Biology subjects

Queitsch, C.

Publications and source records attributed to Queitsch, C..

9 recordsLinked to original sources

bioRxiv2018

Developmental and conditional dynamics of gene expression in single root cells of A. thaliana

Single-cell RNA-seq 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 A. 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.

genetics↗

bioRxiv2018

High-throughput identification of dominant negative polypeptides in yeast

Dominant negative polypeptides can act as inhibitors by binding to the wild type protein or by titrating an essential ligand. Here, we use high-throughput sequencing of DNA libraries composed of fragments of yeast genes to identify dominant negative polypeptides based on their depletion during cell growth. The method uncovers numerous inhibitory polypeptides for a protein and thus is capable of defining interacting domains with exquisite resolution, even pinpointing individual residues that contact ligands.

genomics↗

bioRxiv2018

HSP90 buffers newly induced mutations in massively mutated plant lines

Robustness to both genetic and environmental change is an emergent feature of living systems. Loss of phenotypic robustness can be associated with increased penetrance of genetic variation. In model organisms and in humans, the phenotypic consequences of standing genetic variation can be buffered by the molecular chaperone HSP90. However, it has been argued that HSP90 has the opposite effect on newly introduced genetic variation. To test the buffering effect of HSP90 on new mutations, we introduced vast numbers of mutations into wild-type and HSP90-reduced plants and assessed embryonic lethality and early seedling phenotypes for thousands of offspring. Although the levels of newly introduced mutations were similar in the two backgrounds, the HSP90-reduced plants showed a significantly greater frequency of embryonic lethality and severe phenotypic abnormalities, consistent with higher penetrance and expressivity of newly introduced genetic variation. We further demonstrate that some mutant phenotypes were heritable in an HSP90-dependent manner, and we map candidate HSP90-dependent polymorphisms. Moreover, both sequence and phenotypic analyses of wild-type and HSP90-reduced plants suggest that the HSP90-dependent phenotypes are largely due the newly introduced mutations rather than to an increased mutation rate in HSP90-reduced plants. Taken together, our results support a model in which HSP90 buffers newly introduced mutations, and the phenotypic consequences of such mutations outweigh those of mutations arising de novo in response to HSP90 perturbation.

genetics↗

bioRxiv2018

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. Yet, the intrinsic instigators of DNA damage are poorly understood. Here we identify proteins that promote endogenous DNA damage when overproduced: the DNA-damaging proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six DNA-damage-causing 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 expression in tumors predicts heavy mutagenesis and poor prognosis. Half of tested human homologs, when overproduced in human cells, promote DNA damage and mutation, with DNA-damaging mechanisms like those in E. coli. Together, our work reveals DDP networks that provoke endogenous DNA damage and may indicate functions of many human known and newly implicated cancer-promoting proteins.

cancer biology↗

bioRxiv2018

Mapping and dynamics of regulatory DNA during seed development

The genome is reprogrammed during development to produce diverse cell types, largely through altered expression and activity of key transcription factors. The accessibility and critical functions of epidermal cells have made them a model for connecting transcriptional events to development in a range of model systems. In Arabidopsis thaliana and many other plants, fertilization triggers differentiation of specialized epidermal seed coat cells that have a unique morphology caused by large extracellular deposits of pectin. Here, we used DNase I-seq to generate regulatory landscapes of A. thaliana seeds at two critical time points in seed coat maturation, enriching for seed coat cells with the INTACT method. We found over 3000 developmentally dynamic regulatory DNA elements and explored their relationship with nearby gene expression. The dynamic regulatory elements were enriched for motifs for several transcription factors families; most notably the TCP family at the earlier time point and the MYB family at the later one. To assess the extent to which the observed regulatory sites in seeds added to previously known regulatory sites in A. thaliana, we compared our data to 11 other data sets generated with seven-day-old seedlings for diverse tissues and conditions. Surprisingly, over a quarter of the regulatory, i.e. accessible, bases observed in seeds were novel. Notably, in this comparison, development exerted a stronger effect on the plant regulatory landscape than extreme environmental perturbations, highlighting the importance of extending studies of regulatory landscapes to other tissues and cell types during development.

plant biology↗

bioRxiv2017

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.

plant biology↗

bioRxiv2017

Short tandem repeats with massive variation and functional consequences across strains of Arabidopsis thaliana

Short tandem repeat (STR) mutations may be responsible for more than half of the mutations in eukaryotic coding DNA, yet STR variation is rarely examined as a contributor to complex traits. We assess the scope of this contribution across a collection of 96 strains of Arabidopsis thaliana by massively parallel STR genotyping. We found that 95% of examined STRs are polymorphic, with a median of six alleles per STR in these strains. Modest STR expansions are found in most strains, some of which have evident functional effects. For instance, three of six intronic STR expansions are associated with intron retention. Coding STRs are depleted of variation relative to non-coding STRs, consistent with the action of purifying selection, and some STRs show hypervariable patterns consistent with diversifying selection. Finally, we detect dozens of novel STR-phenotype associations that could not be detected with SNPs alone, validating several with follow-up experiments. Our results demonstrate that STRs comprise a large, unascertained reservoir of functionally relevant genomic variation.

genomics↗

bioRxiv2017

Trait specificity mediated by alternative DNA-binding preferences of a single transcription factor in yeast.

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 co-factor Tec1. Here, we characterize the in vitro binding preferences of Ste12 to identify a defined spacing and orientation of dimeric sites, one that is common in pheromone-regulated genes. We find that single amino acid changes in the DNA-binding domain of Ste12 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. Some exceptional Ste12 mutants promote hyperinvasion in a Tec1-independent manner; these fail to bind cooperative sites with Tec1 and bind to unusual dimeric Ste12 sites that contain one highly degenerate half site. We propose a model for how activation of invasion genes could have evolved with Ste12 alone.

genomics↗

bioRxiv2017

Regulatory DNA in A. thaliana can tolerate high levels of sequence divergence

Variation in regulatory DNA is thought to drive evolution. Cross-species comparisons of regulatory DNA have provided evidence for both weak purifying selection and substantial turnover in regulatory regions. However, disruption of transcription factor binding sites can affect the expression of neighboring genes. Thus, the base-pair level functional annotation of regulatory DNA has proven challenging. Here, we explore regulatory DNA variation and its functional consequences in genetically diverse strains of the plant Arabidopsis thaliana, which largely maintain the positional homology of regulatory DNA. Using chromatin accessibility to delineate regulatory DNA genome-wide, we find that 15% of approximately 50,000 regulatory sites varied in accessibility among strains. Some of these accessibility differences are associated with extensive underlying sequence variation, encompassing many deletions and dramatically hypervariable sequence. For the majority of such regulatory sites, nearby gene expression was similar, despite this large genetic variation. However, among all regulatory sites, those with both high levels of sequence variation and differential chromatin accessibility are the most likely to reside near genes with differential expression among strains. Unexpectedly, the vast majority of regulatory sites that differed in chromatin accessibility among strains show little variation in the underlying DNA sequence, implicating variation in upstream regulators.

genomics↗