Biology subjects

Szleifer, I.

Publications and source records attributed to Szleifer, I..

12 recordsLinked to original sources

bioRxiv2026

Heterochromatin protein 1α acts as an essential stabilizer of chromatin packing domains

Background: The spatial organization of the human genome emerges from an enzyme-guided self-assembly process in which chromatin has a bidirectional relationship with transcription. Central to this architecture are chromatin packing domains, 50-200 nm structures exhibiting mass-scaling behavior. Nascent domains arise through transcription and cohesin/CTCF-mediated loop extrusion, while their maturation depends on conditions that permit differential access of epigenetic enzymes to their dense cores and continuously lower density periphery, resulting in the accumulation of heterochromatic marks in domain cores and euchromatic marks outside of the domain cores. In turn, packing domains support transcription by creating an "ideal zone" peripherally to their cores due to a nonmonotonic dependence of transcription rate on local chromatin density. Results: We investigated the role of heterochromatin protein 1 alpha (HP1), a key chromatin crosslinking factor with affinity to H3K9me3 marks, in maintaining packing domain organization. Paradoxically, short-term HP1 degradation resulted in overall downregulation of genes, contrasting with the canonical view that repression of heterochromatin allows chromatin to become more accessible for transcription. While some packing domains persisted, heterochromatic cores of dense packing domains were disrupted. When exposed to hypoxia, HP1-depleted cells exhibited a dampened transcriptional response, showcasing that when domain maturation is disrupted, stress response pathways are not effectively activated. Conclusions: This work highlights HP1 crosslinking as an essential component of domain maturation, as HP1 loss disrupts domain geometry and inhibits efficient transcriptional reprogramming. Our findings suggest an intriguing perspective on the potential relationship between chromatin architecture and gene regulation that could inform chromatin engineering-based treatments.

genomics↗

bioRxiv2026

One Chromatin, Many Structures: From Ensemble Contact Maps to Single-Cell 3D Organization

Understanding how chromatin folds in three dimensions remains challenging because most experimental assays capture low-dimensional projections of an underlying, highly heterogeneous polymer. Here we present an ensemble-based interpretive framework built on the previously introduced Self-Returning Excluded Volume (SR-EV) model, a minimal generator of nucleosome-resolution chromatin conformations based on stochastic return rules and excluded-volume geometry. Despite its simplicity, SR-EV reproduces key experimental signatures across scales: heterogeneous nanoscale packing domains resembling ChromEMT and ChromSTEM observations, sparse and highly variable single-configuration contact patterns analogous to single-cell chromosome conformation capture (Hi-C), and robust ensemble-level contact enrichment consistent with topologically associating domains (TADs). In this framework, Hi-C loop and TAD signatures are interpreted as ensemble-level statistical enrichments rather than invariant features of single-cell conformations. SR-EV is explicitly designed to generate large ensembles of complete three-dimensional chromatin configurations that can be projected consistently onto two-dimensional contact maps and one-dimensional genomic profiles. By introducing architectural-protein effects only through ensemble selection rather than explicit forces, SR-EV supports a separation between intrinsic polymer geometry and regulatory bias and suggests that TAD-like features can emerge as statistical enrichments rather than deterministic three-dimensional structures. Coordination number and probe-based accessibility computed directly from SR-EV provide a unified link between three-dimensional packing, two-dimensional contact maps, and one-dimensional genomic profiles. Together, these results establish SR-EV as a minimal and physically grounded reference framework for interpreting how heterogeneous chromatin ensembles give rise to multimodal experimental observables, while remaining consistent with the fact that chromatin organization is realized in individual cells. SIGNIFICANCEChromatin domains, boundaries, and contact enrichments are often interpreted as fixed structural entities, even though most experimental measurements average over large and heterogeneous cell populations. The SR-EV framework shows that many of these features can be understood as emerging from minimal geometric rules combined with ensemble-level bias, without requiring explicit molecular interactions or deterministic folding mechanisms. By distinguishing single-configuration heterogeneity from ensemble-level statistical organization - including the emergence of packing domains- SR-EV supports an interpretation in which chromatin organization is realized in individual cells but must be analyzed through ensembles. This perspective clarifies the probabilistic nature of genome architecture and provides a tractable reference framework for interpreting multimodal genomic and imaging data.

biophysics↗

bioRxiv2025

Geometrically encoded positioning of introns, intergenic segments, and exons in the human genome

Human tissues require a mechanism to generate durable, yet modifiable, transcriptional memories to sustain cell function across a lifetime. Previously, we demonstrated that nanoscale packing domains couple heterochromatin (cores) and euchromatin (outer zone) into unified reaction volumes that can generate transcriptional memory. In prior work, this framework demonstrated that RNA synthesis occurred within the ideal zone (intermediate density) portions of the domain. Naturally, this creates a question of where genes are positioned in relation to the packing domain architecture and which genetic material fills the domain core to sustain transcription. Here we propose that this could be solved by the encoded positioning of introns, intergenic segments, and exons as a projection of the functional packing layers of domains. This suggests that introns and intergenic segments are coupled to adjacent exons to generate coherent packing domain volumes. We illustrate how this organization would reconcile contradictions in epigenetic patterns, non-randomness in oncogenic mutations, and produce durable transcriptional memory. We conclude by showing that this genome geometry might have coincided with the rapid evolution of body-plan complexity, suggesting that chromatin geometry could be fundamental to metazoan evolution.

biophysics↗

bioRxiv2024

The Impact of Charge Regulation and Ionic Intranuclear Environment on the Nucleosome Core Particle

We theoretically investigate how the intranuclear environment influences the charge of a nucleosome core particle (NCP) - the fundamental unit of chromatin consisting of DNA wrapped around a core of histone proteins. The molecular-based theory explicitly considers the size, shape, conformations, charges, and chemical states of all molecular species - thereby linking the structural state with the chemical/charged state of the system. We investigate how variations in monovalent and divalent salt concentrations, as well as pH, affect the charge distribution across different regions of an NCP and quantify the impact of charge regulation. The effective charge of an NCP emerges from a delicate and complex balance involving the chemical dissociation equilibrium of the amino acids and the DNA-phosphates, the electrostatic interaction between them, and the translational entropy of the mobile solution ions, i.e., counter ion release and ion condensation. From our results, we note the significant effect of divalent magnesium ions on the charge and electrostatic energy as well as the counterion cloud that surrounds an NCP, as a function of magnesium concentration, charge neutralization, and even charge inversion is predicted - in line with experimental observation of NCPs. The strong Mg-dependence of the nucleosome charge state arises from ion bridges between two DNA-phosphates and one Mg + ion. We demonstrate that to describe and predict the charged state of an NCP properly, it is essential to consider molecular details, such as DNA-phosphate ion condensation and the acid-base equilibrium of the amino acids that comprise the core histone proteins.

biophysics↗

bioRxiv2024

Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure

As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques.

biophysics↗

bioRxiv2020

Nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform bridges 4-D chromatin organization with molecular function

In eukaryotic cells, chromatin structure is linked to transcription processes through the regulation of genome organization. Extending across multiple length-scales - from the nucleosome to higher-order three-dimensional structures - chromatin is a dynamic system which evolves throughout the lifetime of a cell. However, no individual technique can fully elucidate the behavior of chromatin organization and its relation to molecular function at all length- and timescales at both a single-cell and a cell population level. Herein, we present a multi-technique nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform that bridges electron tomography and optical superresolution imaging of chromatin conformation and transcriptional processes, with resolution down to the level of individual nucleosomes, with high-throughput, label-free analysis of chromatin packing and its dynamics in live cells. Utilizing nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nm, sub-Mb genomic size, and an internal fractal structure. The chromatin packing behavior of these domains is directly influenced by active gene transcription. Furthermore, we demonstrated that the chromatin packing domain structure is correlated among progenitor cells and all their progeny, indicating that the organization of chromatin into fractal packing domains is heritable across cell division. Further studies employing the nano-ChIA platform have the potential to provide a more coherent picture of chromatin structure and its relation to molecular function.

genomics↗

bioRxiv2019

Physical and data structure of 3D genome

With the textbook view of chromatin folding based on the 30nm fiber being challenged, it has been proposed that interphase DNA has an irregular 10nm nucleosome polymer structure whose folding philosophy is unknown. Nevertheless, experimental advances suggested that such irregular packing is associated with many nontrivial physical properties that are puzzling from a polymer physics point of view. Here, we show that the reconciliation of these exotic properties necessitates modularizing 3D genome into tree data structures on top of, and in striking contrast to the linear topology of DNA double helix. Such functional modules need to be connected and isolated by an open backbone that results in porous and heterogeneous packing in a quasi-self-similar manner as revealed by our electron and optical imaging. Our multi-scale theoretical and experimental results suggest the existence of higher-order universal folding principles for a disordered chromatin fiber to avoid entanglement and fulfill its biological functions.

biophysics↗

bioRxiv2019

Molecular model of the nuclear pore complex reveals a thermoreversible FG-network with distinct territories occupied by different FG motifs

Despite the intensive study of the nuclear pore complex (NPC), its functional core, the central transporter, remains poorly understood. Here, we investigate this unfolded and dynamic part of the NPC using a molecular theory that addresses both entropic and enthalpic effects of the intrinsically disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Our model shows that the cooperative effect of FG-pairing, specific spacer cohesion, and charge interaction leads to a remarkably elaborate gating structure inside the NPC. In particular, we find sequence-programmed \"phase separation\" between charge-rich and charge-poor regions, and a polarized electrostatic potential throughout the NPC. The model predicts a thermoreversible FG-network with inhomogeneous FG-pairing fraction in space, which features distinct territories of different types of FG motifs. Our theoretical anatomy of the central transporter reveals a clear sequence-structure-function relationship of the FG-Nups, and explains in a self-consistent way how nucleocytoplasmic transport can be efficient yet specific.

cell biology↗

bioRxiv2019

Classification of RNA backbone conformation into rotamers using 13C' chemical shifts: How far we can go?

The conformational space of the ribose-phosphate backbone is very complex as is defined in terms of six torsional angles. To help delimit the RNA backbone conformational preferences 46 rotamers have been defined in terms of the these torsional angles. In the present work, we use the ribose experimental and theoretical 13C' chemical shifts data and machine learning methods to classify RNA backbone conformations into rotamers and families of rotamers. We show to what extent the use of experimental 13C' chemical shifts can be used to identify rotamers and discuss some problem with the theoretical computations of 13C' chemical shifts.

biophysics↗

bioRxiv2018

Multimodal interferometric imaging of nanoscale structure and macromolecular motion uncovers UV induced cellular paroxysm

We present a multimodal label-free interferometric imaging platform for measuring intracellular nanoscale structure and macromolecular dynamics in living cells with a sensitivity to macromolecules as small as 20nm and millisecond temporal resolution. We validate this system by pairing experimental measurements of nanosphere phantoms with a novel interferometric theory. Applying this system in vitro, we explore changes in higher-order chromatin structure and dynamics that occur due to cellular fixation, stem cell differentiation, and ultraviolet (UV) light irradiation. Finally, we discover a new phenomenon, cellular paroxysm, a near-instantaneous, synchronous burst of motion that occurs early in the process of UV induced cell death. Given this platforms ability to obtain nanoscale sensitive, millisecond resolved information within live cells without concerns of photobleaching, it has the potential to answer a broad range of critical biological questions about macromolecular behavior in live cells, particularly about the relationship between cellular structure and function.

biophysics↗

bioRxiv2018

Interphase chromatin as a self-returning random walk: Can DNA fold into liquid trees?

We introduce a self-returning random walk to describe the structure of interphase chromatin. Based on a simple folding algorithm, our de novo model unifies the high contact frequency discovered by genomic techniques, and the high structural heterogeneity revealed by imaging techniques, which two chromatin properties we theoretically prove to be irreconcilable within a fractal polymer framework. Our model provides a holistic view of chromatin folding, in which the topologically associated domains are liquid-tree-like structures, linked and isolated by stretched-out, transcriptionally active DNA to form a secondary structure of chromatin that further folds into a \"3D forest\" under confinement.

biophysics↗

bioRxiv2018

Preservation of cellular nano-architecture by the process of chemical fixation for nanopathology

Transformation in chromatin organization is one of the most universal markers of carcinogenesis. Microscale chromatin alterations have been a staple of histopathological diagnosis of neoplasia, and nanoscale alterations have emerged as a promising marker for cancer prognostication and the detection of predysplastic changes. While numerous methods have been developed to detect these alterations, most methods for sample preparation remain largely validated via conventional microscopy, and have not been examined with nanoscale sensitive imaging techniques. For these nanoscale sensitive techniques to become standard of care screening tools, new histological protocols must be developed that preserve nanoscale information. Partial Wave Spectroscopic (PWS) microscopy has recently emerged as a novel imaging technique sensitive to length scales ranging between 20 and 200 nanometers. As a label-free, high-throughput, and non-invasive imaging technique, PWS microscopy offers many advantages for risk stratification of early cancer, and is an ideal tool to quantify structural information during sample preparation. Therefore, in this work we applied PWS microscopy to systematically evaluate the effects of cytological preparation on the nanoscales changes of chromatin using two cell line models: Hela cells differentially treated with daunorubicin and TP53 differentially mutated ovarian carcinoma cells. Notably, we show that existing cytological preparation can be modified in order to maintain clinically relevant nanoscopic differences, paving the way for the emerging field of nanopathology.

biophysics↗