Biology subjects

Papantonis, A.

Publications and source records attributed to Papantonis, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Mapping Disease Transitions from Premalignant, Asymptomatic to Advanced Myeloma through Integrative Epigenomic and Transcriptional Analyses

AbstractMultiple myeloma (MM) evolves from asymptomatic precursor conditions through progressive genetic and epigenetic remodeling, yet the regulatory mechanisms driving the development toward more active stages of the disease remain poorly understood. Here, we integrated bulk-based paired chromatin accessibility and activation, and transcriptomic profiling across disease stages to map regulatory remodeling during myeloma development. We identified a progressive increase in chromatin accessibility; furthermore, this epigenetic reconfiguration is accompanied by a stage-dependent shift from promoter-centered regulation in precursor states toward enhancer-dominated transcriptional control in active MM. Motif enrichment and regulatory network analyses identified both established and previously underappreciated transcription factors (TFs), including members of the IRF, MEF2, and FOX families, associated with disease-stage-specific transcriptional programs. Among these, MEF2D and FOXK2 emerged as candidate regulators of pathways involved in cell survival and chemotaxis. Functional perturbation demonstrated that MEF2D depletion markedly impaired MM cell viability, whereas inhibition of either MEF2D or FOXK2 reduced chemotactic migration. Together, these findings provide a stage-resolved framework of epigenetic and transcriptional remodeling across myeloma development, revealing regulatory programs established in precursor conditions and progressively reinforced during disease evolution, while identifying candidate transcriptional dependencies with potential biological and therapeutic relevance.

cancer biology↗

bioRxiv2026

The Interaction with Nanotopographical Environment regulates nuclear mechanoresponse in mESCs via Histone Demethylase KDM3A

Cell identity is traditionally viewed as a product of biochemical signalling, yet cells are exposed to defined physical landscapes, whose role in fate control remains unclear. In particular, how pluripotent stem cells integrate nanoscale extracellular cues into gene regulatory programs remains poorly understood. Here, we show that biomimetic substrate nanotopography acts as a potent regulator of naive pluripotency in mouse embryonic stem cells (mESCs). Using supersonic cluster beam deposition, we generate substrates with defined nanotopography that recapitulate native features of extracellular matrix. We demonstrate that nanotopography induces a mechanically relaxed cell state characterised by reduced adhesion, cellular and nuclear softening, and altered nuclear architecture. These mechanical changes are coupled with chromatin remodelling, including reduced H3K27me3 and H3K9me2, redistribution of H3K9me3, and increased H3K4me3. Transcriptomic analyses reveal suppression of adhesion- and cytoskeleton-associated programs together with the activation of a naive pluripotency transcriptional signature, including upregulation of Nanog. Mechanistically, we identify the H3K9 demethylase KDM3A as a mechanosensitive epigenetic regulator required for nanotopography-induced Nanog expression. Together, our findings uncover a novel mechanotransductive pathway directly linking extracellular nanotopography to chromatin state and pluripotency control.

molecular biology↗

bioRxiv2026

LoopBin, a VaDE-based neural network for chromatin loop classification

Classifying chromatin loops from 3D genomics data according to their epigenetic and structural attributes is important for inferring their functional roles. Currently, such classification typically relies on the manual intersection of epigenomic signal peaks and loop anchor locations. To automate this, remove peak-calling biases and include information inherent to 3D genomics signal structure, we developed LoopBin, a framework based on a variational deep embedding (VaDE) neural network. We applied LoopBin to kilobase-resolution Micro-C data and segmented tens of thousands of loops into clusters with distinct features using minimal histone modification and transcription factor-binding data. These features were indicative of apparently distinct biological function by each subgroup of loops. Therefore, LoopBin can provide insights into the dynamic shifts in loop classification that can occur upon perturbation of cell homeostasis or signaling.

bioinformatics↗

bioRxiv2025

Variant U1 snRNAs contribute to cell cycle and differentiation control of human iPS cells

Maintenance of stem cell identity as well as differentiation of stem cells into the various lineages requires intricate regulation of gene expression. Despite intensive research, our understanding of these regulatory networks remains incomplete. Here, we focus on the understudied paralogs of the U1 small nuclear RNA (snRNA) gene known as variant U1 (vU1) snRNAs. By generating isogenic knockout lines of human induced pluripotent stem cells for two different vU1s, we show that their loss profoundly changes both hiPSC gene expression and cell cycle profiles. These changes appear to manifest at the level of alternative splicing choices, including those at recursive splicing sites, and lead to the differential availability of stem cell regulators. Together, our results shed new light on the function of vU1 snRNAs and further our understanding of the programs controlling human pluripotency.

genomics↗

bioRxiv2024

CTCF depletion decouples enhancer-mediated gene activation from chromatin hub formation during cellular differentiation

Enhancers and promoters interact in 3D chromatin structures to regulate gene expression. Here, we characterize the mechanisms that drive the formation of these structures and their function in gene regulation in a lymphoid-to-myeloid transdifferentiation system. Based on analyses at base-pair resolution, we demonstrate a close correlation between binding of regulatory proteins, formation of chromatin interactions, and gene expression. Integration of multi-way interaction analyses and computational modeling shows that tissue-specific gene loci are organized into chromatin hubs, characterized by cooperative interactions between multiple enhancers, promoters, and CTCF-binding sites. Depletion of CTCF strongly impairs the formation of these structures. However, the effects of CTCF depletion on gene expression are modest and can be explained by rewired enhancer-promoter interactions. This demonstrates an instructive role for enhancer-promoter interactions in gene regulation that is independent of cooperative interactions in chromatin hubs. Together, these results contribute to a mechanistic understanding of the structure-function relationship of the genome during cellular differentiation.

genomics↗

bioRxiv2024

Two distinct chromatin modules regulate proinflammatory gene expression

Various mechanisms have been proposed to explain gene activation and co-regulation, including enhancer-promoter interactions via chromatin looping and the enrichment of transcription factors into hubs or condensates. However, these conclusions often stem from analyses of individual loci, and genome-wide studies exploring mechanistic differences with coupled gene expression are lacking. In this study, we dissected the proinflammatory gene expression program induced by TNF in primary human endothelial cells using NGS- and imaging-based techniques. Our findings, enabled by our novel RWireX approach for single-cell ATAC-seq analysis, revealed two distinct regulatory chromatin modules: autonomous links of co-accessibility (ACs) between separated sites, and domains of contiguous co-accessibility (DCs) with increased local transcription factor binding. Genes in ACs and DCs exhibited different transcriptional bursting kinetics, highlighting the existence of two structurally and functionally distinct regulatory chromatin modules in the proinflammatory response. These findings provide a novel mechanistic framework for understanding how cells achieve rapid and precise gene expression control. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/606159v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1f5f7e8org.highwire.dtl.DTLVardef@166b4d7org.highwire.dtl.DTLVardef@1ea9206org.highwire.dtl.DTLVardef@118d534_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LITwo distinct, non-mutually exclusive chromatin modules, ACs and DCs, that regulate proinflammatory gene expression were identified based on deep scATAC-seq. C_LIO_LIACs represent long-range genomic interactions with regulation occurring more by transcription burst frequency. C_LIO_LIDCs are regions of increased local transcription factor binding that can modulate transcription burst size. C_LIO_LIThe AC/DC model integrates sequencing-based evidence for chromatin looping with microscopy observations of transcription factor hubs/condensates into a unified model. C_LIO_LIOur findings provide a novel framework for understanding how cells achieve rapid and precise gene expression control. C_LI

genomics↗

bioRxiv2024

Senescent cells cluster CTCF on nuclear speckles to sustain their splicing program

Senescence --the endpoint of replicative lifespan for normal cells-- is established via a complex sequence of molecular events. One such event is the dramatic reorganization of CTCF into senescence-induced clusters (SICCs). However, the molecular determinants, genomic consequences, and functional purpose of SICCs remained unknown. Here, we combine functional assays, super-resolution imaging, and 3D genomics with computational modelling to dissect SICC emergence. We establish that the competition between CTCF-bound and non-bound loci dictates clustering propensity. Upon senescence entry, cells repurpose SRRM2 --a key component of nuclear speckles-- and BANF1 --a molecular glue for chromosomes-- to cluster CTCF and rewire genome architecture. This CTCF-centric reorganization in reference to nuclear speckles functionally sustains the senescence splicing program, as SICC disruption fully reverts alternative splicing patterns. We therefore uncover a new paradigm, whereby cells translate changes in nuclear biochemistry into architectural changes directing splicing choices so as to commit to the fate of senescence. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/603680v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@cfb561org.highwire.dtl.DTLVardef@8cfdf3org.highwire.dtl.DTLVardef@7485d5org.highwire.dtl.DTLVardef@1fe9051_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIHMGB2-bound loci compete with CTCF-bound ones for nuclear speckle association C_LIO_LISenescent cells repurpose SRRM2 and BANF1 to cluster CTCF on speckles C_LIO_LIBANF1 is essential, but not sufficient for CTCF clustering C_LIO_LIThe SRRM2 RNA-binding domain directs CTCF clustering C_LIO_LISICCs rewire chromatin positioning to sustain the senescence splicing program C_LI

cell biology↗

bioRxiv2024

Epigenetic editing at individual age-associated CpGs affects the genome-wide epigenetic aging landscape

Aging is reflected by genome-wide DNA methylation changes, but it is largely unclear how these epigenetic modifications are regulated. In this study, we explored the possibility to interfere with epigenetic clocks by epigenetic editing at individual CpG sites. CRISPR-guided approaches (dCas9-DNMT3A and CRISPRoff) facilitated targeted methylation at an age-associated genomic region in PDE4C that remained stable for more than three months. Furthermore, epigenetic editing evoked many genome-wide off-target effects, which were highly reproducible and enriched at other age-associated CpGs - thus, they are not random off-target effects, but seem to resemble coregulated epigenetic bystander modifications. 4C chromatin conformation analysis at age-associated sites revealed increased interactions with bystander modifications and other age-associated CpG sites. Subsequently, we multiplexed epigenetic modifications in HEK293T and primary T cells at five genomic regions that become either hypermethylated or hypomethylated upon aging. While epigenetic editing at age-hypomethylated CpGs appeared less stable, it also resulted in a clear enrichment of bystander modifications at other age-associated CpGs. Conversely, epigenetic clocks tend to be accelerated up to ten years after targeted DNA methylation, particularly at hypermethylated CpGs. These results demonstrate that targeted epigenome editing can modulate the epigenetic aging network in its entirety and thereby interfere with epigenetic clocks.

genetics↗

bioRxiv2024

The proximity-based protein interaction landscape of the transcription factor p65 NF-kappaB/RELA and its gene-regulatory logics

The protein interactome of p65 / RELA, the most active subunit of the transcription factor (TF) NF-{kappa}B, has not been previously determined in living cells. Using p65-miniTurbo fusion proteins, we identified by biotin tagging > 350 RELA interactors from untreated and IL-1-stimulated cells, including many TFs (47 % of all interactors) and > 50 epigenetic regulators belonging to different classes of chromatin remodeling complexes. According to point mutants of p65, the interactions primarily require intact dimerization rather than DNA binding properties. A targeted RNAi screen for 38 interactors and subsequent functional transcriptome and bioinformatics studies identified gene regulatory (sub)networks, each controlled by RELA in combination with one of the TFs ZBTB5, GLIS2, TFE3 / TFEB or S100A8 / A9. The remarkably large, dynamic and versatile high resolution interactome of RELA and its gene-regulatory logics provides a rich resource and a new framework for explaining how RELA cooperativity determines gene expression patterns. HighlightsO_LIIdentification of > 350 largely dimerization-dependent interactors of p65 / RELA by miniTurboID C_LIO_LIThe interactome is dominated by transcription factors and epigenetic regulator complexes C_LIO_LIFunctional validation of 38 high confidence interactors by targeted siRNA screen C_LIO_LIIdentification of genetic networks regulated by RELA and six of its interactors in the IL-1 response C_LI

molecular biology↗

bioRxiv2023

A comparative analysis of planarian genomes reveals regulatory conservation in the face of rapid structural divergence

The planarian Schmidtea mediterranea can regenerate its entire body from small tissue fragments and is studied as regeneration model species. The assembly and functional analysis of planarian genomes has proven challenging due its high A/T content (70% A/T), repetitive nature, and limited transferability of routine laboratory protocols due to their divergent biochemistry. Only few and often fragmented genome assemblies are currently available, and open challenges include the provision of well-annotated chromosome-scale reference assemblies of the model species and other planarians for a comparative genome evolution perspective. Here we report a haplotype-phased, chromosome-scale genome assembly and high-quality gene annotations of the sexual S2 strain of S. mediterranea and provide putative regulatory region annotations via optimized ATAC-seq and ChIP-seq protocols. To additionally leverage sequence conservation for regulatory element annotations, we generated chromosome-scale genome assemblies and chromatin accessibility data for the three closest relatives of S. mediterranea: S. polychroa, S. nova, and S. lugubris. We find substantial divergence in protein-coding sequences and regulatory regions, yet reveal remarkable conservation in ChIP-mark bearing open chromatin regions identified as promoters and enhancers in S. mediterranea. The resulting high-confidence set of evolutionary conserved enhancers and promoters provides a valuable resource for the analysis of gene regulatory circuits and their evolution within the taxon. In addition, our four chromosome-scale genome assemblies provide a first comparative perspective on planarian genome evolution. Our analyses reveal frequent retrotransposon-associated chromosomal inversions and inter-chromosomal translocations that lead to a degradation of synteny across the genus. Interestingly, we further find independent and near-complete losses of the ancestral metazoan synteny across Schmidtea and two other flatworm groups, indicating that platyhelminth genomes largely evolve without syntenic constraints. Our work provides valuable genome resources for the planarian research community and sets a foundation for the comparative genomics of planarians. We reveal a contrast between the fast structural evolution of planarian genomes and the conservation of their regulatory elements, suggesting a unique genome evolution in flatworms where gene positioning may not be essential.

genomics↗

bioRxiv2023

Increasingly efficient chromatin binding of cohesin and CTCF supports chromatin architecture formation during zebrafish embryogenesis

The three-dimensional folding of chromosomes is essential for nuclear functions such as DNA replication and gene regulation. The emergence of chromatin architecture is thus an important process during embryogenesis. To shed light on the molecular and kinetic underpinnings of chromatin architecture formation, we characterized biophysical properties of cohesin and CTCF binding to chromatin and their changes upon cofactor depletion using single-molecule imaging in live developing zebrafish embryos. We found that chromatin-bound fractions of both cohesin and CTCF increased significantly between the 1000-cell and shield stages, which we could explain through changes in both their association and dissociation rates. Moreover, increasing binding of cohesin restricted chromatin motion, potentially via loop extrusion, and showed distinct stage-dependent nuclear distribution. Polymer simulations with experimentally derived parameters recapitulated the experimentally observed gradual emergence of chromatin architecture. Our findings suggest a kinetic framework of chromatin architecture formation during zebrafish embryogenesis.

biophysics↗

bioRxiv2023

Spatial tumor immune heterogeneity facilitates subtype co-existence and therapy response via AP1 dichotomy in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) displays a high degree of spatial subtype heterogeneity. This intratumoral co-existence of classical and basal-like programs is evident in multi-scale transcriptomic and spatial analyses of resected, advanced-stage and chemotherapy-treated specimens and reciprocally linked to a diverse stromal immune microenvironment as well as worse clinical outcome. However, the underlying mechanisms of intratumoral subtype heterogeneity remain largely unclear. Here, by combining preclinical models, multi-center clinical, bulk and compartment-specific transcriptomic, proteomic, and bioimaging data from human specimens, we identified an interplay between neoplastic intrinsic AP1 transcription factor dichotomy and extrinsic CD68+ macrophages as a driver of intratumoral subtype co-existence along with an immunosuppressive tumor microenvironment with T cell exclusion. Our ATAC-, ChIP-, and RNA-seq analyses revealed that JUNB/AP1- and HDAC-mediated epigenetic programs repress pro-inflammatory immune signatures in tumor cells, antagonizing cJUN/AP1 signaling to favor a therapy-responsive classical neoplastic identity. Through the tumor microenvironment, this dichotomous regulation was further amplified via regional macrophage populations. Moreover, CD68+/TNF-+ cells associated with a reactive phenotype and reduced CD8+ T cell infiltration in human PDAC tumors. Consequently, combined anti-TNF- immunotherapy and chemotherapy resulted in reduced macrophage counts and promoted CD3+/CD8+ T cell infiltration in basal-like PDAC, leading to improved survival in preclinical murine models. We conclude that tumor cell intrinsic epigenetic programs, together with extrinsic microenvironmental cues, facilitate intratumoral subtype heterogeneity and disease progression.

cancer biology↗

bioRxiv2023

Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor

Cellular senescence is now acknowledged as a key contributor to organismal ageing and late-life disease. Although popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and its paracrine effects. To address these issues, we repurposed the small molecule inhibitor inflachromene (ICM) to induce senescence to human primary cells. Within six days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered significant similarity of the two states. Notably though, ICM suppresses the proinflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM induces a senescence-like phenotype rapidly and synchronously thereby allowing the study of its core regulatory program without any confounding heterogeneity.

genomics↗

bioRxiv2023

Remodelling of the endothelial cell transcriptional program via paracrine and DNA-binding activities of MPO

Myeloperoxidase (MPO) is an enzyme that functions in host defence by catalysing the formation of reactive oxygen intermediates. Synthesized majorly by myeloid progenitor cell types and neutrophils, MPO is released into the vascular lumen during inflammation, where it may adhere and subsequently enter endothelial cells coating vascular walls. Here, we show that MPO actually enters the nucleus of these endothelial cells and binds chromatin independently of its enzymatic activity to cause changes in chromatin structure. At its binding sites, MPO drives chromatin decondensation, while enhancing condensation at flanking regions. We further show that MPO binds loci relevant for the activation of the endothelial-to- mesenchymal transition (EndMT) and the migratory potential of ECs. Finally, MPO interacts with the RNA- binding factor ILF3 affecting its relative abundance between cytoplasm and nucleus. This leads to ILF3:MPO- driven transcriptional and post-transcriptional regulation. Accordingly, MPO-knockout mice show reduced EC numbers at scars formed after myocardial infarction, indicating reduced neovascularization. In summary, we describe a non-enzymatic role for MPO in coordinating EndMT and controlling the fate of endothelial cells through direct chromatin binding and association with such co-factors as ILF3.

genomics↗

bioRxiv2023

A single-cell strategy for the identification of intronic variants related to mis-splicing in pancreatic cancer

Most clinical diagnostic and genomic research setups focus almost exclusively on coding regions and essential splice sites, thereby overlooking other non-coding variants. As a result, intronic variants that can promote mis-splicing events across a range of diseases, including cancer, are yet to be systematically investigated. Such investigations would require both genomic and transcriptomic data, but there currently exist very few datasets that satisfy these requirements. We address this by developing a single-nucleus full-length RNA-sequencing approach that allows for the detection of potentially pathogenic intronic variants. We exemplify the potency of our approach by applying pancreatic cancer tumor and tumor-derived specimens and linking intronic variants to splicing dysregulation. We specifically find that prominent intron retention and pseudo-exon activation events are shared by the tumors and affect genes encoding key transcriptional regulators. Our work paves the way for the assessment and exploitation of intronic mutations as powerful prognostic markers and potential therapeutic targets in cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/539836v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@18ddba7org.highwire.dtl.DTLVardef@4fc53aorg.highwire.dtl.DTLVardef@11d8d0dorg.highwire.dtl.DTLVardef@1178b86_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

Competition between transcription and loop extrusion modulates promoter and enhancer dynamics

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the 4D arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find that alterations in chromatin mobility, not promoter-enhancer distance, is more informative about transcriptional status. Active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.

cell biology↗

bioRxiv2023

Extreme structural heterogeneity rewires glioblastoma chromosomes to sustain patient-specific transcriptional programs

Glioblastoma multiforme (GBM) encompasses brain malignancies marked by phenotypic and transcriptional heterogeneity thought to render these tumors aggressive, resistant to therapy, and inevitably recurrent. However, little is known about how the spatial organization of GBM genomes underlies this heterogeneity and its effects. Here, we compiled a cohort of 28 patient-derived glioblastoma stem cell-like lines (GSCs) known to reflect the properties of their tumor-of-origin; six of these were primary-relapse tumor pairs from the same patient. We generated and analyzed kbp-resolution chromosome conformation capture (Hi-C) data from all GSCs to systematically map >3,100 standalone and complex structural variants (SVs) and the >6,300 neoloops arising as a result. By combining Hi-C, histone modification, and gene expression data with chromatin folding simulations, we explain how the pervasive, uneven, and idiosyncratic occurrence of neoloops sustains tumor-specific transcriptional programs via the formation of new enhancer-promoter contacts. We also show how even moderately recurrent neoloops can help us infer patient-specific vulnerabilities. Together, our data provide a resource for dissecting GBM biology and heterogeneity, as well as for informing therapeutic approaches.

genomics↗

bioRxiv2022

Enhancer-promoter contact formation requires RNAPII and antagonizes loop extrusion

Mammalian chromosomes are folded by converging and opposing forces. Here, we tested the role of RNAPII across different scales of interphase chromatin folding in a cellular system allowing for its auxin-mediated degradation. We used Micro-C and computational modeling to characterize subsets of loops differentially gained or lost upon RNAPII depletion. Gained loops, extrusion of which was antagonized by RNAPII, almost invariably formed by engaging new or rewired CTCF anchors. Lost loops selectively concerned contacts between enhancers and promoters anchored by RNAPII. Surprisingly, promoter-promoter contacts were almost insensitive to polymerase depletion and sustained cohesin occupancy in its absence. Selective loss of enhancer-promoter contacts explains the repression of most genes. Together, our findings reconcile the role of RNAPII in transcription with that in setting-up regulatory 3D chromatin architectures genome-wide, while also revealing a direct impact on cohesin loop extrusion.

genomics↗