Biology subjects

Nemhauser, J. L.

Publications and source records attributed to Nemhauser, J. L..

11 recordsLinked to original sources

bioRxiv2026

Auxin coordinates cell states during Arabidopsis root development

Cell-to-cell variation in gene expression can be highly detrimental and, in some contexts, is actively buffered out; however, in other contexts, it is crucial and actively amplified. For example, variation must be minimized to build organs with consistent size and shape, yet the initiation of organogenesis requires a subset of cells to take on a new fate, a process that often relies on small differences between cells. In plants, much of development is controlled by the hormone auxin, which has been hypothesized to coordinate cell responses by inducing degradation of transcriptional repressors. To quantify the level of cell-to-cell variation and directly test its connection to auxin signaling, we assessed variation in expression of a lateral root founder cell marker GATA23 when auxin levels or responsiveness was modulated. We found that auxin acted as both an amplifier and a constrainer of transcriptional variation during the initiation of a new root. We then extended this work to analysis of root regeneration, where auxin was also found to play a critical role in coordinating cells during fate transitions. ARTICLE SUMMARYDuring organogenesis, cell-to-cell variation is induced, enabling some cells to adopt a new identity and differentiate. Despite this dependence on variation, organogenesis is robust in developmental stages and outcomes. In plants, organogenesis is usually controlled by the hormone auxin, and we hypothesized that auxin ensures coordination among differentiating cells to enable robust development. Using Arabidopsis lateral root development as a model, our results showed that cells receiving the highest auxin dosage both amplify their own auxin response and repress the auxin response in neighboring cells receiving lower auxin dosage, establishing separation between differentiating and non-differentiating cells and ensuring coordinated organogenesis.

developmental biology↗

bioRxiv2024

A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.

molecular biology↗

bioRxiv2022

Expanding the synthetic biology toolbox with a library of constitutive and repressible promoters

Structured AbstractO_ST_ABSBackgroundC_ST_ABSTo support the increasingly complex circuits needed for plant synthetic biology applications, additional constitutive promoters are essential. Reusing promoter parts can lead to difficulty in cloning, increased heterogeneity between transformants, transgene silencing and trait instability. Moreover, the utility of such promoters could be increased by introducing target sequences not found elsewhere in the Arabidopsis thaliana genome and well-suited for Cas9-associated guide RNAs (gRNAs). MethodsWe have developed a pipeline to identify genes that have stable expression across a wide range of Arabidopsis tissues at different developmental stages, and have identified a number of promoters that are well expressed in both transient (Nicotiana benthamiana) and stable (Arabidopsis) transformation assays. We have also introduced two genome-orthogonal gRNA target-sites in a subset of the screened promoters, converting them into NOR logic gates. ResultsOf twenty-two promoters identified in our bioinformatic screen, sixteen drove detectable reporter expression in N. benthamiana. Only three of these promoters were able to produce visible expression of the RUBY reporter in Arabidopsis despite producing RUBY mRNA that could be readily detected by qPCR. We then modified six of these promoters to be repressible, and five of which functioned as NOR gates ConclusionsOne of the major bottlenecks for the ambitious engineering projects currently under development in plants is the lack of well-characterized constitutive promoters. The work here begins to fill this gap. It can also form the basis of constructing more complex information processing circuits in the future.

synthetic biology↗

bioRxiv2022

An integrase toolbox to record gene-expression during plant development.

There are many open questions about the mechanisms that coordinate the dynamic, multicellular behaviors required for organogenesis. Synthetic circuits that can record in vivo signaling networks have been critical in elucidating animal development. Here, we report on the transfer of this technology to plants using orthogonal serine integrases to mediate site-specific and irreversible DNA recombination visualized by switching between fluorescent reporters. When combined with promoters expressed during lateral root initiation, integrases amplified reporter signal and permanently marked all descendants. In addition, we have developed a suite of methods to tune the threshold for integrase switching, including: RNA/protein degradation tags, a nuclear localization signal, and a split-intein system. These tools improved the robustness of integrase-mediated switching with different promoters and the stability of switching behavior over multiple generations. This integrase toolbox can be used to build history-dependent circuits to decode the order of expression during organogenesis in many contexts.

synthetic biology↗

bioRxiv2022

A single helix repression domain is functional across eukaryotes

The corepressor TOPLESS (TPL) and its paralogs coordinately regulate a large number of genes critical to plant development and immunity. As in many members of the larger pan-eukaryotic Tup1/TLE/Groucho corepressor family, TPL contains a Lis1 Homology domain (LisH), whose function is not well understood. We have previously found that the LisH in TPL--and specifically the N-terminal 18 amino acid alpha-helical region (TPL-H1) --can act as an autonomous repression domain. We hypothesized that homologous domains across diverse LisH-containing proteins could share the same function. To test that hypothesis, we built a library of H1s that broadly sampled the sequence and evolutionary space of LisH domains, and tested their activity in a synthetic transcriptional repression assay in Saccharomyces cerevisiae. Using this approach, we found that repression activity was highly conserved and likely the ancestral function of this motif. We also identified key residues that contribute to repressive function. We leveraged this new knowledge for two applications. First, we tested the role of mutations found in somatic cancers on repression function in two human LisH-containing proteins. Second, we validated function of many of our repression domains in plants, confirming that these sequences should be of use to synthetic biology applications across eukaryotes.

plant biology↗

bioRxiv2020

Structure-function analysis of Arabidopsis TOPLESS reveals conservation of repression mechanisms across eukaryotes

The plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli is poorly understood. Previously, we had used the N-terminus of Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle domain subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.

plant biology↗

bioRxiv2019

Auxin promotion of seedling growth via ARF5 is dependent on the brassinosteroid-regulated transcription factors BES1 and BEH4.

Seedlings must continually calibrate their growth in response to the environment. Auxin and brassinosteroids (BRs) are plant hormones that work together to control growth responses during photomorphogenesis. We used our previous analysis of promoter architecture in an auxin and BR target gene to guide our investigation into the broader molecular bases and biological relevance of transcriptional co-regulation by these hormones. We found that the auxin-regulated transcription factor AUXIN RESPONSIVE FACTOR 5 (ARF5) and the brassinosteroid-regulated transcription factor BRI1-EMS-SUPPRESOR 1/BRASSINOZOLERESISTANT 2 (BES1) co-regulated a subset of growth promoting genes via conserved bipartite cis-regulatory elements. Moreover, ARF5 binding to DNA could be enriched by increasing BES1 levels. The evolutionary loss of bipartite elements in promoters results in loss of hormone responsiveness. We also identified another member of the BES1/BZR1 family called BEH4 that acts partially redundantly with BES1 to regulate seedling growth. Double mutant analysis showed that BEH4 and not BZR1 were required alongside BES1 for normal auxin response during early seedling development. We propose that an ARF5-BES1/BEH4 transcriptional module acts to promote growth via modulation of a diverse set of growth-associated genes.

plant biology↗

bioRxiv2018

Accelerating structure-function mapping using the ViVa webtool to mine natural variation

Thousands of sequenced genomes are now publicly available capturing a significant amount of natural variation within plant species; yet, much of this data remains inaccessible to researchers without significant bioinformatics experience. Here, we present a webtool called ViVa (Visualizing Variation) which aims to empower any researcher to take advantage of the amazing genetic resource collected in the Arabidopsis thaliana 1001 Genomes Project (http://1001genomes.org). ViVa facilitates data mining on the gene, gene family or gene network level. To test the utility and accessibility of ViVa, we assembled a team with a range of expertise within biology and bioinformatics to analyze the natural variation within the well-studied nuclear auxin signaling pathway. Our analysis has provided further confirmation of existing knowledge and has also helped generate new hypotheses regarding this well studied pathway. These results highlight how natural variation could be used to generate and test hypotheses about less studied gene families and networks, especially when paired with biochemical and genetic characterization. ViVa is also readily extensible to databases of interspecific genetic variation in plants as well as other organisms, such as the 3,000 Rice Genomes Project (http://snp-seek.irri.org/) and human genetic variation (https://www.ncbi.nlm.nih.gov/clinvar/).

bioinformatics↗

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

Synthetic analysis of natural variants yields insights into the evolution and function of auxin signaling F-box proteins in Arabidopsis thaliana

The evolution of complex body plans in land plants has been paralleled by gene duplication and divergence within nuclear auxin-signaling networks. A deep mechanistic understanding of auxin signaling proteins therefore may allow rational engineering of novel plant architectures. Towards that end, we analyzed natural variation in the auxin receptor F-box family of wild accessions of the reference plant Arabidopsis thaliana and used this information to populate a structure/function map. We employed a synthetic assay to identify natural hypermorphic F-box variants, and then assayed auxin-associated phenotypes in accessions expressing these variants. To more directly measure the impact of the strongest variant in our synthetic assay on auxin sensitivity, we generated transgenic plants expressing this allele. Together, our findings link evolved sequence variation to altered molecular performance and auxin sensitivity. This approach demonstrates the potential for combining synthetic biology approaches with quantitative phenotypes to harness the wealth of available sequence information and guide future engineering efforts of diverse signaling pathways.

plant biology↗

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↗