Proteostasis governs differential temperature sensitivity across embryonic cell types.

Embryonic development is remarkably robust, but temperature stress can degrade its ability to generate animals with invariant anatomy. Phenotypes associated with environmental stress suggest that some cell types are more sensitive to stress than others, but the basis of this sensitivity is unknown. Here, we characterize hundreds of individual zebrafish embryos under temperature stress using whole-animal single-cell RNA sequencing (RNA-seq) to identify cell types and molecular programs driving phenotypic variability. We find that temperature perturbs the normal proportions and gene expression programs of numerous cell types and also introduces asynchrony in developmental timing. The notochord is particularly sensitive to temperature, which we map to a specialized cell type: sheath cells. These cells accumulate misfolded protein at elevated temperature, leading to a cascading structural failure of the notochord and anatomic defects. Our study demonstrates that whole-animal single-cell RNA-seq can identify mechanisms for developmental robustness and pinpoint cell types that constitute key failure points.

Bacteria-to-Human Protein Networks Reveal Origins of Endogenous DNA Damage.

DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.

Genome organization and botanical diversity.

The rich diversity of angiosperms, both the planet's dominant flora and the cornerstone of agriculture, is integrally intertwined with a distinctive evolutionary history. Here, we explore the interplay between angiosperm genome organization and botanical diversity, empowered by genomic approaches ranging from genetic linkage mapping to analysis of gene regulation. Commonality in the genetic hardware of plants has enabled robust comparative genomics that has provided a broad picture of angiosperm evolution and implicated both general processes and specific elements in contributing to botanical diversity. We argue that the hardware of plant genomes-both in content and in dynamics-has been shaped by selection for rather substantial differences in gene regulation between plants and animals such as maize and human, organisms of comparable genome size and gene number. Their distinctive genome content and dynamics may reflect in part the indeterminate development of plants that puts strikingly different demands on gene regulation than in animals. Repeated polyploidization of plant genomes and multiplication of individual genes together with extensive rearrangement and differential retention provide rich raw material for selection of morphological and/or physiological variations conferring fitness in specific niches, whether natural or artificial. These findings exemplify the burgeoning information available to employ in increasing knowledge of plant biology and in modifying selected plants to better meet human needs.

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.

An autoregulatory poison exon in Smndc1 is conserved across kingdoms and influences organism growth.

Many of the most highly conserved elements in the human genome are "poison exons," alternatively spliced exons that contain premature termination codons and permit post-transcriptional regulation of mRNA abundance through induction of nonsense-mediated mRNA decay (NMD). Poison exons are widely assumed to be highly conserved due to their presumed importance for organismal fitness, but this functional importance has never been tested in the context of a whole organism. Here, we report that a poison exon in Smndc1 is conserved across mammals and plants and plays a molecular autoregulatory function in both kingdoms. We generated mouse and A. thaliana models lacking this poison exon to find its loss leads to deregulation of SMNDC1 protein levels, pervasive alterations in mRNA processing, and organismal size restriction. Together, these models demonstrate the importance of poison exons for both molecular and organismal phenotypes that likely explain their extraordinary conservation.

First discovered, long out of sight, finally visible: ribosomal DNA.

With the advent of long-read sequencing, previously unresolvable genomic elements are being revisited in an effort to generate fully complete reference genomes. One such element is ribosomal DNA (rDNA), the highly conserved genomic region that encodes rRNAs. Genomic structure and content of the rDNA are variable in both prokarya and eukarya, posing interesting questions about the biology of rDNA. Here, we consider the types of variation observed in rDNA - including locus structure and number, copy number, and sequence variation - and their known phenotypic consequences. With recent advances in long-read sequencing technology, incorporating the full rDNA sequence into reference genomes is within reach. This knowledge will have important implications for understanding rDNA biology within the context of cell physiology and whole-organism phenotypes.

LTP2 hypomorphs show genotype-by-environment interaction in early seedling traits in Arabidopsis thaliana.

Isogenic individuals can display seemingly stochastic phenotypic differences, limiting the accuracy of genotype-to-phenotype predictions. The extent of this phenotypic variation depends in part on genetic background, raising questions about the genes involved in controlling stochastic phenotypic variation. Focusing on early seedling traits in Arabidopsis thaliana, we found that hypomorphs of the cuticle-related gene LIPID TRANSFER PROTEIN 2 (LTP2) greatly increased variation in seedling phenotypes, including hypocotyl length, gravitropism and cuticle permeability. Many ltp2 hypocotyls were significantly shorter than wild-type hypocotyls while others resembled the wild-type. Differences in epidermal properties and gene expression between ltp2 seedlings with long and short hypocotyls suggest a loss of cuticle integrity as the primary determinant of the observed phenotypic variation. We identified environmental conditions that reveal or mask the increased variation in ltp2 hypomorphs and found that increased expression of its closest paralog LTP1 is necessary for ltp2 phenotypes. Our results illustrate how decreased expression of a single gene can generate starkly increased phenotypic variation in isogenic individuals in response to an environmental challenge.

Anno genominis XX: 20 years of Arabidopsis genomics.

Twenty years ago, the Arabidopsis thaliana genome sequence was published. This was an important moment as it was the first sequenced plant genome and explicitly brought plant science into the genomics era. At the time, this was not only an outstanding technological achievement, but it was characterized by a superb global collaboration. The Arabidopsis genome was the seed for plant genomic research. Here, we review the development of numerous resources based on the genome that have enabled discoveries across plant species, which has enhanced our understanding of how plants function and interact with their environments.

A single-cell view of the transcriptome during lateral root initiation in Arabidopsis thaliana.

Root architecture is a major determinant of plant fitness and is under constant modification in response to favorable and unfavorable environmental stimuli. Beyond impacts on the primary root, the environment can alter the position, spacing, density, and length of secondary or lateral roots. Lateral root development is among the best-studied examples of plant organogenesis, yet there are still many unanswered questions about its earliest steps. Among the challenges faced in capturing these first molecular events is the fact that this process occurs in a small number of cells with unpredictable timing. Single-cell sequencing methods afford the opportunity to isolate the specific transcriptional changes occurring in cells undergoing this fate transition. Using this approach, we successfully captured the transcriptomes of initiating lateral root primordia in Arabidopsis thaliana and discovered many upregulated genes associated with this process. We developed a method to selectively repress target gene transcription in the xylem pole pericycle cells where lateral roots originate and demonstrated that the expression of several of these targets is required for normal root development. We also discovered subpopulations of cells in the pericycle and endodermal cell files that respond to lateral root initiation, highlighting the coordination across cell files required for this fate transition.

Optimization of ATAC-seq in wheat seedling roots using INTACT-isolated nuclei.

BACKGROUND: The genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however, cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification. RESULTS: In this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks and cis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels. CONCLUSIONS: We have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome.

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.

Substitutions Are Boring: Some Arguments about Parallel Mutations and High Mutation Rates.

Extant genomes are largely shaped by global transposition, copy-number fluctuation, and rearrangement of DNA sequences rather than by substitutions of single nucleotides. Although many of these large-scale mutations have low probabilities and are unlikely to repeat, others are recurrent or predictable in their effects, leading to stereotyped genome architectures and genetic variation in both eukaryotes and prokaryotes. Such recurrent, parallel mutation modes can profoundly shape the paths taken by evolution and undermine common models of evolutionary genetics. Similar patterns are also evident at the smaller scales of individual genes or short sequences. The scale and extent of this 'non-substitution' variation has recently come into focus through the advent of new genomic technologies; however, it is still not widely considered in genotype-phenotype association studies. In this review we identify common features of these disparate mutational phenomena and comment on the importance and interpretation of these mutational patterns.

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.

It's not magic - Hsp90 and its effects on genetic and epigenetic variation.

Canalization, or phenotypic robustness in the face of environmental and genetic perturbation, is an emergent property of living systems. Although this phenomenon has long been recognized, its molecular underpinnings have remained enigmatic until recently. Here, we review the contributions of the molecular chaperone Hsp90, a protein that facilitates the folding of many key regulators of growth and development, to canalization of phenotype - and de-canalization in times of stress - drawing on studies in eukaryotes as diverse as baker's yeast, mouse ear cress, and blind Mexican cavefish. Hsp90 is a hub of hubs that interacts with many so-called 'client proteins,' which affect virtually every aspect of cell signaling and physiology. As Hsp90 facilitates client folding and stability, it can epistatically suppress or enable the expression of genetic variants in its clients and other proteins that acquire client status through mutation. Hsp90's vast interaction network explains the breadth of its phenotypic reach, including Hsp90-dependent de novo mutations and epigenetic effects on gene regulation. Intrinsic links between environmental stress and Hsp90 function thus endow living systems with phenotypic plasticity in fluctuating environments. As environmental perturbations alter Hsp90 function, they also alter Hsp90's interaction with its client proteins, thereby re-wiring networks that determine the genotype-to-phenotype map. Ensuing de-canalization of phenotype creates phenotypic diversity that is not simply stochastic, but often has an underlying genetic basis. Thus, extreme phenotypes can be selected, and assimilated so that they no longer require environmental stress to manifest. In addition to acting on standing genetic variation, Hsp90 perturbation has also been linked to increased frequency of de novo variation and several epigenetic phenomena, all with the potential to generate heritable phenotypic change. Here, we aim to clarify and discuss the multiple means by which Hsp90 can affect phenotype and possibly evolutionary change, and identify their underlying common feature: at its core, Hsp90 interacts epistatically through its chaperone function with many other genes and their gene products. Its influence on phenotypic diversification is thus not magic but rather a fundamental property of genetics.

Massive variation of short tandem repeats with functional consequences across strains of Arabidopsis thaliana.

Short tandem repeat (STR) mutations may comprise more than half of the mutations in eukaryotic coding DNA, yet STR variation is rarely examined as a contributor to complex traits. We assessed this contribution across a collection of 96 strains of Arabidopsis thaliana, genotyping 2046 STR loci each, using highly parallel STR sequencing with molecular inversion probes. We found that 95% of examined STRs are polymorphic, with a median of six alleles per STR across these strains. STR expansions (large copy number increases) are found in most strains, several of which have evident functional effects. These include three of six intronic STR expansions we found to be associated with intron retention. Coding STRs were depleted of variation relative to noncoding STRs, and we detected a total of 56 coding STRs (11%) showing low variation consistent with the action of purifying selection. In contrast, some STRs show hypervariable patterns consistent with diversifying selection. Finally, we detected 133 novel STR-phenotype associations under stringent criteria, most of which could not be detected with SNPs alone, and validated some with follow-up experiments. Our results support the conclusion that STRs constitute a large, unascertained reservoir of functionally relevant genomic variation.

Profiling of Accessible Chromatin Regions across Multiple Plant Species and Cell Types Reveals Common Gene Regulatory Principles and New Control Modules.

The transcriptional regulatory structure of plant genomes remains poorly defined relative to animals. It is unclear how many cis-regulatory elements exist, where these elements lie relative to promoters, and how these features are conserved across plant species. We employed the assay for transposase-accessible chromatin (ATAC-seq) in four plant species (Arabidopsis thaliana, Medicago truncatula, Solanum lycopersicum, and Oryza sativa) to delineate open chromatin regions and transcription factor (TF) binding sites across each genome. Despite 10-fold variation in intergenic space among species, the majority of open chromatin regions lie within 3 kb upstream of a transcription start site in all species. We find a common set of four TFs that appear to regulate conserved gene sets in the root tips of all four species, suggesting that TF-gene networks are generally conserved. Comparative ATAC-seq profiling of Arabidopsis root hair and non-hair cell types revealed extensive similarity as well as many cell-type-specific differences. Analyzing TF binding sites in differentially accessible regions identified a MYB-driven regulatory module unique to the hair cell, which appears to control both cell fate regulators and abiotic stress responses. Our analyses revealed common regulatory principles among species and shed light on the mechanisms producing cell-type-specific transcriptomes during development.

Preferences in a trait decision determined by transcription factor variants.

Few mechanisms are known that explain how transcription factors can adjust phenotypic outputs to accommodate differing environments. In Saccharomyces cerevisiae, 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. Here, we determine the range of phenotypic outputs (mating vs. invasion) of thousands of DNA-binding domain variants in Ste12 to understand how preference for invasion may arise. We find that single amino acid changes in the DNA-binding domain can shift the preference of yeast toward either mating or invasion. These mutations define two distinct regions of this domain, suggesting alternative modes of DNA binding for each trait. We characterize the DNA-binding specificity of wild-type Ste12 to identify a strong preference for spacing and orientation of both homodimeric and heterodimeric sites. Ste12 mutants that promote hyperinvasion in a Tec1-independent manner fail to bind cooperative sites with Tec1 and bind to unusual dimeric Ste12 sites composed of one near-perfect and one highly degenerate site. We propose a model in which Ste12 alone may have evolved to activate invasion genes, which could explain how preference for invasion arose in the many fungal pathogens that lack Tec1.

Evolutionary assembly patterns of prokaryotic genomes.

Evolutionary innovation must occur in the context of some genomic background, which limits available evolutionary paths. For example, protein evolution by sequence substitution is constrained by epistasis between residues. In prokaryotes, evolutionary innovation frequently happens by macrogenomic events such as horizontal gene transfer (HGT). Previous work has suggested that HGT can be influenced by ancestral genomic content, yet the extent of such gene-level constraints has not yet been systematically characterized. Here, we evaluated the evolutionary impact of such constraints in prokaryotes, using probabilistic ancestral reconstructions from 634 extant prokaryotic genomes and a novel framework for detecting evolutionary constraints on HGT events. We identified 8228 directional dependencies between genes and demonstrated that many such dependencies reflect known functional relationships, including for example, evolutionary dependencies of the photosynthetic enzyme RuBisCO. Modeling all dependencies as a network, we adapted an approach from graph theory to establish chronological precedence in the acquisition of different genomic functions. Specifically, we demonstrated that specific functions tend to be gained sequentially, suggesting that evolution in prokaryotes is governed by functional assembly patterns. Finally, we showed that these dependencies are universal rather than clade-specific and are often sufficient for predicting whether or not a given ancestral genome will acquire specific genes. Combined, our results indicate that evolutionary innovation via HGT is profoundly constrained by epistasis and historical contingency, similar to the evolution of proteins and phenotypic characters, and suggest that the emergence of specific metabolic and pathological phenotypes in prokaryotes can be predictable from current genomes.

MIPSTR: a method for multiplex genotyping of germline and somatic STR variation across many individuals.

Short tandem repeats (STRs) are highly mutable genetic elements that often reside in regulatory and coding DNA. The cumulative evidence of genetic studies on individual STRs suggests that STR variation profoundly affects phenotype and contributes to trait heritability. Despite recent advances in sequencing technology, STR variation has remained largely inaccessible across many individuals compared to single nucleotide variation or copy number variation. STR genotyping with short-read sequence data is confounded by (1) the difficulty of uniquely mapping short, low-complexity reads; and (2) the high rate of STR amplification stutter. Here, we present MIPSTR, a robust, scalable, and affordable method that addresses these challenges. MIPSTR uses targeted capture of STR loci by single-molecule Molecular Inversion Probes (smMIPs) and a unique mapping strategy. Targeted capture and our mapping strategy resolve the first challenge; the use of single molecule information resolves the second challenge. Unlike previous methods, MIPSTR is capable of distinguishing technical error due to amplification stutter from somatic STR mutations. In proof-of-principle experiments, we use MIPSTR to determine germline STR genotypes for 102 STR loci with high accuracy across diverse populations of the plant A. thaliana. We show that putatively functional STRs may be identified by deviation from predicted STR variation and by association with quantitative phenotypes. Using DNA mixing experiments and a mutant deficient in DNA repair, we demonstrate that MIPSTR can detect low-frequency somatic STR variants. MIPSTR is applicable to any organism with a high-quality reference genome and is scalable to genotyping many thousands of STR loci in thousands of individuals.