Igal Szleifer

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 (HP1α), a key chromatin crosslinking factor with affinity to H3K9me3 marks, in maintaining packing domain organization. Paradoxically, short-term HP1α 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, HP1α-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 HP1α crosslinking as an essential component of domain maturation, as HP1α 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.

Confined spaces at the nanoscale ─ pores, channels, and cavities that alter molecular affinities and restrict molecular motion, orientation and local distribution ─ are emerging as a powerful design principle for next-generation materials. By tailoring the structure and functionality of these confined spaces, it is possible to control reactivity, transport, and material properties in ways that are unattainable in bulk systems. This Perspective highlights nanoconfinement as a unifying principle for programming function and addressing major societal challenges in energy, water, health and sustainability. Drawing inspiration from nature’s remarkable precision in bio-molecular control, we showcase six representative areas in which confined spaces are transforming technology: chromatography, catalysis, nanofluidics, bioinspired pore design, confinement-programmed material states, and data-driven material development. Together, these examples illustrate the power of nanoconfinement as an enabling unifying concept. By integrating orthogonal functionalization strategies, advanced characterization, modeling, and digitalization, programmable nanoconfinement will become a versatile framework for engineering materials and processes that meet the demands of a sustainable and technologically advanced society.

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. SIGNIFICANCE: Chromatin 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.

Human tissues require a mechanism to generate durable, yet modifiable, transcriptional memories to sustain cell function across a lifetime. Previously, it was 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 demonstrates 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, it is proposed that this can 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. How this organization will reconcile contradictions in epigenetic patterns, non‐randomness in oncogenic mutations, and produce durable transcriptional memory is illustrated. The study concludes by showing that this genome geometry may have coincided with the rapid evolution of body‐plan complexity, suggesting that chromatin geometry could be fundamental to metazoan evolution.

Cancer cells exhibit a remarkable resilience to cytotoxic stress, often adapting through transcriptional changes linked to alterations in chromatin structure. In several types of cancer, these adaptations involve epigenetic modifications and restructuring of topologically associating domains. However, the underlying principles by which chromatin architecture facilitates such adaptability across different cancers remain poorly understood. To investigate the role of chromatin in this process, we developed a physics-based model that connects chromatin organization to cell fate decisions, such as survival following chemotherapy. Our model builds on the observation that chromatin forms packing domains, which influence transcriptional activity through macromolecular crowding. The model accurately predicts chemoevasion in vitro, suggesting that changes in packing domains affect the likelihood of survival. Consistent results across diverse cancer types indicate that the model captures fundamental principles of chromatin-mediated adaptation, independent of the specific cancer or chemotherapy mechanisms involved. Based on these insights, we hypothesized that compounds capable of modulating packing domains, termed Transcriptional Plasticity Regulators (TPRs), could prevent cellular adaptation to chemotherapy. We conducted a proof-of-concept compound screen using live-cell chromatin imaging to identify several TPRs that synergistically enhanced chemotherapy-induced cell death. The most effective TPR significantly improved therapeutic outcomes in a patient-derived xenograft model of ovarian cancer. These findings underscore the central role of chromatin in cellular adaptation to cytotoxic stress and present a framework for enhancing cancer therapies, with broad potential across multiple cancer types.

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.

Understanding chromatin organization requires integrating measurements of genome connectivity and physical structure. It is well established that cohesin is essential for TAD and loop connectivity features in Hi-C, but the corresponding change in physical structure has not been studied using electron microscopy. Pairing chromatin scanning transmission electron tomography with multiomic analysis and single-molecule localization microscopy, we study the role of cohesin in regulating the conformationally defined chromatin nanoscopic packing domains. Our results indicate that packing domains are not physical manifestation of TADs. Using electron microscopy, we found that only 20% of packing domains are lost upon RAD21 depletion. The effect of RAD21 depletion is restricted to small, poorly packed (nascent) packing domains. In addition, we present evidence that cohesin-mediated loop extrusion generates nascent domains that undergo maturation through nucleosome posttranslational modifications. Our results demonstrate that a 3D genomic structure, composed of packing domains, is generated through cohesin activity and nucleosome modifications.

In single cells, variably sized nanoscale chromatin structures are observed, but it is unknown whether these form a cohesive framework that regulates RNA transcription. Here, we demonstrate that the human genome is an emergent, self-assembling, reinforcement learning system. Conformationally defined heterogeneous, nanoscopic packing domains form by the interplay of transcription, nucleosome remodeling, and loop extrusion. We show that packing domains are not topologically associated domains. Instead, packing domains exist across a structure-function life cycle that couples heterochromatin and transcription in situ, explaining how heterochromatin enzyme inhibition can produce a paradoxical decrease in transcription by destabilizing domain cores. Applied to development and aging, we show the pairing of heterochromatin and transcription at myogenic genes that could be disrupted by nuclear swelling. In sum, packing domains represent a foundation to explore the interactions of chromatin and transcription at the single-cell level in human health.

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, conformation, charge, and chemical state 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 Mg2+ 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.