Biology subjects

Buitrago, N.

Publications and source records attributed to Buitrago, N..

4 recordsLinked to original sources

bioRxiv2026

Insulators As Dynamic, Tunable Regulators of Enhancer-Promoter Coordination in Living Drosophila Embryos

The three-dimensional organization of the genome enables enhancers and promoters to interact across vast distances and direct transcription. Yet whether architectural elements, such as insulators, serve as rigid, passive barriers or as dynamic, active organizers of this communication remains unclear. Here, using single-cell, live imaging of a Drosophila transgene in which a single enhancer regulates two equidistant promoters, we confirm that the enhancer engages both promoters simultaneously and show that coordinated bursting is intrinsically more productive than uncoordinated activity. Flanking this system with insulators increases coordinated bursting frequency and transcriptional output, indicating that insulator-mediated looping promotes multi-way enhancer-promoter interaction. Further, bidirectionally-paired, homotypic insulators produce stronger coordination than unidirectional pairs. Inserting an intermediate insulator to generate competing loop configurations, together with two-state promoter modeling, we show that these chromatin loops are highly dynamic. This work reframes insulators as active, tunable regulators that shape the frequency, coordination, and productivity of enhancer-promoter communication.

molecular biology↗

bioRxiv2026

Cell-Based Sensor for Extracellular DNA

Detection of molecules with cell-based sensors allows for conversion of binding events into gene expression outputs. Here, we present a cell-based sensor that can detect extracellular double-stranded DNA. This sensor is based on an engineered receptor which we call Luminescent Ultrasensitive Nucleic Acid Reporter, or LUNAR. LUNAR is based on a recently developed Programmable Antigen-gated G-protein-coupled Engineered Receptor (PAGER). PAGERs are a genetic fusion of an auto-inhibitory peptide, a protein-binding domain, and a modified kappa opioid receptor. PAGERs are gated by two binding events. First, a protein ligand displaces an intramolecular inhibitor, Arodyn, then a second ligand activates the receptor. By replacing the protein-binding domain with a DNA binding zinc finger protein (ZFP) we could detect extracellular DNA in a dose-dependent fashion. Here, we show that first-generation LUNAR constructs can detect both oligonucleotides and plasmid double-stranded DNA with nanomolar sensitivity in mammalian cells. Future work will focus on improving sensitivity, fold-change, and multiplexing capabilities for sequence-specific DNA detection.

synthetic biology↗

bioRxiv2025

Modeling synthetic serum marker kinetics for monitoring deep-tissue gene expression.

Serum markers could theoretically enable monitoring of gene expression dynamics with a simple blood draw. However, such markers are typically used to measure long-term changes, such as the progression of disease over multiple days or months. In this theoretical study, we determine the maximum theoretical temporal resolution and precision - the ability to distinguish rapid changes in gene expression and to obtain the true frequency of such changes, respectively. As a model of such processes, we used Released Markers of Activity (RMAs) - a class of synthetic serum markers that are expressed in neurons in the brain but transported into the blood. RMAs are orthogonal to physiological processes and have tunable levels, production rate, and onset time, providing well-defined data for serum marker production, tissue transport, and detection. We explore several scenarios where RMAs were used, including monitoring tissue transduction, changes in endogenous gene expression, and drug-induced marker expression. We demonstrate that the temporal resolution of monitoring primarily depends on the extrinsic noise level of the RMA signal and protein serum half-life. Additionally, we find that decreasing the serum half-life results in improved temporal precision at the cost of signal intensity. To enable broad use of this model, we developed a library and interface for running simulations to approximate marker trajectories to inform experimental design decisions and optimize marker detection. Author SummaryGene expression is a foundational driver of biological processes. We use synthetic serum markers that can report on gene expression with a blood draw. These synthetic markers have well-defined parameters and thus form a useful model for understanding the maximum theoretical temporal resolution and precision of monitoring gene expression. These two variables describe, respectively, the fastest changes in gene expression and how accurately the kinetics can be reflected through a serum marker measurement. Developing more robust reporter systems requires understanding how biological properties of these markers, such as serum half-life or measurement noise, affect their kinetics. Here, we developed computational models describing in vivo behavior of synthetic serum markers to identify the most critical parameters for optimization. We show that serum half-life and measurement noise are the main contributors to the temporal precision and resolution of monitoring. Additionally, we demonstrate the ability to predict marker levels in various contexts, including constitutive and drug-induced expression.

bioengineering↗

bioRxiv2025

Controlled Delivery of a Neurotrophic Factor in the Adult Mouse Brain Using Engineered Microglia

Microglia, the resident immune cells of the central nervous system, have been proposed as vehicles for delivering therapeutic biologics. These cells can be genetically engineered in vitro and transplanted into host animals following ablation of endogenous microglia, enabling repopulation of the brain parenchyma. However, current replacement strategies often rely on radiation or transgenic models, limiting their clinical relevance. CSF1R inhibitors offer a more translational approach to microglia ablation, though surviving host cells can compete with transplanted microglia during repopulation. In this study, we successfully ablated endogenous microglia using a CSF1R inhibitor in adult mice and developed a method to transplant engineered microglia expressing Brain-Derived Neurotrophic Factor (BDNF) in a doxycycline-inducible manner. To enhance engraftment, transplanted cells also expressed a constitutively active CSF1R mutant (caCSF1R). BDNF-expressing transplanted microglia spread through large areas of host mice brains, displayed similar morphology and transcriptional profile to repopulating host microglia, and responded to pro-inflammatory stimuli. Treatment with doxycycline resulted in increased BDNF expression and TrkB phosphorylation in the host brain. Expression of caCSF1R provided transplanted cells with a competitive advantage over endogenous repopulating cells, resulting in the accelerated spread of the transplants. Our results demonstrate the functional integration and therapeutic potential of microglia as vehicles for delivering neurotrophic factors to the brain in a controllable manner. Furthermore, we show that caCSF1R expression is able to enhance the spread of transplanted microglia. SIGNIFICANCEThis study demonstrates the potential of engineered microglia to deliver the protein Brain-Derived Neurotrophic Factor to the brain parenchyma, under the control of orally-administered doxycycline. The technique can be generalized to a wide array of proteins, offering a novel paradigm for neurological therapy.

neuroscience↗