Biology subjects

Ribeiro, A. S.

Publications and source records attributed to Ribeiro, A. S..

13 recordsLinked to original sources

bioRxiv2026

Hierarchical organization in sparse gene regulatory networks shapes structural coherence and emergent regulatory coordination

How large-scale regulatory coordination in biological systems emerges from local signed and directed interactions in sparse gene regulatory networks (GRNs) remains an unanswered fundamental question. We introduce the coherence matrix, a topology-based framework that captures the consistency of regulatory influence between gene pairs by integrating information across all direct and indirect paths. Analysis of synthetic networks reveals that structural coherence - a metric derived from the coherence matrix - dictates global coordination: while highly coherent motifs maintain aligned regulatory coordination across widely varying network sparsity values, motifs with low coherence allow such coordination only at biologically unrealistic sparsity values. Our investigation of six whole-organism GRNs and further analysis of synthetic networks highlighted that hierarchical organization in GRNs a dense middle layer enriched in feedback loops that mediates coordination between input and output layers - serves as a structural buffer to allow regulatory coordination even for sparse networks. Finally, comparison with Escherichia coli transcriptomic modules further shows that the coherence matrix accurately predicts the sign of coordinated gene contribution, emphasizing its biological application, while also serving as a unifying descriptor integrating local interactions and global network architecture to explain the emergent regulatory coordination.

systems biology↗

bioRxiv2025

Thermal shifts can override the effects of Transcription-targeting Antibiotics

Bacteria traverse diverse stresses but rarely experience them in isolation. Thermal fluctuations and antibiotic exposure often coincide. Yet, how cells transduce these concurrent cues into gene regulatory programs remains unresolved. Here, we show that in Escherichia coli, cold and heat shocks exert dominance over antibiotic stresses across multiple levels of bacterial physiology. First, combined exposure to antibiotics and thermal shifts produced transcriptomes that consistently converged to temperature-defined transcriptomic states. This dominance emerges through the effects of thermal shifts on metabolism, nucleoid organization, engagement of antibiotic targets with DNA, over-expression of global transcription regulators, drug-target conformational plasticity, and regulatory induction of efflux pathways during combined exposure. Finally, cross-species simulations and phylogenetic analysis revealed that thermal disruption of antibiotic efficiency is conserved across evolutionary distant bacterial species. Together, these results identify temperature as a critical determinant of antibiotic efficacy.

microbiology↗

bioRxiv2025

Single-cell Analysis of Attenuation-Driven Transcription Reveals New Principles of Bacterial Gene Regulation

Transcription attenuation fine-tunes biosynthetic gene expression in bacteria via premature termination upon metabolic signals. In transcription initiation-controlled bacterial systems, promoter architecture and transcription factor binding sets the size of transcriptional bursts at {sigma}70 promoters, while distal enhancer elements and associated transcriptional activators modulate burst frequency at {sigma}54 promoters. Using the tryptophan biosynthesis operon as a model, we show that transcription attenuation, acting post-initiation and alongside transcriptional repression, simultaneously modulates both burst size and frequency from a {sigma}70 promoter. This challenges the view that frequency modulation requires distal enhancer input and reveals that post-initiation mechanisms can shape divergent transcriptional bursting. We also uncover that bacteria use cross-feeding as a previously unrecognised strategy for controlling cell-to-cell variation in gene expression, with implications for metabolic coordination among cells. These findings redefine transcription dynamics within cell populations and suggest new principles by which bacteria regulate gene expression to adapt to environmental change.

microbiology↗

bioRxiv2025

Mechanisms shaping the transcriptome of E. coli to non-lethal rifampicin stress

Rifampicin, by hampering transcription, perturbs bacteria even at non-lethal concentrations. In response, Escherichia coli adapts its phenotype to minimize mortality, which is followed by beneficial mutations. Most genome-wide transcriptional regulatory mechanisms controlling the adaptations remain unidentified. We studied the genome-wide, time-resolved, transcriptional program of susceptible E. coli cells under non-lethal rifampicin stress. Dynamically, the transcriptome widely diverged from the control, but later partially realigned. The mechanisms were changes in RNAP and Gyrase levels, promoter sequences, transcription factor network, intergenic distance, sensitivity to DNA supercoiling buildup, {sigma} factor specificity, (p)ppGpp, and a few global regulators. These results show that the genome-wide response dynamics to rifampicin is influenced by the structure of the gene regulatory network. Next, we compared the evolutionarily distant pathogen Mycobacterium tuberculosis. In both species, adjacent genes on the DNA exhibited similar response strengths. Also, the response strengths of orthologous genes were correlated, suggesting that both species implement similar (likely beneficial) phenotypic adaptations. In support, E. coli orthologs were enriched in the mechanisms identified as influential. Overall, E. coli, and likely other bacteria, have mechanisms influencing specific gene cohort responses to non-lethal rifampicin stress, which likely enhances survivability, thus facilitating the emergence of resistance.

systems biology↗

bioRxiv2024

Deciphering the impact of cancer cells secretome and its derived-peptide VGF on breast cancer brain metastasis

Brain metastases (BM) are one of the most serious clinical problems in breast cancer (BC) progression, associated with lower survival rates and a lack of effective therapies. Thus, to dissect the early stages of the brain metastatic process, we have searched for a brain-tropic metastatic signature on BC cells secretome, as a promising source for the discovery of new biomarkers involved in brain metastatic progression. Therefore, six specifically deregulated peptides were found to be enriched in the secretome of brain organotropic BC cells. Importantly, these secretomes caused significant blood-brain barrier (BBB) disruption, as well as microglial activation, in vitro and in vivo. We identified the VGF nerve growth factor inducible as a brain-specific peptide, promoting BBB dysfunction similar to the secretome of brain organotropic BC cells. Concerning microglial activation, a slight increase was also observed upon VGF treatment. In a series of human breast tumors, VGF was found to be expressed in both cancer cells and in the adjacent stroma. VGF-positive tumors showed a significant worse prognosis and were associated with HER2 overexpression and triple-negative molecular signatures. Finally, in a cohort including primary breast tumors and their corresponding metastatic locations to the lung, bone, and brain, we found that VGF significantly correlates with the brain metastatic site. In conclusion, we found a specific BC brain metastatic signature, where VGF was identified as a key mediator in this process. Importantly, its expression was associated with poor prognosis for BC patients, probably due to its associated increased risk of developing BM.

cancer biology↗

bioRxiv2023

Transcription attenuation in synthetic promoters in tandem formation

Closely spaced promoters are ubiquitous in prokaryotic and eukaryotic genomes. How their structure and dynamics relate remains unclear, particularly for tandem formations. To study their transcriptional interference, we engineered two pairs and one trio of synthetic promoters in non-overlapping, tandem formation, in single-copy plasmids. From in vivo measurements in E. coli cells, we found that promoters in tandem formation have attenuated transcription rates. The attenuation strength can be widely fine-tuned by the promoters positioning, natural regulatory mechanisms, and other factors, including the antibiotic rifampicin, which hampers RNAP promoter escape. From this, and supported by in silico models, we concluded that the attenuation emerges from premature terminations generated by collisions between RNAPs elongating from upstream promoters and RNAPs occupying downstream promoters. Moreover, we found that these collisions can cause one or both RNAPs to fall-off. The broad spectrum of possible, externally regulated, attenuation strengths in synthetic tandem promoters should make these structures valuable internal regulators of future synthetic circuits.

synthetic biology↗

bioRxiv2023

A library of reporters of the global regulators of gene expression of Escherichia coli

The topology of the transcription factor network (TFN) of E. coli is far from uniform, with 22 global regulator (GR) proteins controlling one-third of all genes. So far, their production rates cannot be tracked by comparable fluorescent proteins. We developed a library of fluorescent reporters for 16 GRs for this purpose. Each consists of a single-copy plasmid coding for GFP fused to the full-length copy of the native promoter. We tracked their activity in exponential and stationary growth, as well as under weak and strong stresses. We show that the reporters have high sensitivity and specificity to all stresses tested and detect single-cell variability in transcription rates. Given the influence of GRs on the TFN, we expect that the new library will contribute to dissecting global transcriptional stress-response programs of E. coli. Moreover, the library can be invaluable in bioindustrial applications that tune those programs to, instead of cell growth, favor productivity while reducing energy consumption. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/568972v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@15d7ef4org.highwire.dtl.DTLVardef@139d856org.highwire.dtl.DTLVardef@aa0517org.highwire.dtl.DTLVardef@344a2b_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Dynamics of bacterial operons during genome-wide stresses

Bacterial gene networks have operons, each coordinating several genes under a primary promoter. Half of the operons in Escherichia coli have been reported to also contain internal promoters. We studied their role during genome-wide stresses targeting key transcription regulators, RNAP and gyrase. Our results suggest that operons responses are influenced by stress-related changes in premature elongation terminations and internal promoters activity. Globally, this causes the responses of genes in the same operon to differ with the distance between them in a wavelike pattern. Meanwhile, premature terminations are affected by positive supercoiling buildup, collisions between elongating and promoter-bound RNAPs, and local regulatory elements. We report similar findings in E. coli under other stresses and in evolutionarily distant bacteria Bacillus subtilis, Corynebacterium glutamicum, and Helicobacter pylori. Our results suggest that the strength, number, and positioning of operons internal promoters might have evolved to compensate for premature terminations, providing distal genes similar response strengths.

systems biology↗

bioRxiv2023

Unveiling the role of osteosarcoma-derived secretome in premetastatic lung remodeling

Lung metastasis represents the leading cause of osteosarcoma-related death. Progress in preventing lung metastasis is pretty modest due to the inherent complexity of the metastatic process and the lack of suitable models. Herein, we provide mechanistic insights into how osteosarcoma systemically reprograms the lung microenvironment for metastatic outgrowth using metastatic mouse models and a multi-omics approach. We found that osteosarcoma-bearing mice or those preconditioned with cell-secretome harbour profound lung structural alteration with airways damage, inflammation, neutrophil infiltration, and remodelling of the extracellular matrix with deposition of fibronectin and collagen by stromal activated fibroblasts for tumour cell adhesion. These changes, supported by transcriptomic and histological data, promoted and accelerated the development of lung metastasis. Comparative proteome profiling of the cell secretome and mouse plasma identified a large number of proteins engaged in the extracellular-matrix organization, cell-matrix adhesion, neutrophil degranulation, and cytokine-mediated signalling, which were consistent with the observed lung microenvironmental changes. Moreover, we identified EFEMP1, a secreted extracellular matrix glycoprotein, as a potential risk factor for lung metastasis and a poor prognosis factor in osteosarcoma patients.

cancer biology↗

bioRxiv2022

Polo-like kinase 4 (Plk4) potentiates anoikis-resistance of p53KO mammary epithelial cells by inducing a hybrid EMT phenotype

Polo-like kinase 4 (Plk4), the major regulator of centriole biogenesis, has emerged as a putative therapeutic target in cancer due to its abnormal expression in human carcinomas, leading to centrosome number deregulation, mitotic defects and chromosomal instability. Moreover, Plk4 deregulation promotes tumor growth and metastasis in mouse models and is significantly associated with poor patient prognosis. Here, we further investigate the role of Plk4 in carcinogenesis and show that its overexpression significantly potentiates resistance to cell death by anoikis of non-tumorigenic p53 knock-out (p53KO) mammary epithelial cells. Importantly, this effect is independent of Plk4s role in centrosome biogenesis, suggesting that this kinase has additional cellular functions. Interestingly, the Plk4-induced anoikis resistance is associated with the induction of a stable hybrid epithelial-mesenchymal phenotype and is partially dependent on P-cadherin upregulation. Furthermore, we found that the conditioned media of Plk4-induced p53KO mammary epithelial cells also induces anoikis resistance of breast cancer cells in a paracrine way, being also partially dependent on soluble P-cadherin secretion. Our work shows, for the first time, that high expression levels of Plk4 induce anoikis resistance of both mammary epithelial cells with p53KO background, as well as of breast cancer cells exposed to their secretome, which is partially mediated through P-cadherin upregulation. These results reinforce the idea that Plk4, independently of its role in centrosome biogenesis, functions as an oncogene, by impacting the tumor microenvironment to promote malignancy.

cancer biology↗

bioRxiv2022

The transcription factor network of E. coli steers global responses to shifts in RNAP concentration

The robustness and sensitivity of gene networks to environmental changes is critical for cell survival. How gene networks produce specific, chronologically ordered responses to genome-wide perturbations, while robustly maintaining homeostasis, remains an open question. We analysed if short- and mid-term genome-wide responses to shifts in RNA polymerase (RNAP) concentration are influenced by the known topology and logic of the transcription factor network (TFN) of Escherichia coli. We found that, at the gene cohort level, the magnitude of the single-gene, mid-term transcriptional responses to changes in RNAP concentration can be explained by the absolute difference between the genes numbers of activating and repressing input transcription factors (TFs). Interestingly, this difference is strongly positively correlated with the number of input TFs of the gene. Meanwhile, short-term responses showed only weak influence from the TFN. Our results suggest that the global topological traits of the TFN of E. coli shape which gene cohorts respond to genome-wide stresses.

systems biology↗

bioRxiv2021

Analytical kinetic model of native tandem promoters in E. coli

Closely spaced promoters in tandem formation are abundant in bacteria. We investigated the evolutionary conservation, biological functions, and the RNA and single-cell protein expression of genes regulated by tandem promoters in E. coli. We also studied the sequence (distance between transcription start sites dTSS, pause sequences, and distances from oriC) and potential influence of the input transcription factors of these promoters. From this, we propose an analytical model of gene expression based on measured expression dynamics, where RNAP-promoter occupancy times and dTSS are the key regulators of transcription interference due to TSS occlusion by RNAP at one of the promoters (when dTSS [≤] 35 bp) and RNAP occupancy of the downstream promoter (when dTSS > 35 bp). Occlusion and downstream promoter occupancy are modeled as linear functions of occupancy time, while the influence of dTSS is implemented by a continuous step function, fit to in vivo data on mean single-cell protein numbers of 30 natural genes controlled by tandem promoters. The best-fitting step is at 35 bp, matching the length of DNA occupied by RNAP in the open complex formation. This model accurately predicts the squared coefficient of variation and skewness of the natural single-cell protein numbers as a function of dTSS. Additional predictions suggest that promoters in tandem formation can cover a wide range of transcription dynamics within realistic intervals of parameter values. By accurately capturing the dynamics of these promoters, this model can be helpful to predict the dynamics of new promoters and contribute to the expansion of the repertoire of expression dynamics available to synthetic genetic constructs. Author SummaryTandem promoters are common in nature, but investigations on their dynamics have so far largely relied on synthetic constructs. Thus, their regulation and potentially unique dynamics remain unexplored. We first performed a comprehensive exploration of the conservation of genes regulated by these promoters in E. coli and the properties of their input transcription factors. We then measured protein and RNA levels expressed by 30 Escherichia coli tandem promoters, to establish an analytical model of the expression dynamics of genes controlled by such promoters. We show that start site occlusion and downstream RNAP occupancy can be realistically captured by a model with RNAP binding affinity, the time length of open complex formation, and the nucleotide distance between transcription start sites. This study contributes to a better understanding of the unique dynamics tandem promoters can bring to the dynamics of gene networks and will assist in their use in synthetic genetic circuits.

systems biology↗

bioRxiv2019

Transcription closed and open complex formation coordinate expression of genes with a shared promoter region

Many genes are spaced closely, allowing coordination without explicit control through shared regulatory elements and molecular interactions. We study the dynamics of a stochastic model of a gene-pair in a head-to-head configuration, sharing promoter elements, which accounts for the rate-limiting steps in transcription initiation. We find that only in specific regions of the parameter space of the rate-limiting steps is orderly co-expression exhibited, suggesting that successful cooperation between closely spaced genes requires the co-evolution of compatible rate-limiting step configuration. The model predictions are validated by in vivo single-cell, single-RNA measurements of the dynamics of pairs of genes sharing promoter elements. Our results suggest that, in E. coli, the kinetics of the rate-limiting steps in active transcription can play a central role in the dynamics of pairs of genes sharing promoter elements.

biophysics↗