Biology subjects

Levine, Z. A.

Publications and source records attributed to Levine, Z. A..

6 recordsLinked to original sources

bioRxiv2026

Targeted ATAC-see (tATAC-see): A Visual Assay for Target-Specific Chromatin Profiling

We present targeted ATAC-see (tATAC-see), a visual genomics assay for site-specific chromatin profiling. By integrating the in situ visualization of ATAC-see with antibody-tethered tagmentation, tATAC-see captures chromatin states at defined protein-occupied domains with the simplicity of standard immunofluorescence. Modulating the spatial interaction radius of Tn5 via salt titration enables extended tagmentation of local chromatin environments near the target. We validated this imaging-based method by capturing the expanded chromatin neighborhoods of active euchromatin (H3K27ac, H3K4me3) alongside the dense structural lamina-associated domains (LADs) of lamin A/C and lamin B1. Using an HDAC inhibitor, we tested the assays sensitivity to detect dynamic structural remodeling. We not only tracked chromatin decompaction but also demonstrated the robust structural resistance of LADs. As a proof-of-principle biological application, we applied tATAC-see to models of replicative, chronological, and pathological (Hutchinson-Gilford progeria syndrome) aging to assess its ability to detect well-characterized peripheral heterochromatin and LAD remodeling. We observed divergent chromatin trajectories at the nuclear envelope, reflective of the lamins distinct roles. lamin B1 domains exhibit increased local accessibility consistent with age-associated heterochromatin erosion, while lamin A/C-associated domains physically detach from their scaffold. Ultimately, tATAC-see provides a robust, accessible platform for mechanistic and population-level studies to uncover spatial epigenome dynamics. Summary StatementtATAC-see is a tunable visual genomics assay that can capture target-specific chromatin remodeling across diverse biological contexts.

cell biology↗

bioRxiv2026

Visualizing the Epigenetic Landscape of Aging and Cellular Reprogramming: Optimized ATAC-see for Cells and Tissues

Spatial chromatin organization dictates cellular function and resilience, yet scalable imaging methods to quantify chromatin states in situ across aging and interventions are lacking. While ATAC-see can visualize accessible chromatin, its broader application is hindered by protocol variability, low throughput, and incompatibility with complex tissues. Here, we systematically optimize the ATAC-see workflow for robust, high-throughput quantitative imaging in fixed, adherent mammalian cells and fresh frozen tissues. We validate the platforms sensitivity to pharmacologic remodeling and apply it to replicative, chronological, and pathological aging in primary human fibroblasts, revealing progressive age-associated chromatin opening and heterochromatin remodeling. Furthermore, we demonstrate that our optimized ATAC-see captures rapid, reversible chromatin reorganization during OSK(M)-driven partial reprogramming of aged fibroblasts. Finally, we extend a cost-effective and accessible protocol to murine tissue sections, quantifying in situ age-dependent remodeling. This standardized framework establishes chromatin accessibility as a highly scalable, sequencing-compatible imaging biomarker for evaluating aging and rejuvenation. Summary StatementATAC-see was optimized for scalable, quantitative imaging of chromatin remodeling during aging and cellular reprogramming, and extended to characterize age-associated epigenetic changes across organs.

cell biology↗

bioRxiv2026

Inhibition of ApoE4 Endocytosis with LDLR-LA Peptides

The Apolipoprotein E4 (ApoE4) genotype is the most significant genetic risk factor for late-onset Alzheimers disease (AD). A key driver of ApoE4 cellular toxicity is the endo-lysosomal burden resulting from the excessive receptor-mediated uptake of ApoE4 lipoparticles. The high-affinity interaction between lipidated ApoE4 and the Low-Density Lipoprotein Receptor (LDLR) saturates the cellular degradation machinery, correlating with lysosomal alkalinization, lipid accumulation, and cell death. To target this critical interaction interface, which consists of 7 tandem ligand-binding type-A (LA) modules in the human LDLR, we present the design and evaluation of recombinant LDLR minireceptors comprising combinations of these LA modules to competitively antagonize ApoE4 endocytosis. We observe a distinct isoform-dependent uptake dynamic across multiple central nervous system (CNS) cell models, with ApoE4 showing significantly greater total intracellular accumulation than ApoE2. Furthermore, engineered LA peptides selectively bind ApoE4 over human serum LDL and differentially inhibit its uptake, revealing a distinct structural efficacy hierarchy of LA3456 [~] LA345 > LA456 [~] LA45 >> LA34. We establish the resilience of the LA45 minireceptor under physiological serum conditions and identify LA345 as the most stable truncated construct in vitro. Notably, molecular tagging orientation is critical for therapeutic engineering; C-terminal tagging completely preserves the inhibitory function of the minireceptors, whereas N-terminal tagging drastically reduces it. These findings provide a framework for scalable, deliverable inhibition of the ApoE4-LDLR interaction as a potential therapeutic target to mitigate endo-lysosomal accumulation in AD.

biochemistry↗

bioRxiv2025

ApoE lipidation, not isoform, is the key modulator of Aβ interaction, uptake, and cytotoxicity

BackgroundInherited variations in the Apolipoprotein E (APOE) gene are the largest genetic determinant for late-onset Alzheimers Disease, with the APOE{varepsilon}4 allele conferring the highest risk. While APOE was shown to modulate amyloid beta (A{beta}) pathology in a genotype-specific manner (APOE{varepsilon}4>APOE{varepsilon}3>APOE{varepsilon}2), it remains an important open question whether these differences are due directly to isoform-specific interactions between ApoE and A{beta} or indirect effects on A{beta} clearance. This is further complicated because ApoE exists in lipidated or unlipidated states, which influence its biochemical properties. To disentangle how single ApoE mutations confer vastly different effects on A{beta} pathology, we investigated how both the ApoE isoform and lipidation modulate its interaction with inert and cytotoxic A{beta} species, and its effect on A{beta} uptake and cytotoxicity in human astrocytes. MethodsWe prepared A{beta} in distinct aggregation states (monomers, oligomers, and fibrils) and ApoE in different lipidation states to characterize their size and affinity for one another using fluorescence correlation spectroscopy and fluorescence polarization, respectively. We then utilized flow cytometry and live cell imaging to quantify the uptake of A{beta} in different aggregation states by primary and immortalized human astrocytes in the presence of different isoforms of lipidated or unlipidated ApoE. ResultsThis study revealed that ApoE lipidation, not isoform, has the biggest impact on its interaction with A{beta}, on the uptake of A{beta} by astrocytes, and on A{beta}-induced cytotoxicity. Specifically, unlipidated ApoE preferentially interacts with A{beta} oligomers and fibrils, which substantially inhibits their uptake by astrocytes. Conversely, lipidated ApoE showed no interaction with A{beta} oligomers and had a reduced ability to inhibit A{beta} uptake. ConclusionsOur observations provide important molecular details that suggest previously observed ApoE isoform-specific differences in AD risk are likely driven primarily by in vivo differences in ApoE lipidation, rather than biophysical differences between ApoE isoforms in their interaction with A{beta}. We propose that the impaired lipidation of ApoE4 in the brain increases levels of unlipidated ApoE, which then bind toxic A{beta} oligomers and reduce their clearance by astrocytes. These findings underscore the therapeutic potential of interventions aimed at increasing ApoE lipidation and decreasing interactions between toxic A{beta} oligomers and ApoE.

biophysics↗

bioRxiv2025

Consensus DNA Inhibits p53 Aggregation but Fails to Rescue Mutants

The tumor suppressor p53 plays a crucial role in regulating gene expression under cellular stress. Somatic mutations to its DNA-binding domain are common in lethal cancers and lead to lost regulatory function and pathological hallmarks, such as misfolded p53 aggregates and amyloid-like fibrils. Despite decades of these observations, the molecular determinants driving p53 aggregation and its role in cancer incidence, progression, and lethality remain poorly defined. Identifying these determinants, however, is critical for developing therapeutic interventions that modulate protein solubility in cancer and understanding disparities in cancer outcomes. Therefore, we investigated whether consensus DNA sequences that stabilize wild-type p53 are sufficient to regulate the aggregation of oncogenic p53 mutants with reduced binding affinities. Using a combination of probe-based and label-free spectroscopic and microscopic techniques, we examined how consensus (p21, Bax) and non-consensus (p21-scramble, Poly-GC) DNA oligonucleotides regulate the aggregation of wild-type p53 in comparison to three cancer-associated mutants (R248Q, R273H, and R175H). We find that equimolar p21 consensus sequences significantly inhibits wild-type p53 aggregation, while other DNA sequences do not. In contrast, oncogenic p53 mutants evaded DNA regulation of protein aggregation, and some oligonucleotides even enhanced aggregation at low concentrations, which suggests concentration-dependent DNA-p53 interactions. These findings emphasize that DNA response elements are sufficient for regulating wild-type p53 aggregation in solution. However, key somatic mutations in cancer promote aggregation at the expense of DNA-binding, directly leading to the loss of p53 solubility through biochemical interactions. Taken together, these observations suggest that potent regulators of p53 aggregation should aim to restore affinity between oncogenic p53 mutants and regulatory DNA to minimize the pathological hallmarks of lethal tumors. Significance StatementOur research investigates whether DNA sequences that bind tumor suppressor protein p53 regulate its aggregation behavior in wild-type and somatically mutated forms linked to cancer. While specific DNA sequences regulate p53 aggregation, protein mutations that reduce affinity for consensus DNA or destabilize p53s conformation result in persistent, likely dysfunctional aggregates, even in the presence of regulatory nucleotides. Unsurprisingly, p53 and its mutants exhibit increased aggregation when there is substantially more protein to DNA, suggesting insufficient regulatory DNA or excess unregulated p53 may inevitably lead to pathological aggregation. These findings clarify our understanding of p53s molecular behavior and suggest that cancer-linked somatic mutations enable p53 to evade DNAs regulatory effects and subsequently aggregate, leading to downstream losses in function.

biophysics↗

bioRxiv2024

Drugging Disordered Proteins by Conformational Selection to Inform Therapeutic Intervention

Drugging intrinsically disordered proteins (IDPs) has historically been a major challenge due to their lack of stable binding sites, conformational heterogeneity, and rapid ability to self-associate or bind non-specific neighbors. Furthermore, it is unclear whether binders of disordered proteins i) induce entirely new conformations or ii) target transient pre-structured conformations via stabilizing existing states. To distinguish between these two mechanisms, we utilize molecular dynamics simulations to induce structured conformations in islet amyloid polypeptide (IAPP), a disordered endocrine peptide implicated in Type II Diabetes. Using umbrella sampling, we measure conformation-specific affinities of molecules previously shown to bind IAPP to determine if they can discriminate between two distinct IAPP conformations (fixed in either -helix or {beta}-sheet). We show our two-state model of IAPP faithfully predicts the experimentally observed selectivity of two classes of IAPP binders while revealing differences in their molecular mechanisms of binding. Specifically, the binding preferences of foldamers designed for human IAPP was not fully accounted for by conformational selection, unlike {beta}-breaking peptides designed to mimic IAPP self-assembly sequences. Furthermore, the binding of these foldamers, but not {beta}-breaking peptides, was disrupted by changes in the rat IAPP sequence. Taken together, our data quantifies the sequence and conformational specificity for IAPP binders and reveals conformational selection sometimes overrides sequence-level specificity. This work highlights the important role of conformational selection in stabilizing IDPs, and it reveals how fixed conformations can provide a tractable target for developing disordered protein binders.

biophysics↗