Plant enhancers exhibit both cooperative and additive interactions among their functional elements.

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. Here, we use Plant STARR-seq to characterize 3 light-responsive plant enhancers-AB80, Cab-1, and rbcS-E9-derived from genes associated with photosynthesis. Saturation mutagenesis revealed mutations, many of which clustered in short regions, that strongly reduced enhancer activity in the light, in the dark, or in both conditions. When tested in the light, these mutation-sensitive regions did not function on their own; rather, cooperative interactions with other such regions were required for full activity. Epistatic interactions occurred between mutations in adjacent mutation-sensitive regions, and the spacing and order of mutation-sensitive regions in synthetic enhancers affected enhancer activity. In contrast, when tested in the dark, mutation-sensitive regions acted independently and additively in conferring enhancer activity. Taken together, this work demonstrates that plant enhancers show evidence for both cooperative and additive interactions among their functional elements. This knowledge can be harnessed to design strong, condition-specific synthetic enhancers.

Arabidopsis and Maize Terminator Strength is Determined by GC Content, Polyadenylation Motifs and Cleavage Probability.

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. Terminator activity is species-specific, differing in tobacco leaf and maize protoplast assays. While recapitulating known biology, our results reveal the relative contributions of polyadenylation motifs to terminator strength. We built a computational model to predict terminator strength and used it to conduct in silico evolution that generated optimized synthetic terminators. Additionally, we discover alternative polyadenylation sites across tens of thousands of terminators; however, the strongest terminators tend to have a dominant cleavage site. Our results establish features of plant terminator function and identify strong naturally occurring and synthetic terminators.

Impact on splicing in Saccharomyces cerevisiae of random 50-base sequences inserted into an intron.

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 Saccharomyces cerevisiae, 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 S. cerevisiae 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. More than 70% of approximately 140,000 N50 elements reduced splicing by at least 20%. N50 features, including higher GC content, presence of GU repeats, and stronger predicted secondary structure of its pre-mRNA, correlated with reduced splicing efficiency. A likely basis for the reduced splicing of such a large proportion of variants is the formation of RNA structures that pair N50 bases-such as the GU repeats-with other bases specifically within the reporter pre-mRNA analyzed. However, multiple models were unable to explain more than a small fraction of the variance in splicing efficiency across the library, suggesting that complex nonlinear interactions in RNA structures are not accurately captured by RNA structure prediction methods. Our results imply that the specific context of a pre-mRNA may determine the bases allowable in an intron to prevent secondary structures that reduce splicing. This large data set can serve as a resource for further exploration of splicing mechanisms.

Mapping functional regions of essential bacterial proteins with dominant-negative protein fragments.

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 Escherichia coli, applying it to a set of 10 essential proteins. This approach yielded single amino acid resolution maps of inhibitory activity, with peaks localized to functionally important interaction sites, including oligomerization interfaces and folding contacts. Leveraging these data, we performed a systematic analysis to uncover principles of fragment-based inhibition. We determined a robust negative correlation between susceptibility to inhibition and cellular protein concentration, demonstrating that inhibitory fragments likely act primarily by titrating native protein interactions. We also characterized a series of trade-offs related to fragment length, showing that shorter peptides allow higher-resolution mapping but suffer from lower inhibitory activity. We employed an unsupervised statistical analysis to show that the inhibitory activities of protein fragments are largely driven not by generic properties such as charge, hydrophobicity, and secondary structure, but by the more specific characteristics of their bespoke macromolecular interactions. Overall, this work demonstrates fundamental characteristics of inhibitory protein fragment function and provides a foundation for understanding and controlling protein interactions in vivo.

Comprehensive Mutational Analysis of the Lasso Peptide Klebsidin.

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 Klebsiella pneumoniae that inhibits certain bacterial RNA polymerases. We develop a high-throughput assay based on growth inhibition of Escherichia coli to analyze the mutational tolerance of klebsidin. We transform a library of klebsidin variants into E. coli and use next-generation DNA sequencing to count the frequency of each variant before and after its expression, thereby generating functional scores for 320 of 361 single amino acid changes. We identify multiple positions in the macrocyclic ring and the C-terminal tail region of klebsidin that are intolerant to mutation, as well as positions in the loop region that are highly tolerant to mutation. Characterization of selected peptide variants scored as active reveals that each adopts a threaded lasso conformation; active loop variants applied extracellularly as peptides slow the growth of E. coli and K. pneumoniae. We generate an E. coli strain with a mutation in RNA polymerase that confers resistance to klebsidin and similarly carry out a selection with the klebsidin library. We identify a single variant, klebsidin F9Y, that maintains activity against the resistant E. coli when expressed intracellularly. This finding supports the utility of this method and suggests that comprehensive mutational analysis of lasso peptides can identify unique and potentially improved variants.

Effects of sequence motifs in the yeast 3' untranslated region determined from massively parallel assays of random sequences.

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. We determine relative protein expression levels from the fitness of transformants in a growth selection. We find that the consensus 3' UTR efficiency element significantly boosts expression, independent of sequence context; on the other hand, the consensus positioning element has only a small effect on expression. Some sequence motifs that are binding sites for Puf proteins substantially increase expression in the library, despite these proteins generally being associated with post-transcriptional downregulation of native mRNAs. Our measurements also allow a systematic examination of the effects of point mutations within efficiency element motifs across diverse sequence backgrounds. These mutational scans reveal the relative in vivo importance of individual bases in the efficiency element, which likely reflects their roles in binding the Hrp1 protein involved in cleavage and polyadenylation. CONCLUSIONS: The regulatory effects of some 3' UTR sequence features, like the efficiency element, are consistent regardless of sequence context. In contrast, the consequences of other 3' UTR features appear to be strongly dependent on their evolved context within native genes.

Expanding the binding specificity for RNA recognition by a PUF domain.

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. We use sequencing to score the binding of each variant, identifying many variants with highly repeat-specific interactions. From these data, we generate an RNA binding code specific to each repeat and base. We use this code to design PUF domains against 16 RNAs, and find that some of these domains recognize RNAs with two, three or four changes from the wild type sequence.

Synthetic promoter designs enabled by a comprehensive analysis of plant core promoters.

Targeted engineering of plant gene expression holds great promise for ensuring food security and for producing biopharmaceuticals in plants. However, this engineering requires thorough knowledge of cis-regulatory elements to precisely control either endogenous or introduced genes. To generate this knowledge, we used a massively parallel reporter assay to measure the activity of nearly complete sets of promoters from Arabidopsis, maize and sorghum. We demonstrate that core promoter elements-notably the TATA box-as well as promoter GC content and promoter-proximal transcription factor binding sites influence promoter strength. By performing the experiments in two assay systems, leaves of the dicot tobacco and protoplasts of the monocot maize, we detect species-specific differences in the contributions of GC content and transcription factors to promoter strength. Using these observations, we built computational models to predict promoter strength in both assay systems, allowing us to design highly active promoters comparable in activity to the viral 35S minimal promoter. Our results establish a promising experimental approach to optimize native promoter elements and generate synthetic ones with desirable features.

The regulatory landscape of Arabidopsis thaliana roots at single-cell resolution.

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. We leverage this shared information on cell identity to integrate accessibility and transcriptome data to characterize developmental progression, endoreduplication and cell division. We further use the combined data to characterize cell type-specific motif enrichments of transcription factor families and link the expression of family members to changing accessibility at specific loci, resolving direct and indirect effects that shape expression. Our approach provides an analytical framework to infer the gene regulatory networks that execute plant development.

MaveRegistry: a collaboration platform for multiplexed assays of variant effect.

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.varianteffect.org. MaveRegistry source code: https://github.com/kvnkuang/maveregistry-front-end.

Balance between promiscuity and specificity in phage λ host range.

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. Phages harboring amino acid substitutions were subjected to selection for infectivity on wild type E. coli, revealing a highly restrictive fitness landscape, in which most substitutions completely abrogate function. A subset of positions that are tolerant of mutation in this assay, but diverse over evolutionary time, are associated with host range expansion. Imposing selection for phage infectivity on three λ-resistant hosts, each harboring a different missense mutation in the λ receptor, reveals hundreds of adaptive variants in λ. We distinguish λ variants that confer promiscuity, a general ability to overcome host resistance, from those that drive host-specific infectivity. Both processes may be important in driving adaptation to a novel host.

Identification of Plant Enhancers and Their Constituent Elements by STARR-seq in Tobacco Leaves.

Genetic engineering of cis-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 cis-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 (Nicotiana benthamiana) leaves. We demonstrate that the optimal placement in the reporter construct of enhancer sequences from a plant virus, pea (Pisum sativum) and wheat (Triticum aestivum), was just upstream of a minimal promoter and that none of these four known enhancers was active in the 3' untranslated region of the reporter gene. The optimized assay sensitively identified small DNA regions containing each of the four enhancers, including two whose activity was stimulated by light. Furthermore, we coupled the assay to saturation mutagenesis to pinpoint functional regions within an enhancer, which we recombined to create synthetic enhancers. Our results describe an approach to define enhancer properties that can be performed in potentially any plant species or tissue transformable by Agrobacterium and that can use regulatory DNA derived from any plant genome.

Distinct patterns of mutational sensitivity for λ resistance and maltodextrin transport in Escherichia coli LamB.

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 Escherichia coli LamB with respect to sensitivity to phage λ 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. However, individual mutations could be found that specifically disrupt λ-sensitivity without affecting maltodextrin transport. We identify and individually assay nine such mutations, whose spatial positions implicate loop L6 of LamB in λ binding. Although missense mutations that lead to λ-resistance are rare, they were approximately as likely to be maltodextrin-utilizing (Mal+) as not (Mal-), implying that E. coli can adapt to λ while conserving the receptor's native function. We propose that in order for E. coli and λ to stably cohabitate, selection for λ-resistance and maltose transport must be spatially or temporally separated.

Dimensionality reduction by UMAP to visualize physical and genetic interactions.

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. This approach is more sensitive than previous methods and should be broadly useful as additional transcriptome datasets become available for other organisms.

Binding and Regulation of Transcription by Yeast Ste12 Variants To Drive Mating and Invasion Phenotypes.

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. Mating-defective variants led to decreased expression of distinct subsets of genes necessary for mating. Hyper-invasive variants also decreased expression of subsets of genes involved in mating, but increased the expression of other subsets of genes associated with the cellular response to osmotic stress. While single-amino acid changes in the coding region of this transcription factor result in complex regulatory reconfiguration, the major phenotypic consequences for the cell appear to depend on changes in the expression of a small number of genes with related functions.

High-throughput identification of dominant negative polypeptides in yeast.

Dominant negative polypeptides can inhibit protein function by binding to a wild-type subunit or by titrating a ligand. Here we use high-throughput sequencing of libraries composed of fragments of yeast genes to identify polypeptides that act in a dominant negative manner, in that they are depleted during cell growth. The method can uncover numerous inhibitory polypeptides for a protein and thereby define small inhibitory regions, even pinpointing individual residues with critical functional roles.

Dynamics of Gene Expression in Single Root Cells of Arabidopsis thaliana.

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 (Arabidopsis thaliana) 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. We identify hundreds of genes with cell-type-specific expression, with pseudotime analysis of several cell lineages revealing both known and novel genes that are expressed along a developmental trajectory. We identify transcription factor motifs that are enriched in early and late cells, together with the corresponding candidate transcription factors that likely drive the observed expression patterns. We assess and interpret changes in total RNA expression along developmental trajectories and show that trajectory branch points mark developmental decisions. Finally, by applying heat stress to whole seedlings, we address the longstanding question of possible heterogeneity among cell types in the response to an abiotic stress. Although the response of canonical heat-shock genes dominates expression across cell types, subtle but significant differences in other genes can be detected among cell types. Taken together, our results demonstrate that single cell transcriptomics holds promise for studying plant development and plant physiology with unprecedented resolution.

A Multiplex Homology-Directed DNA Repair Assay Reveals the Impact of More Than 1,000 BRCA1 Missense Substitution Variants on Protein Function.

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. Here, we describe a multiplex HDR reporter assay for concurrently measuring the effects of hundreds of variants of BRCA1 for their role in DNA repair. Using this assay, we characterized the effects of 1,056 amino acid substitutions in the first 192 residues of BRCA1. Benchmarking these results against variants with known effects on DNA repair function or on cancer predisposition, we demonstrate accurate discrimination of loss-of-function versus benign missense variants. We anticipate that this assay can be used to functionally characterize BRCA1 missense variants at scale, even before the variants are observed in results from genetic testing.

Engineered Biosensors from Dimeric Ligand-Binding Domains.

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. The introduction of a destabilizing mutation to the dimer interface increased biosensor dynamic range by an order of magnitude. Computational redesign of the dimer interface and functional selections were used to create heterodimeric pairs with further improved dynamic range. A heterodimeric biosensor built from the digoxigenin and progesterone ligand-binding domains functioned as a synthetic "AND"-gate, with 20-fold stronger response to the two ligands in combination than to either one alone. We also identified mutations that increase the sensitivity or selectivity of the biosensors to chemically similar ligands. These dimerizing biosensors provide additional flexibility for the construction of logic gates and other applications.