Biology subjects

Alamos, S.

Publications and source records attributed to Alamos, S..

13 recordsLinked to original sources

bioRxiv2026

Genome-scale mapping of plant transcription factor effector domains reveals conserved regulatory principles across eukaryotes

Eukaryotic life spans species that diverged over a billion years ago, yet our molecular view of gene regulation remains heavily skewed toward Opisthokonta model systems, particularly yeast and humans. The degree to which the protein features governing transcriptional regulation are conserved across eukaryotes or have diversified within individual eukaryotic supergroups remains unresolved. To provide a distinct evolutionary reference point, we used a massively parallel reporter assay in plant cells to identify transcriptional effector domains and define their sequence constraints, measuring ~26,000 protein fragments tiling nearly all Arabidopsis transcription factors (1,859 proteins). Comparison with the activity of the same peptide sequences in yeast revealed that activation is governed by a mixture of deeply conserved biochemical features and lineage-dependent functional differences across widely divergent eukaryotic lineages. Likewise, plant repression domains are governed by flexible sequence grammars that extend beyond conserved repression motifs. Together, these results reveal both broadly conserved and clade-specific principles governing eukaryotic transcriptional regulation.

plant biology↗

bioRxiv2026

A toolkit for programmable transcriptional engineering across eukaryotic kingdoms

Chromatin is essential for eukaryotic life. Tens of thousands of chromatin regulator (CR) proteins exist in eukaryotic genomes that are predicted to modulate chromatin states; however, their molecular functions remain largely untested experimentally. Here, we construct a library of over 300 full-length CRs from humans, plants, yeast, protozoa, and virus, each fused to DNA-binding domains, and test their direct effect on transcriptional repression and activation in plants and human cells. We discover CRs with cross-kingdom functionality when transferred across eukaryotes, including CRs that outperform existing tools for programmable transcriptional repression and activation in plants and human cells. Using pooled CRISPR screens, we demonstrate a suite of CRISPR repressors that titrate gene expression at intermediate levels. Finally, we identify RCOR1 and MTA2 as universal eukaryotic repressors that retain repressive activity in plants, yeast, and human cells. Our toolkit advances synthetic eukaryotic engineering and expands our understanding of CR functionality across eukaryotes.

synthetic biology↗

bioRxiv2025

Causes and consequences of experimental variation in Nicotiana benthamiana transient expression

Infiltration of Agrobacterium tumefaciens into Nicotiana benthamiana has become a foundational technique in plant biology, enabling efficient delivery of transgenes in planta with technical ease, robust signal, and relatively high throughput. Despite transient expressions prevalence in disciplines such as synthetic biology, little work has been done to describe and address the variability inherent in this system, a concern for experiments that rely on highly quantitative readouts. In a comprehensive analysis of N. benthamiana agroinfiltration experiments, we model sources of variability that affect transient expression. Our findings emphasize the need to validate normalization methods under the specific conditions of each study, as distinct normalization schemes do not always reduce variation either within or between experiments. Using a dataset of 1,915 plants collected over three years, we develop a model of variation in N. benthamiana transient expression, using power analysis to determine the number of individual plants required for a given effect size. Drawing on our longitudinal data, these findings inform practical guidelines for minimizing variability through strategic experimental design and power analysis, providing a foundation for more robust and reproducible use of N. benthamiana in quantitative plant biology and synthetic biology applications.

plant biology↗

bioRxiv2025

Multiplexed profiling of transcriptional regulators in plant cells

Transcriptional regulators play key roles in plant growth, development, and environmental responses; however, understanding how their regulatory activity is encoded at the protein level has been hindered by a lack of multiplexed large-scale methods to characterize protein libraries in planta. Here, we present ENTRAP-seq (Enrichment of Nuclear Trans-elements Reporter Assay in Plants), a high-throughput method that introduces protein-coding libraries into plant cells to drive a nuclear magnetic sorting-based reporter, enabling multiplexed measurement of regulatory activity from thousands of protein variants. Using ENTRAP-seq and machine learning, we screened 1,495 plant viruses and identified hundreds of novel putative transcriptional regulatory domains found in structural proteins and enzymes not associated with gene regulation. In addition, we combined ENTRAP-seq with machine-guided design to engineer the activity of a plant transcription factor in a semi-rational fashion. Our findings demonstrate how scalable protein function assays deployed in planta will enable the characterization of natural and synthetic coding diversity in plants.

plant biology↗

bioRxiv2024

Predictable modulation of plant root development using engineered cytokinin regulators

Achieving precise control over quantitative developmental phenotypes is a key objective in plant biology. Recent advances in synthetic biology have enabled tools to reprogram entire developmental pathways; however, the complexity of designing synthetic genetic programs and the inherent interactions between various signaling processes remains a critical challenge. Here, we leverage Type-B response regulators to modulate the expression of genes involved in cytokinin-dependent growth and development processes. We rationally engineered these regulators to modulate their transcriptional activity (i.e., repression or activation) and potency while reducing their sensitivity to cytokinin. By localizing the expression of these engineered transcription factors using tissue-specific promoters, we can predictably tune cytokinin-regulated traits. As a proof of principle, we deployed this synthetic system in Arabidopsis thaliana to either decrease or increase the number of lateral roots. The simplicity and modularity of our approach makes it an ideal system for controlling other developmental phenotypes of agronomic interest in plants.

plant biology↗

bioRxiv2023

DRMY1 promotes robust morphogenesis by sustaining translation of a hormone signaling protein

Robustness is the invariant development of phenotype despite environmental changes and genetic perturbations. In the Arabidopsis flower bud, four sepals robustly initiate and grow to constant size to enclose and protect the inner floral organs. We previously characterized the mutant development related myb-like1 (drmy1), where 3-5 sepals initiate variably and grow to different sizes, compromising their protective function. The molecular mechanism underlying this loss of robustness was unclear. Here, we show that drmy1 has reduced TARGET OF RAPAMYCIN (TOR) activity, ribosomal content, and translation. Translation reduction decreases the protein level of ARABIDOPSIS RESPONSE REGULATOR7 (ARR7) and ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), two cytokinin signaling inhibitors that are normally rapidly produced before sepal initiation. The resultant upregulation of cytokinin signaling disrupts robust auxin patterning and sepal initiation. Our work shows that the homeostasis of translation, a ubiquitous cellular process, is crucial for the robust spatiotemporal patterning of organogenesis.

developmental biology↗

bioRxiv2022

Optogenetic dissection of transcriptional repression in a multicellular organism

Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can repress or activate specific genes, how these functions are implemented at the molecular level has remained elusive. Here we combine optogenetics, single-cell live-imaging, and mathematical modeling to study how a zinc-finger repressor, Knirps, induces switch-like transitions into long-lived quiescent states. Using optogenetics, we demonstrate that repression is rapidly reversible ([~]1 minute) and memoryless. Furthermore, we show that the repressor acts by decreasing the frequency of transcriptional bursts in a manner consistent with an equilibrium binding model. Our results provide a quantitative framework for dissecting the in vivo biochemistry of eukaryotic transcriptional regulation.

biophysics↗

bioRxiv2021

Chromatin changes in PIF-regulated genes parallel their rapid transcriptional response to light

As sessile organisms, plants must adapt to a changing environment, sensing variations in resource availability and modifying their development in response. Light is one of the most important resources for plants, and its perception by sensory photoreceptors (e.g. phytochromes) and subsequent transduction into long-term transcriptional reprogramming have been well characterized. Chromatin changes have been shown to be involved in photomorphogenesis. However, the initial short-term transcriptional changes produced by light and what factors enable these rapid changes are not well studied. Here, we identify rapidly light-responsive, PIF (Phytochrome Interacting Factor) direct-target genes (LRP-DTGs). We found that a majority of these genes also show rapid changes in Histone 3 Lysine-9 acetylation (H3K9ac) in response to the light signal. Detailed time-course analysis of transcriptional and chromatin changes showed that, for light-repressed genes, H3K9 deacetylation parallels light-triggered transcriptional repression, while for light-induced genes, H3K9 acetylation appeared to somewhat precede light-activated transcription. However, real-time imaging of transcription elongation revealed that, in fact, H3K9 acetylation also parallels transcriptional induction. Collectively, the data raise the possibility that light-induced transcriptional and chromatin-remodeling processes are mechanistically intertwined. Histone modifying proteins involved in long term light responses do not seem to have a role in this fast response, indicating that different factors might act at different stages of the light response. This work not only advances our understanding of plant responses to light, but also unveils a system in which rapid chromatin changes in reaction to an external signal can be studied under natural conditions.

plant biology↗

bioRxiv2021

Minimal synthetic enhancers reveal control of the probability of transcriptional engagement and its timing by a morphogen gradient

How enhancers interpret morphogen gradients to generate spatial patterns of gene expression is a central question in developmental biology. Although recent studies have begun to elucidate that enhancers can dictate whether, when, and at what rate a promoter will engage in transcription, the complexity of endogenous enhancers calls for theoretical models with too many free parameters to quantitatively dissect these regulatory strategies. To overcome this limitation, we established a minimal synthetic enhancer system in embryos of the fruit 2y Drosophila melanogaster. Here, a gradient of the Dorsal activator is read by a single Dorsal binding site. By quantifying transcriptional activity using live imaging, our experiments revealed that this single Dorsal binding site is capable of regulating whether promoters engage in transcription in a Dorsal concentration-speci1c manner. By modulating binding-site aZnity, we determined that a genes decision to engage in transcription and its transcriptional onset time can be explained by a simple theoretical model where the promoter has to traverse multiple kinetic barriers before transcription can ensue. The experimental platform developed here pushes the boundaries of live-imaging in studying gene regulation in the early embryo by enabling the quanti1cation of the transcriptional activity driven by a single transcription factor binding site, and making it possible to build more complex enhancers from the ground up in the context of a dialogue between theory and experiment.

developmental biology↗

bioRxiv2020

Quantitative characterization of the eukaryotic transcription cycle using live imaging and statistical inference

The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel application of Bayesian inference techniques to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters as well as single-cell correlations between these parameters. These measurements, combined with theoretical modeling, suggest a substantial variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.

biophysics↗

bioRxiv2020

Quantitative imaging of RNA polymerase II activity in plants reveals the single-cell basis of tissue-wide transcriptional dynamics

The responses of plants to their environment often hinge on the spatiotemporal dynamics of transcriptional regulation. While live-imaging tools have been used extensively to quantitatively capture rapid transcriptional dynamics in living animal cells, lack of implementation of these technologies in plants has limited concomitant quantitative studies. Here, we applied the PP7 and MS2 RNA-labeling technologies for the quantitative imaging of RNA polymerase II activity dynamics in single cells of living plants as they respond to experimental treatments. Using this technology, we count nascent RNA transcripts in real-time in Nicotiana benthamiana (tobacco) and Arabidopsis thaliana (Arabidopsis). Examination of heat shock reporters revealed that plant tissues respond to external signals by modulating the number of cells engaged in transcription rather than the transcription rate of active cells. This switch-like behavior, combined with cell-to-cell variability in transcription rate, results in mRNA production variability spanning three orders of magnitude. We determined that cellular heterogeneity stems mainly from the stochasticity intrinsic to individual alleles. Taken together, our results demonstrate that it is now possible to quantitatively study the dynamics of transcriptional programs in single cells of living plants.

plant biology↗

bioRxiv2018

Loop Assembly: a simple and open system for recursive fabrication of DNA circuits

High efficiency methods for DNA assembly are based on sequence overlap between fragments or Type IIS restriction endonuclease cleavage and ligation. These have enabled routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customisation, elaborate vector sets and serial manipulations for the different stages of assembly. We present Loop assembly, based on a recursive approach to DNA fabrication. Alternate use of two Type IIS restriction endonucleases and corresponding vector sets allows efficient and parallel assembly of large DNA circuits. Plasmids containing standard Level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. The vectors also contain modular sites for hybrid assembly using sequence overlap methods. Loop assembly provides a simple generalised solution for DNA construction with standardised parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access.

synthetic biology↗

bioRxiv2017

Variation in traction forces during cell cycle progression

Tissue morphogenesis results from the interplay between cell growth and mechanical forces. While the impact of forces on cell proliferation has been fairly well characterized, the inverse relationship is much less understood. Here we investigated how traction forces vary during cell cycle progression. Cell shape was constrained on micropatterned substrates in order to distinguish variations in cell contractility from cell size increase. We performed traction force measurements of asynchronously dividing cells expressing a cell-cycle reporter, to obtain measurements of contractile forces generated during cell division. We found that forces tend to increase as cells progress through G1, before reaching a plateau in S phase, and then decline during G2. This biphasic behaviour revealed a previously undocumented specific and opposite regulation of cell contractility during each cell cycle stage.

cell biology↗