activity.

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Intron splicing is a key regulatory step in gene expression in eukaryotes. Three sequence elements required for splicing-5' and 3' splice sites and a branchpoint-are especially well-characterized in , but our understanding of additional intron features that impact splicing in this organism is incomplete, due largely to its small number of introns. To overcome this limitation, we constructed a library in of random 50-nt (N50) elements individually inserted into the intron of a reporter gene and quantified canonical splicing and the use of cryptic splice sites by sequencing analysis.

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The 3' end of a gene, often called a terminator, modulates mRNA stability, localization, translation, and polyadenylation. Here, we adapted Plant STARR-seq, a massively parallel reporter assay, to measure the activity of over 50,000 terminators from the plants and . We characterize thousands of plant terminators, including many that outperform bacterial terminators commonly used in plants.

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Massively parallel measurements of dominant-negative inhibition by protein fragments have been used to map protein interaction sites and discover peptide inhibitors. However, the underlying principles governing fragment-based inhibition have thus far remained unclear. Here, we adapted a high-throughput inhibitory fragment assay for use in , applying it to a set of 10 essential proteins.

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Antibiotic resistance is a growing threat to public health, making the development of antibiotics of critical importance. One promising class of potential new antibiotics are ribosomally synthesized and post-translationally modified peptides (RiPPs), which include klebsidin, a lasso peptide from that inhibits certain bacterial RNA polymerases. We develop a high-throughput assay based on growth inhibition of to analyze the mutational tolerance of klebsidin.

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Background: The 3' untranslated region (UTR) plays critical roles in determining the level of gene expression through effects on activities such as mRNA stability and translation. Functional elements within this region have largely been identified through analyses of native genes, which contain multiple co-evolved sequence features.

Results: To explore the effects of 3' UTR sequence elements outside of native sequence contexts, we analyze hundreds of thousands of random 50-mers inserted into the 3' UTR of a reporter gene in the yeast Saccharomyces cerevisiae.

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The ability to design a protein to bind specifically to a target RNA enables numerous applications, with the modular architecture of the PUF domain lending itself to new RNA-binding specificities. For each repeat of the Pumilio-1 PUF domain, we generate a library that contains the 8,000 possible combinations of amino acid substitutions at residues critical for RNA contact. We carry out yeast three-hybrid selections with each library against the RNA recognition sequence for Pumilio-1, with any possible base present at the position recognized by the randomized repeat.

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The scarcity of accessible sites that are dynamic or cell type-specific in plants may be due in part to tissue heterogeneity in bulk studies. To assess the effects of tissue heterogeneity, we apply single-cell ATAC-seq to Arabidopsis thaliana roots and identify thousands of differentially accessible sites, sufficient to resolve all major cell types of the root. We find that the entirety of a cell's regulatory landscape and its transcriptome independently capture cell type identity.

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Article Synopsis
  • Targeted engineering of plant gene expression is important for food security and biopharmaceutical production, requiring a deep understanding of cis-regulatory elements to control gene activity.
  • Researchers used a massively parallel reporter assay to analyze promoter activity in Arabidopsis, maize, and sorghum, finding that core promoter elements and transcription factor binding sites significantly affect promoter strength.
  • By testing in two different plant systems, the study revealed species-specific variations and led to the creation of computational models for predicting promoter strength, ultimately enabling the design of effective native and synthetic promoters.
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Summary: Multiplexed assays of variant effect (MAVEs) are capable of experimentally testing all possible single nucleotide or amino acid variants in selected genomic regions, generating 'variant effect maps', which provide biochemical insight and functional evidence to enable more rapid and accurate clinical interpretation of human variation. Because the international community applying MAVE approaches is growing rapidly, we developed the online MaveRegistry platform to catalyze collaboration, reduce redundant efforts, allow stakeholders to nominate targets and enable tracking and sharing of progress on ongoing MAVE projects.

Availability And Implementation: MaveRegistry service: https://registry.

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As hosts acquire resistance to viruses, viruses must overcome that resistance to re-establish infectivity, or go extinct. Despite the significant hurdles associated with adapting to a resistant host, viruses are evolutionarily successful and maintain stable coevolutionary relationships with their hosts. To investigate the factors underlying how pathogens adapt to their hosts, we performed a deep mutational scan of the region of the λ tail fiber tip protein that mediates contact with the receptor on λ's host, Escherichia coli.

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Genetic engineering of -regulatory elements in crop plants is a promising strategy to ensure food security. However, such engineering is currently hindered by our limited knowledge of plant -regulatory elements. Here, we adapted self-transcribing active regulatory region sequencing (STARR-seq)-a technology for the high-throughput identification of enhancers-for its use in transiently transformed tobacco () leaves.

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Bacteria can evade cohabiting phages through mutations in phage receptors, but these mutations may come at a cost if they disrupt the receptor's native cellular function. To investigate the relationship between these two conflicting activities, we generated sequence-function maps of LamB with respect to sensitivity to phage and transport of maltodextrin. By comparing 413 missense mutations whose effect on both traits could be analysed, we find that these two phenotypes were correlated, implying that most mutations affect these phenotypes through a common mechanism such as loss of protein stability.

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Dimensionality reduction is often used to visualize complex expression profiling data. Here, we use the Uniform Manifold Approximation and Projection (UMAP) method on published transcript profiles of 1484 single gene deletions of Saccharomyces cerevisiae. Proximity in low-dimensional UMAP space identifies groups of genes that correspond to protein complexes and pathways, and finds novel protein interactions, even within well-characterized complexes.

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Amino acid substitutions are commonly found in human transcription factors, yet the functional consequences of much of this variation remain unknown, even in well-characterized DNA-binding domains. Here, we examine how six single-amino acid variants in the DNA-binding domain of Ste12-a yeast transcription factor regulating mating and invasion-alter Ste12 genome binding, motif recognition, and gene expression to yield markedly different phenotypes. Using a combination of the "calling-card" method, RNA sequencing, and HT-SELEX (high throughput systematic evolution of ligands by exponential enrichment), we find that variants with dissimilar binding and expression profiles can converge onto similar cellular behaviors.

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Article Synopsis
  • Dominant negative polypeptides can interfere with normal protein function by either attaching to a healthy subunit or absorbing a necessary ligand.
  • The study utilizes high-throughput sequencing of yeast gene fragments to find polypeptides that exhibit dominant negative behavior, indicated by their reduced levels during cell growth.
  • This approach helps identify multiple inhibitory polypeptides and highlights specific regions and individual residues crucial for protein function.
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Single cell RNA sequencing can yield high-resolution cell-type-specific expression signatures that reveal new cell types and the developmental trajectories of cell lineages. Here, we apply this approach to Arabidopsis () root cells to capture gene expression in 3,121 root cells. We analyze these data with Monocle 3, which orders single cell transcriptomes in an unsupervised manner and uses machine learning to reconstruct single cell developmental trajectories along pseudotime.

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Loss-of-function pathogenic variants in BRCA1 confer a predisposition to breast and ovarian cancer. Genetic testing for sequence changes in BRCA1 frequently reveals a missense variant for which the impact on cancer risk and on the molecular function of BRCA1 is unknown. Functional BRCA1 is required for the homology-directed repair (HDR) of double-strand DNA breaks, a critical activity for maintaining genome integrity and tumor suppression.

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Biosensors are important components of many synthetic biology and metabolic engineering applications. Here, we report a second generation of Saccharomyces cerevisiae digoxigenin and progesterone biosensors based on destabilized dimeric ligand-binding domains that undergo ligand-induced stabilization. The biosensors, comprising one ligand-binding domain monomer fused to a DNA-binding domain and another fused to a transcriptional activation domain, activate reporter gene expression in response to steroid binding and receptor dimerization.

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Few mechanisms are known that explain how transcription factors can adjust phenotypic outputs to accommodate differing environments. In , the decision to mate or invade relies on environmental cues that converge on a shared transcription factor, Ste12. Specificity toward invasion occurs via Ste12 binding cooperatively with the cofactor Tec1.

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Article Synopsis
  • Researchers developed a new method to create biosensors in E. coli that can detect environmental chemicals by stabilizing a transcriptional activator in response to specific ligands.
  • They constructed a biosensor by fusing the lac repressor protein (LacI) to a DNA-binding domain and a transcription-activating domain, achieving a significant 7-fold increase in bacterial growth rate in response to the ligand IPTG.
  • The strategy, which involves mutating the lacI gene and testing its efficacy, was also successfully applied to engineer another protein, MphR, demonstrating its potential for wider use in creating biosensors from natural proteins.
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