Biology subjects

Di Stefano, M.

Publications and source records attributed to Di Stefano, M..

15 recordsLinked to original sources

bioRxiv2026

Albumin-coated VS1 nanocrystals enable STARD3 inhibition and potentiate fluoropyrimidine therapy in colorectal cancer

Poor aqueous solubility remains a major obstacle to the translational development of targeted anticancer compounds. VS1, a first-in-class inhibitor of the cholesterol-transfer protein STARD3, has emerged as a promising chemosensitizing agent in colorectal cancer (CRC), but its clinical applicability is limited by its poor water solubility. Here, we combine structural biology, nanotechnology, and functional pharmacology to establish STARD3 inhibition as a delivery-enabled strategy to potentiate fluoropyrimidine therapy. To define the molecular basis of STARD3 inhibition, we solved the crystal structure of VS1 bound to the STARD3 ligand-binding domain at 2.1 [A] resolution, revealing direct occupation of the sterol-binding cavity. Molecular dynamics simulations confirmed a stable binding mode and identified the {Omega}1 loop as a dynamic gate regulating ligand binding and dissociation. To overcome the formulation barrier of VS1, we engineered carrier-free, albumin-coated nanocrystals through sonication-assisted nanocrystallization followed by surfactant exchange with human serum albumin. The resulting rod-shaped nanocrystals displayed nanometric size, narrow size distribution, sustained release, and improved aqueous dispersibility, increasing the apparent solubility of VS1 by more than 14-fold while preserving its molecular integrity and crystallinity. Biologically, VS1 selectively potentiated 5-fluorouracil (5-FU) in CRC cells, with synergistic effects restricted to 5-FU-sensitive models and associated with enhanced reactive oxygen species accumulation. Albumin-coated formulation retained the chemosensitizing activity of the free compound. In HCT-116 xenografts, combined treatment with albumin-coated VS1 nanocrystals and 5-FU significantly reduced tumor growth, prolonged tumor doubling time, and increased intratumoral necrosis without exacerbating systemic toxicity. Together, these findings establish that albumin-coated nanocrystals can overcome the delivery limitations of an insoluble STARD3 inhibitor and provide a formulation-enabled strategy to enhance fluoropyrimidine therapy in colorectal cancer. HIGHLIGHTSO_LIA 2.1 [A] crystal structure shows VS1 in the STARD3 sterol-binding cavity C_LIO_LIAlbumin-coated nanocrystals make the insoluble STARD3 inhibitor VS1 deliverable C_LIO_LINanocrystallization raises VS1 apparent solubility >14-fold with sustained release C_LIO_LIVS1 synergizes with 5-FU selectively in 5-FU-sensitive colorectal cancer lines C_LIO_LIAlbumin-nanocrystal VS1 plus 5-FU curbs xenograft growth without added toxicity C_LI

cancer biology↗

bioRxiv2026

Loss of SUMOylation drives aberrant PRC1 clustering and 3D genome rewiring independent of H3K27me3

Polycomb Repressive Complex 1 (PRC1) forms nuclear condensates that organize target chromatin domains. SUMOylation modulates PRC1 clustering, but its impact on condensate properties and 3D genome architecture remains unclear. Here, we show that depletion of SUMO in Drosophila wing imaginal discs transforms PRC1 condensates into large structures with reduced molecular dynamics. Strikingly, this biophysical reorganization occurs without global loss of the H3K27me3 mark. Instead, Hi-C reveals widespread rewiring of topologically associating domain (TAD) interactions. PRC1-bound TADs lose specific long-range contacts with each other while gaining ectopic interactions with active chromatin. These topological shifts correlate with gene misregulation independently of changes in canonical Polycomb histone modifications. Our results establish SUMOylation as a critical regulator of PRC1 condensates, demonstrating that post-translational control of biomolecular condensation dictates 3D genome architecture and transcriptional output through mechanisms separable from histone mark deposition.

genomics↗

bioRxiv2025

Looping specificity of Polycomb response elements requires GAF and a combinatorial code of looping factors

Chromatin looping between cis-regulatory elements is essential for precise developmental gene expression, and its disruption is frequently linked to disease. Polycomb Response Elements (PREs) are specialized tethering elements that mediate chromatin loops and are bound by transcription factors like GAGA-associated factor (GAF), contributing to the recruitment of Polycomb group (PcG) proteins. While both PcG proteins and GAF have been implicated in looping, their specific roles and the mechanisms of loop specificity remain unresolved. Using genome-wide and locus-specific approaches, we show that high GAF occupancy is required for chromatin looping and gene regulation. However, GAF alone cannot establish loops without additional factors. Surprisingly, PRE looping does not require the PcG subunit Polyhomeotic (PH) or the repressive histone marks H3K27me3. Intriguingly, orthologous PRE sequences can rescue looping, while unrelated PREs with similar GAF levels cannot. This indicates that looping specificity depends on both GAF levels and compatible factor combinations at loop anchors. Our results support a combinatorial model in which GAF collaborates with additional looping factors, to drive PRE-specific interactions. We propose the existence of a "looping code" as a mechanistic basis that might explain why only a subset of PREs form loops and contribute to Polycomb-mediated gene silencing.

molecular biology↗

bioRxiv2024

Transient histone deacetylase inhibition induces cellular memory of gene expression and three-dimensional genome folding

Epigenetic memory enables the stable propagation of gene expression patterns in response to transient developmental and environmental stimuli. Although three-dimensional (3D) organisation is emerging as a key regulator of genome function, it is unknown whether it contributes to cellular memory. Here, we establish that acute perturbation of the epigenome can induce cellular memory of gene expression in mouse embryonic stem cells (mESCs). Specifically, we uncover how a pulse of histone deacetylase inhibition translates to changes in histone acetylation and methylation, as well as global and local genome folding. While most epigenomic and transcriptional changes are readily reversible once the perturbation is removed, genome architecture partially maintains its perturbed conformation. This is significant, as a second transient pulse of hyperacetylation induces continued gene expression deregulation at hundreds of loci. Using ultra-deep Micro-C, we associate memory of gene expression with enhancer-promoter contacts and repressive chromatin topology mediated by Polycomb. These results demonstrate how cells are able to record a transient stress in their 3D genome architecture, enabling them to respond more robustly in a second bout of the same perturbation.

genomics↗

bioRxiv2024

A Scaffolding Element Rewires Local 3D Chromatin Architecture During Differentiation

1Upon differentiation chromatin rewires to reflect its new cellular identity and function. While it is widely known that this process involves cooperative changes in transcription, chromatin composition and 3D conformation, it is unclear what exactly drives these changes and how they influence one another. Here we used ESC-to-NPC differentiation to study rewiring at a 3 Mb large neuronal Zfp608 locus. During this process, this large chromatin domain splits in half right at the Zfp608 promoter, local chromatin gets littered with activating marks, compacts in 3D space and Zfp608 abounds in transcription. We investigated the cis and trans elements using capture Hi-C (cHi-C), extensive biophysical modelling, and 3-colour 3D-FISH with technical and analytical breakthroughs and found that transcription abundance modulates the contacts in the region as well as the insulation at the domain split. Furthermore, we found a genetic element we named scaffolding element, with a dual enhancer and architectural function that is essential for chromatin rewiring and loop formation at the NPC stage. The loss of this element disrupts the formation of all local NPC-loops irrespective if they are anchored in this element or not, highlighting the hierarchical relationship between elements that act as loop anchors. Furthermore, we uncovered that the scaffolding function, although driven by multiple mechanisms, can form loops independent of loop-extrusion and that other molecular attractions were necessary to form NPC-specific contacts in the region. Together, these results demonstrate that a hierarchy of genetic elements in cis allows successful rewiring during differentiation and that multiple trans acting elements contribute to make this rewiring efficient.

cell biology↗

bioRxiv2023

Transient loss of Polycomb components induces an epigenetic cancer fate

Cell fate depends on genetic, epigenetic and environmental inputs that are interconnected, making it difficult to disentangle their respective contributions to cell fate decisions1-3, and epigenetic reprogramming is a major contributor to tumor plasticity and adaptation4-6. Although cancer initiation and progression are generally associated with the accumulation of somatic mutations7,8, substantial epigenomic alterations underlie many aspects of tumorigenesis and cancer susceptibility9-18, suggesting that genetic mechanisms alone may not be sufficient to drive malignant transformations19-23. However, whether purely non-genetic reprogramming mechanisms are sufficient to initiate tumorigenesis irrespective of mutations is unknown. Here, we show that a transient perturbation of transcriptional silencing mediated by Polycomb-Group proteins is sufficient to induce an irreversible switch to a cancer cell fate in Drosophila. This is linked to the irreversible derepression of genes that can drive tumorigenesis, including JNK and JAK-STAT signalling pathways and zfh1, the fly homolog of the ZEB1 oncogene, which we show to be a necessary driver of the cancer fate. These data show that a reversible perturbation of Polycomb-Group protein levels can induce cancer in the absence of driver mutations and suggest that this is achieved through epigenetic inheritance of altered cell fates.

developmental biology↗

bioRxiv2021

Spatial organization of chromosomes leads to heterogeneous chromatin motion and drives the liquid- or gel-like behavior of chromatin

Chromosome organization and dynamics are involved in regulating many fundamental processes such as gene transcription and DNA repair. Experiments unveiled that chromatin motion is highly heterogeneous inside cell nuclei, ranging from a liquid-like, mobile state to a gel-like, rigid regime. Using polymer modeling, we investigate how these different physical states and dynamical heterogeneities may emerge from the same structural mechanisms. We found that the formation of topologically-associating domains (TADs) is a key driver of chromatin motion heterogeneity. In particular, we demonstrated that the local degree of compaction of the TAD regulates the transition from a weakly compact, fluid state of chromatin to a more compact, gel state exhibiting anomalous diffusion and coherent motion. Our work provides a comprehensive study of chromosome dynamics and a unified view of chromatin motion enabling to interpret the wide variety of dynamical behaviors observed experimentally across different biological conditions, suggesting that the liquid or solid behaviour of chromatin are in fact two sides of the same coin.

biophysics↗

bioRxiv2020

3D reconstruction of genomic regions from sparse interaction data

Chromosome Conformation Capture (3C) technologies measure the interaction frequency between pairs of chromatin regions within the nucleus in a cell or a population of cells. Some of these 3C technologies retrieve interactions involving non-contiguous sets of loci, resulting in sparse interaction matrices. One of such 3C technologies is Promoter Capture Hi-C (pcHi-C) that is tailored to probe only interactions involving gene promoters. As such, pcHi-C provides sparse interaction matrices that are suitable to characterise short- and long-range enhancer-promoter interactions. Here, we introduce a new method to reconstruct the chromatin structural (3D) organisation from sparse 3C-based datasets such as pcHi-C. Our method allows for data normalisation, detection of significant interactions, and reconstruction of the full 3D organisation of the genomic region despite of the data sparseness. Specifically, it produces reliable reconstructions, in line with the ones obtained from dense interaction matrices, with as low as the 2-3% of the data from the matrix. Furthermore, the method is sensitive enough to detect cell-type-specific 3D organisational features such as the formation of different networks of active gene communities.

bioinformatics↗

bioRxiv2020

Polymer modelling unveils the roles of heterochromatin andnucleolar organizing regions in shaping 3D genome organization inArabidopsis thaliana

The 3D genome is characterized by a complex organization made of genomic and epigenomic layers with profound implications on gene regulation and cell function. However, the understanding of the fundamental mechanisms driving the crosstalk between nuclear architecture and (epi)genomic information is still lacking. The plant Arabidopsis thaliana is a powerful model organism to address these questions owing to its compact genome for which we have a rich collection of microscopy, Chromosome Conformation Capture (Hi-C), and ChIP-seq experiments. Using polymer modelling, we investigate the roles of nucleolus formation and epigenomics-driven interactions in shaping the 3D genome of A. thaliana. By validation of several predictions with published data, we demonstrate that self-attracting nucleolar organizing regions and repulsive constitutive heterochromatin are major mechanisms to regulate the organization of chromosomes. Simulations also suggest that interphase chromosomes maintain a partial structural memory of the V-shapes, typical of (sub)metacentric chromosomes in anaphase. Additionally, self-attraction between facultative heterochromatin regions facilitates the formation of Polycomb bodies hosting H3K27me3-enriched gene-clusters. Since nucleolus and heterochromatin are highly-conserved in eukaryotic cells, our findings pave the way for a comprehensive characterization of the generic principles that are likely to shape and regulate the 3D genome in many species.

biophysics↗

bioRxiv2019

Dynamics of genome architecture and chromatin function during human B cell differentiation and neoplastic transformation

Despite recent advances, the dynamics of genome architecture and chromatin function during human cell differentiation and its potential reorganization upon neoplastic transformation remains poorly characterized. Here, we integrate in situ Hi-C and nine additional omic layers to define and biologically characterize the dynamic changes in three-dimensional (3D) genome architecture across normal B cell differentiation and in neoplastic cells from different subtypes of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) patients. Beyond conventional active (A) and inactive (B) compartments, an integrative analysis of Hi-C data reveals the presence of a highly-dynamic intermediate compartment enriched in poised and polycomb-repressed chromatin. During B cell development, we detect that 28% of the compartments change at defined maturation stages and mostly involve the intermediate compartment. The transition from naive to germinal center B cells is associated with widespread chromatin activation, which mostly reverts into the naive state upon further maturation of germinal center cells into memory B cells. The analysis of CLL and MCL neoplastic cells points both to entity and subtype-specific alterations in chromosome organization. Remarkably, we observe that large chromatin blocks containing key disease-specific genes alter their 3D genome organization. These include the inactivation of a 2Mb region containing the EBF1 gene in CLL and the activation of a 6.1Mb region containing the SOX11 gene in clinically aggressive MCL. This study indicates that 3D genome interactions are extensively modulated during normal B cell differentiation and that the genome of B cell neoplasias acquires a tumor-specific 3D genome architecture.

genomics↗

bioRxiv2019

Hierarchical chromatin organization detected by TADpole.

The rapid development of chromosome conformation capture (3C-based) techniques as well as super-resolution imaging together with bioinformatics analyses has been fundamental for unveiling that chromosomes are organized into the so-called topologically associating domains or TADs. While these TADs appear as nested patterns in the 3C-based interaction matrices, the vast majority of available computational methods are based on the hypothesis that TADs are individual and unrelated chromatin structures. Here we introduce TADpole, a computational tool designed to identify and analyze the entire hierarchy of TADs in intra-chromosomal interaction matrices. TADpole combines principal component analysis and constrained hierarchical clustering to provide an unsupervised set of significant partitions in a genomic region of interest. TADpole identification of domains is robust to the data resolution, normalization strategy, and sequencing depth. TADpole domain borders are enriched in CTCF and cohesin binding proteins, while the domains are enriched in either H3K36me3 or H3k27me3 histone marks. We show TADpole usefulness by applying it to capture Hi-C experiments in wild-type and mutant mouse strains to pinpoint statistically significant differences in their topological structure.

bioinformatics↗

bioRxiv2019

Dynamic simulations of transcriptional control during cell reprogramming reveal spatial chromatin caging

Chromosome structure is a crucial regulatory factor for a wide range of nuclear processes. Chromosome Conformation Capture (3C)-based experiments combined with computational modelling are pivotal for unveiling 3D chromosome structure. Here, we introduce TADdyn, a new tool that integrates time-course 3C data, restraint-based modelling, and molecular dynamics to simulate the structural rearrangements of genomic loci in a completely data-driven way. We applied TADdyn on in-situ Hi-C time-course experiments studying the reprogramming of murine B cells to pluripotent cells, and characterized the structural rearrangements that take place upon changes in the transcriptional state of 11 genomic loci. TADdyn simulations show that structural cages form around the transcription starting site of active loci to stabilize their dynamics, by initiating (hit) and maintaining (stick) interactions with regulatory regions. Consistent findings with TADdyn for all loci under study suggest that this hit-and-stick mechanism may represent a general mechanism to trigger and stabilize transcription.

bioinformatics↗

bioRxiv2017

The spatial position of budding yeast chromosomes affects gene expression

The three-dimensional organization of chromosomes can influence transcription. However, the frequency and magnitude of these effects remains debated. To determine how changes in chromosome positioning affect transcription across thousands of genes with minimal perturbation, we characterized nuclear organization and global gene expression in budding yeast containing chromosome fusions. We used computational modelling and single cell imaging to determine chromosome position and integrated these data with genome-wide transcriptional profiles from RNA sequencing. We find that chromosome fusions dramatically alter 3D nuclear organization without leading to strong genome-wide changes in transcription. However, we observe a mild but significant and reproducible increase in expression of genes near fusion sites. Modeling suggests that this is due to both disruption of telomere-associated silencing and the displacement of genes relative to the nuclear periphery. A 10% decrease in the predicted time a gene spends near the nuclear periphery is associated with a 10% increase in expression. These data suggest that basal transcriptional activity is sensitive to radial changes on gene position, and provide insight into the functional relevance of budding yeast chromosome-level three-dimensional organization in gene expression.

cell biology↗

bioRxiv2017

Single-cell absolute contact probability detection reveals that chromosomes are organized by modulated stochasticity

At the kilo- to mega-base pair scales, eukaryotic genomes are partitioned into self-interacting modules or topologically associated domains (TADs) that associate to form nuclear compartments. Here, we combined high-content super-resolution microscopies with state-of-the-art DNA labeling methods to reveal the variability in the multiscale organization of the Drosophila genome. We found that association frequencies within TADs and between TAD borders are below ~10%, independently of TAD size, epigenetic state, or cell type. Critically, despite this large heterogeneity, we were able to visualize nanometer-sized epigenetic domains at the single-cell level. In addition, absolute contact frequencies within and between TADs were to a large extent defined by genomic distance, higher-order chromosome architecture, and epigenetic identity. We propose that TADs and compartments are organized by multiple, small frequency, yet specific interactions that are regulated by epigenetics and transcriptional state.

biophysics↗

bioRxiv2016

Hi-C-constrained physical models of human chromosomes recover functionally-related properties of genome organization

Combining genome-wide structural models with phenomenological data is at the forefront of efforts to understand the organizational principles regulating the human genome. Here, we use chromosome-chromosome contact data as knowledge- based constraints for large-scale three-dimensional models of the human diploid genome. The resulting models remain minimally entangled and acquire several functional features that are observed in vivo and that were never used as input for the model. We find, for instance, that gene-rich, active regions are drawn towards the nuclear center, while gene poor and lamina-associated domains are pushed to the periphery. These and other properties persist upon adding local contact constraints, suggesting their compatibility with non-local constraints for the genome organization. The results show that suitable combinations of data analysis and physical modelling can expose the unexpectedly rich functionally-related properties implicit in chromosome-chromosome contact data. Specific directions are suggested for further developments based on combining experimental data analysis and genomic structural modelling.

biophysics↗