Biology subjects

Almassalha, L. M.

Publications and source records attributed to Almassalha, L. M..

15 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↗

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

Leveraging chromatin packing domains to target chemoevasion in vivo

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 (TADs). 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 mechanistic model that connects chromatin organization to cell fate decisions, specifically survival following chemotherapy. Our model builds on the observation that chromatin forms packing domains, which influence transcriptional efficiency 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. Using live-cell chromatin imaging, we conducted a compound screen that identified several TPRs which synergistically enhanced chemotherapyinduced cell death. The most effective TPR significantly improved therapeutic outcomes in a patient-derived xenograft (PDX) model of ovarian cancer. These findings underscore the central role of chromatin in cellular adaptation to cytotoxic stress and present a novel framework for enhancing cancer therapies, with broad potential across multiple cancer types.

biophysics↗

bioRxiv2024

Nuclear blebs are associated with destabilized chromatin packing domains

Disrupted nuclear shape is associated with multiple pathological processes including premature aging disorders, cancer-relevant chromosomal rearrangements, and DNA damage. Nuclear blebs (i.e., herniations of the nuclear envelope) have been induced by (1) nuclear compression, (2) nuclear migration (e.g., cancer metastasis), (3) actin contraction, (4) lamin mutation or depletion, and (5) heterochromatin enzyme inhibition. Recent work has shown that chromatin transformation is a hallmark of bleb formation, but the transformation of higher-order structures in blebs is not well understood. As higher-order chromatin has been shown to assemble into nanoscopic packing domains, we investigated if (1) packing domain organization is altered within nuclear blebs and (2) if alteration in packing domain structure contributed to bleb formation. Using Dual-Partial Wave Spectroscopic microscopy, we show that chromatin packing domains within blebs are transformed both by B-type lamin depletion and the inhibition of heterochromatin enzymes compared to the nuclear body. Pairing these results with single-molecule localization microscopy of constitutive heterochromatin, we show fragmentation of nanoscopic heterochromatin domains within bleb domains. Overall, these findings indicate that translocation into blebs results in a fragmented higher-order chromatin structure. SUMMARY STATEMENTNuclear blebs are linked to various pathologies, including cancer and premature aging disorders. We investigate alterations in higher-order chromatin structure within blebs, revealing fragmentation of nanoscopic heterochromatin domains.

bioengineering↗

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↗

bioRxiv2024

Chromatin packing domains persist after RAD21 depletion in 3D.

Understanding chromatin organization requires integrating measurements of genome connectivity and physical structure. Prior work demonstrates that RAD21 depletion results in the complete loss of topologically associated and loop domains on Hi-C, but the corresponding change in physical structure has not been studied using electron microscopy. Pairing chromatin scanning transmission electron tomography with Hi-C, we study the role of cohesin in regulating the spatially resolved, conformationally defined chromatin packing domains. We find that only 20% of packing domains are lost on electron microscopy upon RAD21 depletion with the effect primarily on small, poorly packed (nascent) domains. Overall, this contrasts with the prevailing understanding of genome regulation, indicating that while cohesin influences domain formation, non-cohesin mediated mechanisms predominantly regulate the 3D genomic physical structure.

genomics↗

bioRxiv2023

Differentiation-dependent chromosomal organization changes in normal myogenic cells are absent in rhabdomyosarcoma cells

Myogenesis, the progression of proliferating skeletal myoblasts to terminally differentiated myotubes, regulates thousands of target genes. Uninterrupted linear arrays of such genes are differentially associated with specific chromosomes, suggesting chromosome specific regulatory roles in myogenesis. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle, shares common features with normal muscle cells. We hypothesized that RMS and myogenic cells possess differences in chromosomal organization related to myogenic gene arrangement. We compared the organizational characteristics of chromosomes 2 and 18, chosen for their difference in myogenic gene arrangement, in cultured RMS cell lines and normal myoblasts and myotubes. We found chromosome-specific differences in organization during normal myogenesis, with increased area occupied and a shift in peripheral localization specifically for chromosome 2. Most strikingly, we found a differentiation-dependent difference in positioning of chromosome 2 relative to the nuclear axis, with preferential positioning along the major nuclear axis present only in myotubes. RMS cells demonstrated no preference for such axial positioning, but induced differentiation through transfection of the pro-myogenic miRNA miR-206 resulted in an increase of major axial positioning of chromosome 2. Our findings identify both a differentiation-dependent, chromosome-specific change in organization in normal myogenesis, and highlight the role of chromosomal spatial organization in myogenic differentiation.

cell biology↗

bioRxiv2023

Active Transcription and Epigenetic Reactions Synergistically RegulateMeso-Scale Genomic Organization

In interphase nuclei, chromatin is organized into interspersed dense domains with characteristic sizes, both in the nuclear interior and periphery. However, the quantitative impact of transcription and histone modifications on the size and distribution of these domains remains unclear. Here, we introduce a mesoscale theoretical model that investigates the relationship between heterochromatic domain sizes and loop extrusion rates from these domains. The model considers chromatin-chromatin and chromatin-lamina interactions, methylation and acetylation kinetics, and diffusion of epigenetic marks and nucleoplasm. Our model generates testable predictions that help reveal the biophysics underlying chromatin organization in the presence of transcription-driven loop extrusion. This process is kinetically captured through the conversion of heterochromatin to euchromatin in response to RNAPII activity. We discovered that a balance between diffusive and reactive fluxes governs the steady-state sizes of heterochromatin domains. Using theory and simulations, we predicted that a loss of transcription results in increased chromatin compaction and larger heterochromatin domain sizes. To validate our predictions, we employed complementary super-resolution and nano-imaging techniques on five different cell lines with impaired transcription. We quantitatively assessed how domain sizes scale with loop extrusion rates at the hetero-euchromatin interfaces. Our analysis of previously obtained super-resolution images of nuclei revealed that excessive loop extrusion leads to smaller heterochromatin domains. The model successfully recapitulated these observations, explaining how transcription loss can counteract the effects of cohesin overloading. As the general biophysical mechanisms regulating heterochromatin domain sizes are independent of cell type, our findings have significant implications for understanding the role of transcription in global genome organization.

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

Quantifying Three-dimensional Chromatin Organization Utilizing Scanning Transmission Electron Microscopy: ChromSTEM

Chromatin organization over a wide range of length scales plays a critical role in the regulation of gene expression and deciphering these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein we introduce ChromSTEM, a method which utilizes high angle annular dark field imaging and tomography in scanning transmission electron microscopy in combination with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and tissue biopsies through local DNA distribution and the scaling behavior of chromatin polymer. We observed that chromatin is densely packed with an average volume concentration of over 30% with heterochromatin having a two-fold higher density compared to euchromatin. Chromatin was arranged into spatially well-defined nanoscale packing domains with fractal internal structure and genomic size between 100 and 400 kb, comparable to that of topologically associated domains. The packing domains varied in DNA concentration and fractal dimension and had one of the distinct states of chromatin packing with differential ratio of DNA content to the chromatin volume concentration. Finally, we observed a significant intercellular heterogeneity of chromatin organization even within a genetically uniform cell population, which demonstrates the imperative for high-throughput characterization of chromatin structure at the single cell level.

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↗

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

Physicochemical mechanotransduction alters nuclear shape and mechanics via heterochromatin formation

The nucleus houses, organizes, and protects chromatin to ensure genome integrity and proper gene expression, but how the nucleus adapts mechanically to changes in the extracellular environment is poorly understood. Recent studies have revealed that extracellular chemical or physical stresses induce chromatin compaction via mechanotransductive processes. We report that increased extracellular multivalent cations lead to increased heterochromatin levels through mechanosensitive ion channels. This increase in heterochromatin results in increased chromatin-based nuclear rigidity, which suppresses nuclear blebbing in cells with perturbed chromatin or lamins. Furthermore, transduction of elevated extracellular cations rescues nuclear morphology in model and patient cells of human diseases, including progeria and the breast cancer model cell line MDA-MB-231. We conclude that nuclear mechanics and morphology, including abnormal phenotypes found in human diseases, can be modulated by cell sensing of the extracellular environment and consequent changes to histone modification state and chromatin-based nuclear rigidity, without requiring direct mechanical perturbations to the cell interior.

cell biology↗

bioRxiv2018

Spectral Contrast Optical Coherence Tomography Angiography Enables Single-Scan Vessel Imaging

Optical coherence tomography angiography relies on motion for contrast and requires at least two data acquisitions per pointwise scanning location. We present a method termed spectral contrast optical coherence tomography angiography using visible light that relies on the spectral signatures of blood for angiography from a single scan using endogenous contrast. We demonstrate the molecular sensitivity of this method, which enables lymphatic vessel, blood, and tissue discrimination.

biophysics↗

bioRxiv2017

Chromatin histone modifications and rigidity affect nuclear morphology independent of lamins

Nuclear shape and architecture influence gene localization, mechanotransduction, transcription, and cell function. Abnormal nuclear morphology and protrusions termed \"blebs\" are diagnostic markers for many human afflictions including heart disease, aging, progeria, and cancer. Nuclear blebs are associated with both lamin and chromatin alterations. A number of prior studies suggest that lamins dictate nuclear morphology, but the contributions of altered chromatin compaction remain unclear. We show that chromatin histone modification state dictates nuclear rigidity, and modulating it is sufficient to both induce and suppress nuclear blebs. Treatment of mammalian cells with histone deacetylase inhibitors to increase euchromatin or histone methyltransferase inhibitors to decrease heterochromatin results in a softer nucleus and nuclear blebbing, without perturbing lamins. Oppositely, treatment with histone demethylase inhibitors increases heterochromatin and chromatin nuclear rigidity, which results in reduced nuclear blebbing in lamin B1 null nuclei. Notably, increased heterochromatin also rescues nuclear morphology in a model cell line for the accelerated aging disease Hutchinson-Gilford progeria syndrome caused by mutant lamin A, as well as cells from patients with the disease. Thus, chromatin histone modification state is a major determinant of nuclear blebbing and morphology via its contribution to nuclear rigidity.

cell biology↗