Biology subjects

Poirier, M. G.

Publications and source records attributed to Poirier, M. G..

At least 19 recordsLinked to original sources

bioRxiv2026

Enhanced DNA Scanning and Target Specificity via Motif Sequestration

Sequence-specific DNA-binding proteins must efficiently locate their target sequences within large, structurally heterogeneous genomes while avoiding nonproductive off-target interactions. Here, we show that an autoinhibitory conformational state of the site-specific recombinase Cre lowers nonspecific DNA-binding affinity to promote one-dimensional DNA scanning while suppressing off-target assembly of recombinogenic complexes. Recent studies indicate that the C-terminal segment of Cre transiently adopts a sequestered conformation that occludes the catalytic domain's DNA-binding surface. Using NMR spectroscopy, single-molecule fluorescence microscopy, and optical trapping, we investigated how this conformational equilibrium regulates target search. NMR measurements reveal that the sequestered state predominates in the absence of DNA and persists at noncognate sites, whereas binding to cognate loxP sequences relieves autoinhibition. Single-molecule tracking demonstrates that full-length Cre undergoes processive one-dimensional diffusion and transitions to stable binding upon cognate-site recognition, whereas C-terminal truncation impairs scanning efficiency by arresting diffusion. These findings demonstrate how transient motif sequestration coordinates target search with site-specific complex assembly, suggesting a general mechanism by which DNA-modifying enzymes balance search efficiency and target specificity.

biophysics↗

bioRxiv2026

Fusion Oncoprotein EWSR1::FLI1 Invades Nucleosomes at Consensus ETS Motifs and GGAA Microsatellites

Ewing sarcoma is an aggressive bone malignancy occurring in children, adolescents, and young adults. Most cases are caused by expression of the fusion oncoprotein EWSR1::FLI1, which contains the low complexity domain (LCD) of EWSR1 and the DNA-binding domain (DBD) of FLI1. Previous genomic studies indicate EWSR1::FLI1 accesses GGAA microsatellites in chromatin to function as a potent transcriptional regulator. Due to the technical challenges of purifying full-length EWSR1::FLI1, mechanistic studies biochemically characterizing its pioneer activities have been lacking. Here, we purified both full-length EWSR1::FLI1 and truncated DBD constructs to conduct biochemical and fluorescence-based experiments investigating interactions with different motifs in free DNA and nucleosomes. Both truncated and full-length EWSR1::FLI1 show efficient target binding in nucleosomes, and that the fourth alpha-helix in the FLI1 DBD enhances nucleosome-binding efficiency. Surprisingly, we also observe differences in both free DNA binding affinity and sequence preference between truncated and full-length proteins, though these changes are not apparent in nucleosome-binding assays. These findings reveal that EWSR1::FLI1 possesses a key pioneer factor property, efficiently targeting its binding site within nucleosomes, and that full-length EWSR1::FLI1 binding shifts to preferentially target GGAA repeats, even on motifs that bind a single EWSR1::FLI1 protein.

biochemistry↗

bioRxiv2026

In-Situ ssDNA Isolation from dsDNA Sources as a Streamlined Pathway to DNA Origami Assembly and Testing

Scaffolded DNA origami has become a valuable nanoscale tool for applications in biomedical and physical sciences. Critical to leveraging the modular and programmable properties of DNA origami nanodevices is access to the scaffold strand, a long single-stranded DNA (ssDNA) of precise length and sequence, which is folded into a compact shape via piecewise base-pairing with many staple strands, short ssDNA oligonucleotides. Current methods to produce and manipulate long ssDNA scaffolds can be costly, time-consuming, and cumbersome. In contrast, methods to produce and manipulate the sequence of double-stranded DNA (dsDNA) are efficient and scalable. Here, we present a method for the rapid isolation of target ssDNA sequences from a variety of dsDNA sources using oligonucleotides as blocking strands that bind continuously to the undesired strand, thereby releasing the target scaffold strand. We report successful ssDNA isolation from linear and supercoiled dsDNAs of various sequences and lengths, ranging from 769 to 15,101 nucleotides. In addition to isolating ssDNA, we demonstrated this approach enables folding of DNA origami directly from dsDNA templates using both blocking and staple strands in a single-pot thermally controlled reaction. Furthermore, we explore multi-scaffold and gene-encoding DNA origami structures, expanding the framework for application-based designs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/709872v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@f7ada7org.highwire.dtl.DTLVardef@a9bab8org.highwire.dtl.DTLVardef@121b695org.highwire.dtl.DTLVardef@5a1dea_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Cohesin Facilitates Nucleosome Invasion by Transcription Factors

Nucleosomes present a major barrier to transcription factor (TF) binding. However, a subgroup of TFs known as pioneer factors (PFs) can recognize motifs covered by nucleosomes and initiate chromatin opening. PFs also bind nucleosomal substrates with high affinity in vitro, which may facilitate their nucleosome invasion in vivo. Here, we show that LexA, a bacterial TF with poor nucleosome binding in vitro, can rapidly invade into a well-positioned nucleosome from a motif positioned at the dyad when expressed ectopically in yeast. This striking contrast between LexA binding in vitro and in vivo raises the possibility that TFs can exploit nucleosome dynamics in vivo to access occluded sites. Surprisingly, we find that LexA-mediated chromatin opening can occur in the absence of DNA replication, chromatin remodeling, histone turnover, and a few histone chaperones. Instead, nucleosome invasion by LexA and a native PF, Cbf1, is promoted by the cohesin complex, illustrating intriguing a connection between cohesin and TF binding. Together, our results demonstrate that even non-pioneer TFs like LexA can bind and displace nucleosomes in vivo, through a process facilitated by cohesin.

molecular biology↗

bioRxiv2025

Free energy spectroscopy reveals the mechanistic landscape of chromatin compaction

Eukaryotic genomic DNA is repeatedly wrapped into nucleosome spools: the basic building block of chromatin. This organization regulates the physical accessibility of the genome to gene transcription, replication, and repair regulatory factors. Chromatin compaction is controlled by multivalent weak interactions, resulting in a complicated conformational landscape that remains challenging to characterize. This work reports a method for characterizing chromatin compaction, Free Energy Spectroscopy (FES), which is based on DNA nanotechnology and transmission electron microscopy. This method experimentally determines the chromatin compaction free energy landscape in terms of end-to-end distance and nucleosome stacking interactions. By deconvolving the free energy landscapes of partially and fully compact tetranucleosomes, FES revealed three separate mechanisms by which linker histones reshape the compaction energetics to condense chromatin. This study establishes FES as a method with the potential to help answer a broad range of mechanistic questions about genome and epigenome function.

biophysics↗

bioRxiv2025

Linker histone H1.0 loads onto nucleosomes through multiple pathways that are facilitated by histone chaperones

Linker histone H1 is an essential chromatin architectural protein that compacts chromatin into transcriptionally silent regions by interacting with nucleosomal and linker DNA, while rapidly exchanging in vivo. How H1 targets nucleosomes while being dynamic remains unanswered. Using a single-molecule strategy, we investigated human H1.0 interactions with DNA and nucleosomes. H1.0 directly binds nucleosomes and naked DNA with a preference toward nucleosomes. DNA-bound H1.0 exhibited a range of bound lifetimes with both mobile and immobile states, where the mobile H1.0 did not load onto nucleosomes. The histone chaperone Nap1 facilitated H1.0-nucleosome loading by enabling H1.0 loading through DNA sliding, reducing DNA resident times without impacting nucleosome resident times, increasing mobility along DNA, and targeting H1.0 loading onto the nucleosome dyad. These findings reveal linker histones load onto nucleosomes through multiple distinct mechanisms that are facilitated by chaperones to regulate chromatin accessibility.

biophysics↗

bioRxiv2025

Native nucleosome-positioning elements for the investigation of nucleosome repositioning

Nucleosome repositioning is essential for establishing nucleosome-depleted regions (NDRs) to initiate transcription. This process has been extensively studied using structural, biochemical, and single-molecule approaches, which require homogenously positioned nucleosomes. This is often achieved using the Widom 601 sequence, a highly efficient nucleosome positioning element (NPE) selected for its unusually strong binding to the H3-H4 histone tetramer. Due to the artificial nature of 601, native NPEs are needed to explore the role of DNA sequence in nucleosome repositioning. Here, we characterize the position distributions and nucleosome formation free energy for a set of yeast native nucleosomes (YNNs) from Saccharomyces cerevisiae. We show these native NPEs can be used in biochemical studies of nucleosome repositioning by transcription factors (TFs) and the chromatin remodeler Chd1. TFs could directly reposition a fraction of nucleosomes containing native NPEs, but not 601-containing nucleosomes. In contrast, partial unwrapping was similar for 601 and native NPE sequences, and the rate of ATP-dependent remodeling by Chd1 was within the range of the fast and slow directions of the 601 nucleosomes. This set of native NPEs provides an alternative to the 601 NPE that can be used for probing the repositioning of nucleosomes that contain native DNA sequences.

molecular biology↗

bioRxiv2024

Aberrant expression of histone H2B variants reshape chromatin and alter oncogenic gene expression programs

Chromatin architecture governs DNA accessibility and gene expression. Thus, any perturbations to chromatin can significantly alter gene expression programs and promote disease. Prior studies demonstrate that every amino acid in a histone is functionally significant, and that even a single amino acid substitution can drive specific cancers. We previously observed that naturally occurring H2B variants are dysregulated during the epithelial to mesenchymal transition (EMT) in bronchial epithelial cells. Naturally occurring H2B variants differ from canonical H2B by only a few amino acids, yet single amino acid changes in other histone variants (e.g., H3.3) can drive cancer. We therefore hypothesized that H2B variants might function like oncohistones, and investigated how they modify chromatin architecture, dynamics, and function. We find that H2B variants are frequently dysregulated in many cancers, and correlate with patient prognosis. Despite high sequence similarity, mutations in each H2B variant tend to occur at specific "hotspots" in cancer. Some H2B variants cause tighter DNA wrapping around nucleosomes, leading to more compact chromatin structures and reduced transcription factor accessibility to nucleosomal DNA. They also altered genome-wide accessibility to oncogenic regulatory elements and genes, with concomitant changes in oncogenic gene expression programs. Although we did not observe changes in cell proliferation or migration in vitro, our Gene Ontology (GO) analyses of ATAC-seq peaks and RNA-seq data indicated significant changes in oncogenic pathways. These findings suggest that H2B variants may influence early-stage, cancer-associated regulatory mechanisms, potentially setting the stage for oncogenesis later on. Thus, H2B variant expression could serve as an early cancer biomarker, and H2B variants might be novel therapeutic targets.

cancer biology↗

bioRxiv2024

Recycling Materials for Sustainable DNA Origami Manufacturing

DNA origami nanotechnology has great potential in multiple fields including biomedical, biophysical, and nanofabrication applications. However, current production pipelines lead to single-use devices incorporating a small fraction of initial reactants, resulting in a wasteful manufacturing process. Here, we introduce two complementary approaches to overcome these limitations by recycling the strand components of DNA origami nanostructures (DONs). We demonstrate reprogramming entire DONs into new devices, re-using scaffold strands. We validate this approach by reprogramming DONs with complex geometries into each other, using their distinct geometries to verify successful scaffold recycling. We reprogram one DON into a dynamic structure and show both pristine and recycled structures display similar properties. Second, we demonstrate the recovery of excess staple strands post-assembly and fold DONs with these recycled strands, showing these structures exhibit the expected geometry and dynamic properties. Finally, we demonstrate the combination of both approaches, successfully fabricating DONs solely from recycled DNA components.

bioengineering↗

bioRxiv2024

Piggybacking functionalized DNA nanostructures into live cell nuclei

DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.

bioengineering↗

bioRxiv2023

ATAC and SAGA histone acetyltransferase modules facilitate transcription factor binding to nucleosomes in an acetylation independent manner

Transcription initiation involves the coordination of multiple events, starting with activators binding specific DNA target sequences, which recruits transcription co-activators to open chromatin and enable binding of general transcription factors and RNA polymerase II to promoters. Two key human transcriptional coactivator complexes, ATAC (ADA-Two-A-Containing) and SAGA (Spt-Ada-Gcn5-acetyltransferase), target genomic loci to increase promoter accessibility. To better understand the function of ATAC and SAGA histone acetyltransferase (HAT) complexes, we used in vitro biochemical and biophysical assays to characterize human ATAC and SAGA HAT module interactions with nucleosomes and how a transcription factor (TF) coordinates these interactions. We found that ATAC and SAGA HAT modules bind nucleosomes with high affinity, independent of post-translational modifications (PTMs) and TFs. ATAC and SAGA HAT modules directly interact with the VP16 activator domain and a TF containing this domain enhances HAT module acetylation activity. Surprisingly, ATAC and SAGA HAT modules increase TF binding to its DNA target site within the nucleosome by an order of magnitude independent of histone acetylation. Altogether, our results reveal synergistic coordination between HAT modules and a TF, where ATAC and SAGA HAT modules: (i) acetylate histones to open chromatin, and (ii) facilitate TF targeting within nucleosomes independently of their acetylation activity.

molecular biology↗

bioRxiv2023

Cooperative control of a DNA origami force sensor

Most biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces. These techniques, however, are not optimized for studies in constrained or crowded environments as they typically require micron-scale beads in the case of magnetic or optical tweezers, or direct attachment to a cantilever in the case of atomic force microscopy. We implement a nanoscale force-sensing device using a DNA origami which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, functions as a binary (open or closed) force sensor that undergoes a structural transition under an external force. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). This actuation of the NanoDyn is reversible and the design parameters strongly influence the efficiency of resetting the initial state, with higher stability devices ({gtrsim}10 pN) resetting more reliably during repeated force-loading cycles. Finally, we show that the opening force can be adjusted in real time by the addition of a single DNA oligonucleotide. These results establish the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties.

bioengineering↗

bioRxiv2023

Mechanism of DNA origami folding elucidated by mesoscopic simulations

DNA nanotechnology leverages the canonical base-pairing rules and geometry of DNA to create highly precise nanoscale structures with many potential applications. While the design and fabrication of DNA nanostructures is well-established, the self-assembly process that produces these structures is still poorly understood, especially for DNA origami that involve the assembly of hundreds of strands. Many experimental and computational efforts have sought to better understand DNA origami folding, but the small length and time scales of individual binding events and the long timescale over which folding occurs have posed significant challenges. Here, we present a new mesoscopic model that uses a switchable force field to capture the mechanical behavior of single- and double-stranded DNA motifs and transition between them at a coarseness level of up to 8 nucleotides per particle, allowing access to the long assembly timescales of DNA origami up to several kilobases in size. Brownian dynamics simulations of 4-helix bundle (4HB) structures using this model reveal a hierarchical folding process involving the zipping of structural domains into a partially folded precursor structure followed by gradual crystallization into the final structure. We elucidate the role of hybridization strength, scaffold routing, and staple design in the folding order and kinetics. Simulation of larger 32HB structures reveals heterogeneous staple incorporation kinetics and frequent trapping in metastable states, as opposed to smaller, more accessible structures like the 4HB, which exhibit first-order kinetics and virtually defect-free folding. The development of this model opens an avenue to better understand and design DNA nanostructures for improved yield and folding performance.

biophysics↗

bioRxiv2023

Versatile Computer Aided Design of Freeform DNA Nanostructures and Assemblies

Recent advances in structural DNA nanotechnology have been facilitated by design tools that continue to push the limits of structural complexity while simplifying an often-tedious design process. We recently introduced the software MagicDNA, which enables design of complex 3D DNA assemblies with many components; however, the design of structures with freeform features like vertices or curvature still required iterative design guided by simulation feedback and user intuition. Here, we present an updated design tool, MagicDNA 2.0, that automates the design of freeform 3D geometries, leveraging design models informed by coarse-grained molecular dynamics simulations. Our GUI-based, stepwise design approach integrates a high level of automation with versatile control over assembly and sub-component design parameters. We experimentally validated this approach by fabricating a range of DNA origami assemblies with complex freeform geometries, including a 3D Nozzle, G-clef, and Hilbert and Trifolium curves, confirming excellent agreement between design input, simulation, and structure formation.

bioengineering↗

bioRxiv2022

Steric Communication between Dynamic Components on DNA nanodevices

Biomolecular nanotechnology has helped emulate basic robotic capabilities such as defined motion, sensing, and actuation in synthetic nanoscale systems. DNA origami is an attractive approach for nanorobotics, as it enables creation of devices with complex geometry, programmed motion, rapid actuation, force application, and various kinds of sensing modalities. Advanced robotic functions like feedback control, autonomy, or programmed routines also require the ability to transmit signals among sub-components. Prior work in DNA nanotechnology has established approaches for signal transmission, for example through diffusing strands or structurally coupled motions. However, soluble communication is often slow and structural coupling of motions can limit the function of individual components, for example to respond to the environment. Here, we introduce a novel approach inspired by protein allostery to transmit signals between two distal dynamic components through steric interactions. These components undergo separate thermal fluctuations where certain conformations of one arm will sterically occlude conformations of the distal arm. We implement this approach in a DNA origami device consisting of two stiff arms each connected to a base platform via a flexible hinge joint. We demonstrate the ability for one arm to sterically regulate both the range of motion as well as the conformational state (latched or freely fluctuating) of the distal arm, results that are quantitatively captured by mesoscopic simulations using experimentally informed energy landscapes for hinge-angle fluctuations. We further demonstrate the ability to modulate signal transmission by mechanically tuning the range of thermal fluctuations and controlling the conformational states of the arms. Our results establish a communication mechanism well-suited to transmit signals between thermally fluctuating dynamic components and provide a path to transmitting signals where the input is a dynamic response to parameters like force or solution conditions.

bioengineering↗

bioRxiv2022

DNA-caged Nanoparticles via Electrostatic Self-Assembly

DNA-modified nanoparticles enable DNA sensing and therapeutics in nanomedicine and are also crucial for nanoparticle self-assembly with DNA-based materials. However, methods to conjugate DNA to nanoparticle surfaces are limited, inefficient, and lack control. Inspired by DNA tile nanotechnology, we demonstrate a new approach to nanoparticle modification based on electrostatic attraction between negatively charged DNA tiles and positively charged nanoparticles. This approach does not disrupt nanoparticle surfaces and leverages the programmability of DNA nanotechnology to control DNA presentation. We demonstrated this approach using a variety of nanoparticles, including polymeric micelles, polystyrene beads, gold nanoparticles, and superparamagnetic iron oxide nanoparticles with sizes ranging from 5-20 nm in diameter. DNA cage formation was confirmed through transmission electron microscopy (TEM), neutralization of zeta potential, and a series of fluorescence experiments. DNA cages present "handle" sequences that can be used for reversible target attachment or self-assembly. Handle functionality was verified in solution, at the solid-liquid interface, and inside fixed cells, corresponding to applications in biosensing, DNA microarrays, and erasable immunocytochemistry. These experiments demonstrate the versatility of the electrostatic DNA caging approach and provide a new pathway to nanoparticle modification with DNA that will empower further applications of these materials in medicine and materials science.

bioengineering↗

bioRxiv2022

Thermally Reversible Pattern Formation in Arrays of Molecular Rotors

Control over the mesoscale to microscale patterning of materials is of great interest to the soft matter community. Inspired by DNA origami rotors, we introduce a 2D nearest-neighbor lattice of spinning rotors that exhibit discrete orientational states and interactions with their neighbors. Monte Carlo simulations of rotor lattices reveal that they exhibit a variety of interesting ordering behaviors and morphologies that can be modulated through rotor design parameters. The rotor arrays exhibit diverse patterns including closed loops, radiating loops, and bricklayer structures in their ordered states. They exhibit specific heat peaks at very low temperatures for small system sizes, and some systems exhibit multiple order-disorder transitions depending on inter-rotor interaction design. We devise an energy-based order parameter and show via umbrella sampling and histogram reweighting that this order parameter captures well the order-disorder transitions occurring in these systems. We fabricate real DNA origami rotors which themselves can order via programmable DNA base-pairing interactions and demonstrate both ordered and disordered phases, illustrating how rotor lattices may be realized experimentally and used for responsive organization. This work establishes the feasibility of realizing structural nanomaterials that exhibit locally mediated microscale patterns which could have applications in sensing and precision surface patterning.

biophysics↗

bioRxiv2022

Basic helix-loop-helix pioneer factors interact with the histone octamer to invade nucleosomes and generate nucleosome depleted regions

Nucleosomes drastically limit transcription factor (TF) occupancy, while pioneer transcription factors (PFs) somehow circumvent this nucleosome barrier. In this study, we compare nucleosome binding of two conserved S. cerevisiae basic helix-loop-helix (bHLH) TFs, Cbf1 and Pho4. A Cryo-EM structure of Cbf1 in complex with the nucleosome reveals that the Cbf1 HLH region can electrostatically interact with exposed histone residues within a partially unwrapped nucleosome. Single molecule fluorescence studies show that the Cbf1 but not the Pho4 HLH region facilitates efficient nucleosome invasion by slowing its dissociation rate relative to DNA through interactions with histones. In vivo studies show that this enhanced binding provided by the Cbf1 HLH region enables nucleosome invasion and ensuing repositioning. These structural, single molecule, and in vivo studies reveal the mechanistic basis of dissociation rate compensation by pioneer factors and how this translates to facilitating chromatin opening inside cells.

biophysics↗