Biology subjects

Taddei, A.

Publications and source records attributed to Taddei, A..

16 recordsLinked to original sources

bioRxiv2026

SAD-6/ATRX enables broad genome surveillance and defense in fungi

The chromatin remodeler ATRX and its orthologs maintain genome function by regulating repetitive DNA and dynamic chromatin, and their activities have been canonically associated with replication-independent deposition of the histone H3.3 variant. This model is difficult to reconcile with fungi, which encode ATRX orthologs but lack H3 variants that may separately support replication-coupled and replication-independent deposition. Here we show that the fungal ATRX ortholog SAD-6 instead relies on a highly divergent histone H4 variant (H4v) to mediate broad genome surveillance and defense. Deposition of H4v is strictly SAD-6-dependent and thus provides a sensitive genome-wide readout of SAD-6 activity, revealing its functions at telomeres, tRNA and rDNA loci, AT-rich DNA, artificial transgenes, decaying mobile elements, and many genic regions. We further show that SAD-6 is required for a pathway of repeat-induced point mutation (RIP) that also requires DIM-5, a conserved SUV39 methyltransferase that mediates trimethylation of histone H3 lysine-9 in heterochromatin. Together, these findings establish ATRX-like remodelers as broad regulators of genome surveillance and defense in fungi that act through a highly divergent histone H4 variant rather than H3.3. Given that RIP is proposed to recognize repetitive DNA via recombination-independent homologous pairing, the requirement for SAD-6 in RIP suggests that ATRX-like remodelers may couple DNA pairing to heterochromatin nucleation on repeats.

molecular biology↗

bioRxiv2026

A radial map of the budding yeast genome reveals novel organizational principles

The eukaryotic genome is non-randomly organized within the nucleus, with positioning linked to function. Still, genome-wide radial maps are missing for the majority of experimental model systems. We adapted Genomic loci Positioning by Sequencing (GPSeq) to Saccharomyces cerevisiae, enabling high-resolution mapping along the nuclear center-periphery axis. GPSeq confirms known spatial features and shows that peripheral telomeres and centromeres impose long-range constraints extending up to 200 kb, restricting short chromosome arms from the nuclear interior. Telomere repositioning to the nuclear center, either artificially or during quiescence, reorganizes much of the genome through inward movement of sub telomeric regions and compensatory shifts of mid-arm chromatin outward. In quiescence, reduced centromere peripheral localization further alters genome organization. While transcription has a modest impact on radial positioning in all studied conditions, we uncover that in the absence of centromere or telomere constraints, GC-content functionally organizes chromatin in the nucleus. Graphical abstractThe budding yeast genome is spatially organized in a manner highly dependent on the positioning of centromeres (CENs) and telomeres (TELs). Anchoring of these chromosome landmarks constrains the positioning of adjacent chromatin up to 200 kb within the same radial zone. Beyond this range, genome organization is non-random, with processes like transcription and features such as GC- content associated with specific radial positions in the nucleus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/722996v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1c0a383org.highwire.dtl.DTLVardef@2f1e91org.highwire.dtl.DTLVardef@17ff2ecorg.highwire.dtl.DTLVardef@1257f64_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Integration of artificial intelligence and high-content screening enabled identification of drugs for long-term treatment of cerebral cavernous malformation disease

BackgroundAdults and children with cerebral cavernous malformations (CCMs) are at risk of experiencing lifelong complications such as hemorrhagic strokes, neurological deficits, and epileptic seizures. These complications can severely reduce quality of life. At present, there is no safe or effective therapeutic option for the long-term treatment of CCMs. MethodsUsing advanced artificial intelligence (AI) and machine learning models, powered by the Benevolent Platform, we aimed to identify therapeutic drug targets for CCM pathology (e.g., CCM1, CCM2, CCM3). An AI integrative approach utilized various data types from biomedical entities, including diseases, genes, tissues, and biological mechanisms, together with CCM transcriptomic experimental data. High-throughput drug screening of AI-selected FDA-approved medications, analyses of mitochondrial morphology, and studies on pharmacokinetics, pharmacodynamics, and toxicology were conducted in CCM animal models to identify drugs that could potentially be repurposed for the long-term treatment of CCM disease. ResultsAI predicted the AMPK (AMP-activated protein kinase) and mTOR (mammalian target of rapamycin) pathways as potential therapeutic targets that contribute to CCM pathology. High-content screening validation revealed that the FDA-approved drug metformin, which acts as an AMPK agonist and mTOR inhibitor, can reverse changes in cell-cell junction organization and increase KLF4 expression, a marker for CCM, in human CCM endothelial cells in cultured assays. In addition, pharmacodynamic markers of metformin were observed in CCM mouse models (Slco1c1-iCreERT2;Krit1fl/fl;Ptenfl/wt and Slco1c1-iCreERT2;Pdcd10fl/fl) including reduced S6 kinase or ribosomal protein phosphorylation, a marker of decrease mTOR signaling, and increased AMPK phosphorylation, a marker of AMPK activation, that corresponded to reduced lesion burden. Pharmacokinetic and toxicological studies in CCM animal models showed that that metformin penetrates the brain and long-term administration has a favorable safety profile. We also demonstrated that brain endothelial cells in chronic CCM mouse models exhibit increased levels of the inflammatory marker VCAM-1, which is associated with altered mitochondrial phenotypes, as observed by immunofluorescence, MITO-tagging, and electron microscopy analysis. Additionally, we discovered that metformin and a potent AMPK activator, PF-06409577, can reverse mitochondrial phenotypic changes in brain endothelial cells and reduce the elevation of VCAM-1 expression associated with chronic CCM disease. Therefore, metformin can provide cytoprotection and may reverse the CCM endothelial phenotype by activating AMPK. ConclusionsPredictions using AI technology and high-throughput drug screening, combined with pharmacokinetic, pharmacodynamic, and toxicological studies in CCM animal models, identified metformin as a promising drug candidate for repurposing for the long-term treatment of CCM disease. We propose that metformin enhances metabolic adaptation to brain vascular malformations by activating AMPK, which helps reverse mitochondrial fragmentation in brain endothelial cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/693036v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@17bfadforg.highwire.dtl.DTLVardef@936708org.highwire.dtl.DTLVardef@15171ceorg.highwire.dtl.DTLVardef@69029_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2025

Esc1-mediated anchoring regulates telomere clustering in response to metabolic changes.

Spatial organization of yeast telomeres is highly dynamic and regulated by growth conditions. In rich medium, the 32 telomeres group in 3 to 5 foci at the nuclear periphery. This organization is drastically rearranged in long-lived quiescent (Q) cells forming upon carbon source exhaustion: telomere foci assemble into a hypercluster containing most telomeres and located in the center of the nucleus contributing to their long-term viability. Here we explore the mechanisms regulating telomere distribution during this transition. We rule out a modification of telomere-telomere interactions via the telomeric protein Sir3 as the main factor regulating hypercluster formation. However, our physical modeling predicts that telomere anchoring antagonizes telomere clustering. Systematic deletion of all known telomere anchors identifies the inner nuclear membrane-associated protein Esc1, as a key telomeric anchor after the diauxic shift. Our data support a model where Esc1 mediated anchoring is progressively lost upon entry into quiescence through dephosphorylation of a single residue of Esc1, resulting in the formation of telomere hyperclusters in the center of the nucleus in Q cells.

cell biology↗

bioRxiv2024

Rad52 Acts as an Assembly Chaperone to Form and Stabilize Rad51 Filaments Through a Large C-Terminus 85-Residue Segment

Homologous recombination (HR) is essential for the repair of DNA double-strand breaks and the restart of stalled replication forks. A critical step in HR is the formation of Rad51 nucleofilaments, which perform homology search and strand invasion of a homologous DNA sequence required for repair synthesis. In the yeast Saccharomyces cerevisiae, Rad52 facilitates Rad51 nucleofilament formation by mediating Rad51 loading onto ssDNA and counteracting the dissociation of Rad51 filaments by the DNA translocase Srs2. The molecular basis of these two Rad52 functions remains unclear. Our integrative structural analyses of the Rad51-Rad52 interaction, combining NMR, SAXS, and modeling, reveal that an 85-residue segment of Rad52, conserved in fungi, folds upon binding to a broad surface of a Rad51 monomer. Notably, it includes an FxxA motif conserved in the BRC repeats of BRCA2 and at the Rad51-Rad51 interface. This binding mode was validated through an extensive set of mutations. Using in vivo assays and a functional fluorescent GFP-Rad51 fusion protein, we demonstrated that this entire segment is critical for Rad51 filament formation. These findings highlight how Rad52 functions as an assembly chaperone, preventing Rad51 oligomerization, promoting nucleation of Rad51 nucleofilaments on ssDNA, and protecting Rad51 filaments from destabilization by Srs2.

molecular biology↗

bioRxiv2024

Rad52 sorts and stacks Rad51 at the DNA junction to promote homologous recombination

Homologous recombination (HR) repairs double-stranded DNA breaks (DSBs). The DSBs are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Rad51 is a recombinase and catalyzes strand invasion and the search for homology. However, it binds to ssDNA with lower affinity than RPA. Thus, mediator proteins such as Rad52/BRCA2 are required to promote Rad51 binding to RPA-coated ssDNA, but the underlying mechanisms remain poorly understood. Saccharomyces cerevisiae Rad52 interacts with Rad51 through two distinct binding modes. We here uncover that the Rad51-binding site in the disordered C-terminus of Rad52 (mode-1) sorts polydisperse Rad51 into discrete monomers. The second Rad51 binding site resides in the ordered N-terminal ring of Rad52 (mode-2), but this interaction occurs at only one position on the ring. In single molecule confocal fluorescence microscopy combined with optical tweezer analysis, we directly visualize filament formation using fluorescent-Rad51. Rad52 catalyzes Rad51 loading onto RPA-coated ssDNA, with a distinct preference for junctions, but no filament growth is observed. Deletion of the C-terminus of Rad52 results in loss of Rad51 sorting and abrogates Rad51 binding to RPA-coated DNA. While BRCA2 and Rad52 are structurally unrelated, many of these functional features are conserved. We describe a concerted Sort & Stack mechanism for mediator proteins in promoting HR.

biochemistry↗

bioRxiv2024

Single-nucleosome imaging reveals principles of transient multiscale chromatin unfolding triggered by histone ADP-ribosylation at DNA lesions

Timely access to DNA lesions is crucial for genome integrity. This process requires profound remodeling of densely packed chromatin to establish a repair-competent architecture. However, limited resolution has made it impossible to fully understand these remodeling events. Here, combining microirradiation with live-cell multiscale imaging, we report that DNA damage-induced changes in genome packing rely on the conformational behaviour of the chromatin fiber. Immediately after damage, a transient increase in nucleosome mobility switches chromatin from a densely-packed state to a looser conformation, making it accessible to repair. While histone poly-ADP-ribosylation is required to trigger this switch, mono-ADP-ribosylation is sufficient to maintain the open-chromatin state. The removal of these histone marks by the ARH3 hydrolase then leads to chromatin recondensation. Together, our multiscale study of chromatin dynamics establishes a global model: distinct waves of histone ADP-ribosylation control nucleosome mobility, triggering a transient breathing of chromatin, crucial for initiating the DNA damage response.

cell biology↗

bioRxiv2024

Kynurenine monooxygenase blockade reduces endometriosis-like lesions, improves visceral hyperalgia, and rescues mice from a negative behavioural phenotype in experimental endometriosis

Endometriosis is a common and debilitating neuro-inflammatory disorder that is associated with chronic pain. Definitive diagnosis is based on the presence of endometrial-like tissue (lesions) in sites outside the uterus. Kynurenine monooxygenase (KMO) is a mitochondrial enzyme of tryptophan metabolism that regulates inflammation and immunity. Here, we show that KMO is expressed in epithelial cells in human endometriosis tissue lesions and in corresponding lesions in a mouse model of endometriosis. In mice, oral treatment with the potent KMO inhibitor KNS898 induced a biochemical state of KMO blockade with accumulation of kynurenine, diversion to kynurenic acid and ablation of 3-hydroxykynurenine production. In the mouse model of endometriosis, KMO inhibition improved histological outcomes and endometriosis pain-like behaviours, even when KNS898 treatment commenced one week after initiation of lesions. Taken together, these results suggest that KMO blockade is a promising new non-hormonal therapeutic modality for endometriosis.

pathology↗

bioRxiv2023

Anchoring of parasitic plasmids to inactive regions of eukaryotic chromosomes through nucleosome signal

Natural plasmids are common in prokaryotes but few have been documented in eukaryotes. The natural 2{micro} plasmid present in budding yeast Saccharomyces cerevisiae is one of the most well characterized. This highly stable genetic element coexists with its host for millions of years, efficiently segregating at each cell division through a mechanism that remains poorly understood. Using proximity ligation (Hi-C, Micro-C) to map the contacts between the 2{micro} and yeast chromosomes under dozens of different biological conditions, we found that the plasmid tether preferentially on regions with low transcriptional activity, often corresponding to long inactive genes. Common players in chromosome structure such as members of the structural maintenance of chromosome complexes (SMC) are not involved in these contacts which depend instead on a nucleosomal signal associated with a depletion of RNA Pol II. These contacts are stable throughout the cell cycle, and can be established within minutes. This strategy may involve other types of DNA molecules and species other than S. cerevisiae, as suggested by the binding pattern of the natural plasmid along the silent regions of the chromosomes of Dictyostelium discoideum.

genomics↗

bioRxiv2023

Human colorectal cancer: upregulation of the adaptor protein Rai in TILs leads to cell dysfunction by sustaining GSK-3 activation and PD-1 expression

BackgroundThe immunosuppressive tumor microenvironment (TME) of colorectal cancer (CRC) is a major hurdle for immune checkpoint inhibitor-based therapies. Hence characterization of the signaling pathways driving T cell exhaustion within TME is a critical need for the discovery of novel therapeutic targets and the development of effective therapies. We previously showed that i) the adaptor protein Rai is a negative regulator of T cell receptor signaling and T helper 1 (Th1)/Th17 cell differentiation; and ii) Rai deficiency is implicated in the hyperactive phenotype of T cells in autoimmune diseases. MethodsThe expression level of Rai was measured by qRT-PCR in paired peripheral blood T cells and T cells infiltrating tumor tissue and the normal adjacent tissue in CRC patients. The impact of HIF-1 on Rai expression was evaluated in T cells exposed to hypoxia and by performing chromatin immunoprecipitation assays and RNA interference assays. The mechanism by which upregulation of Rai in T cells promotes T cell exhaustion were evaluated by flow cytometric, qRT-PCR and western blot analyses. ResultsWe show that Rai is a novel HIF-1-responsive gene that is upregulated in tumor infiltrating lymphocytes of CRC patients compared to patient-matched circulating T cells. Rai upregulation in T cells promoted PD-1 expression and impaired antigen-dependent degranulation of CD8+ T cells by inhibiting phospho-inactivation of glycogen synthase kinase (GSK)-3, a central regulator of PD- 1 expression and T cell-mediated anti-tumor immunity. ConclusionsOur data identify Rai as a hitherto unknown regulator of the TME-induced exhausted phenotype of human T cells. What is already known on this topicO_LITumor hypoxia contributes to establish an immunosuppressive microenvironment that unleashes T cell function and limits the efficacy of immunotherapy in CRC. C_LIO_LIThe molecular mechanisms underlying the impact of hypoxia on the signaling pathways controlling PD-1 expression are unknown. C_LI What this study addsO_LIA Rai/Akt/GSK-3 axis regulates PD-1 expression following TCR/CD28 co-stimulation C_LIO_LIThis study uncovers the signaling pathway controlling hypoxia-dependent T cell exhaustion. C_LI How this study might affect research, practice or policyRai expression levels in TILs of CRC patients might be explored as a potential new biomarker of T cell exhaustion and a predictive biomarker for anti-PD-1 response.

cancer biology↗

bioRxiv2022

Exogenous chromosomes reveal how sequence composition drives chromatin assembly, activity, folding and compartmentalization

Genomic sequences co-evolve with DNA-associated proteins to ensure the multiscale folding of long DNA molecules into functional chromosomes. In eukaryotes, different molecular complexes organize the chromosomes hierarchical structure, ranging from nucleosomes and cohesin- mediated DNA loops to large scale chromatin compartments. To explore the relationships between the DNA sequence composition and the spontaneous loading and activity of these DNA-associated complexes in the absence of co-evolution, we characterized chromatin assembly and activity in yeast strains carrying exogenous bacterial chromosomes that diverged from eukaryotic sequences over 1.5 billion years ago. We show that nucleosome assembly, transcriptional activity, cohesin-mediated looping, and chromatin compartmentalization can occur in a bacterial chromosome with a largely divergent sequence integrated in a eukaryotic host, and that the chromatinization of bacterial chromosomes is highly correlated with their sequence composition. These results are a step forward in understanding how foreign sequences are interpreted by a host nuclear machinery during natural horizontal gene transfers, as well as in synthetic genomics projects.

genetics↗

bioRxiv2021

Physical observables to determine the nature of membrane-less cellular sub-compartments

The spatial organization of complex biochemical reactions is essential for the regulation of cellular processes. Membrane-less structures called foci containing high concentrations of specific proteins have been reported in a variety of contexts, but the mechanism of their formation is not fully understood. Several competing mechanisms exist that are difficult to distinguish empirically, including liquid-liquid phase separation, and the trapping of molecules by multiple binding sites. Here we propose a theoretical framework and outline observables to differentiate between these scenarios from single molecule tracking experiments. In the binding site model, we derive relations between the distribution of proteins, their diffusion properties, and their radial displacement. We predict that protein search times can be reduced for targets inside a liquid droplet, but not in an aggregate of slowly moving binding sites. These results are applicable to future experiments and suggest different biological roles for liquid droplet and binding site foci.

biophysics↗

bioRxiv2020

The silencing factor Sir3 is a molecular bridge that sticks together distant loci

Physical contacts between distant loci contribute to regulate genome function. However, the molecular mechanisms responsible for settling and maintaining such interactions remain poorly understood. Here we investigate the well conserved interactions between heterochromatin loci. In budding yeast, the 32 telomeres cluster in 3-5 foci in exponentially growing cells. This clustering is functionally linked to the formation of heterochromatin in subtelomeric regions through the recruitment of the silencing complex SIR composed of Sir2/3/4. Combining microscopy and Hi-C on strains expressing different alleles of SIR3, we show that the binding of Sir3 directly promotes long range contacts between distant regions, including the rDNA, telomeres, and internal Sir3 bound sites. Furthermore, we unveil a new property of Sir3 in promoting rDNA compaction. Finally, using a synthetic approach we demonstrate that Sir3 can bond loci belonging to different chromosomes together, when targeted to these loci, independently of its interaction with its known partners (Rap1, and Sir4), Sir2 activity or chromosome context. Altogether these data suggest that Sir3 represents an uncommon example of protein able to bridge directly distant loci.

cell biology↗

bioRxiv2020

Single molecule microscopy reveals key physical features of repair foci in living cells

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus possibly reflecting the existence of a liquid droplet around damaged DNA.

biophysics↗

bioRxiv2020

Significant and conflicting correlation of IL-9 with Prevotella and Bacteroides in human colorectal cancer

BackgroundColorectal cancer (CRC) is a widespread disease that represents an example of chronic inflammation-associated tumor. In fact, the immune system, besides protecting the host from developing tumors, can support the CRC progression. In this scenario, the gut microbiota (GM) is essential to modulate immune responses and a dysbiotic condition can favor chronic/abnormal immune activation that support the tumor growth. GM can elicit the production of cytokines, influencing the immunostimulatory or immunosuppressive reactions, such as the tendency to mount Th1, Th17, Tregs or Th9 responses that play different roles towards colon cancer. Paradigmatic is the role of IL-9 that can both promote tumor progression in hematological malignancies and inhibit tumorigenesis in solid cancers. Therefore, to investigate the microbiota-immunity axis in CRC patients is crucial to well understand the cancer development with positive relapses in prevention and treatment. AimThe cellular and molecular characterization of the immune response and the evaluation of GM composition in healthy and tumor mucosa, focusing on the correlation between cytokines profile and GM signature. MethodsWe collected tumoral (CRC) and healthy (CRC-S) mucosa samples of 45 CRC patients. For each sample, we characterized the Tissue Infiltrating Lymphocytes (TIL)s subset profile and the GM composition. In addition, in 14 CRC patients, we evaluated the CRC and CRC-S molecular inflammatory response (26 cytokines/chemokines) and we correlated this profile with GM composition using the Dirichlet Multinomial Regression. ResultsThe analysis of T cells subsets distribution showed that CRC samples displayed higher percentages of Th17, Th2, Tregs, Tc17, Tc1/Tc17, and Tcreg, compared to CRC-S. Notably, also the number of Th9 was higher, even if not significantly, in CRC tissue compared to healthy one. In addition, we found that MIP-1, IL-1{beta}, IL-2, IP-10, IL-6, IL-8, IL-17A, IFN-{gamma}, TNF-, MCP-1, IL-1, P-selectin and IL-9 were significantly increased in CRC compared to CRC-S. Moreover, the GM analysis revealed that CRC samples had significantly higher levels of Fusobacteria, Proteobacteria, Fusobacterium, Ruminococcus2 (Lachnospiraceae family) and Ruminococcus (Ruminococcaceae family) than CRC-S. Finally, we found that the abundance of Prevotella spp in CRC samples was negatively correlated with IL-17A and positively with IL-9. In addition, the abundance of Bacteroides and Escherichia/Shigella species in CRC samples showed a negative association with IL-9 and IP-10 respectively. ConclusionsOur data show a clear dissimilarity of inflammatory profile and GM composition between the tumor and the adjacent healthy tissue, displaying the generation of a peculiar CRC microenvironment. Interestingly, relating the tissue cytokine profile with the GM composition, we confirmed the presence of a bidirectional crosstalk between the immune response and the hosts commensal microorganisms; in detail, we documented for the first time that Prevotella spp. and Bacteroides spp. are correlated (positively and negatively, respectively) with the IL-9, whose role in CRC development is still debated.

immunology↗

bioRxiv2017

Dose-dependency of heterochromatin domains reveals subtelomeric structuration in budding yeast

The eukaryotic genome is divided into chromosomal domains of heterochromatin and euchromatin. Transcriptionally silent heterochromatin is found at subtelomeric regions, leading to the telomeric position effect (TPE) in yeast, fly and man. Heterochromatin generally initiates and spreads from defined loci, and diverse mechanisms prevent the ectopic spread of heterochromatin into euchromatin. Here, we overexpressed the silencing factor Sir3 at various levels in yeast, and found that Sir3 spreading into Extended Silent Domains (ESD) eventually reached saturation at subtelomeres. We observed that Sir3 spreading into ESDs covered zone associated with specific histone marks in wild-type cells and stopped at zones of histone mark transitions including H3K79 tri-methylation levels. The conserved enzyme Dot1 deposits H3K79 methylation, and we found that it is essential for viability upon overexpression of Sir3, but not of a spreading-defective mutant Sir3A2Q. These data suggest that H3K79 methylation actively blocks Sir3 spreading. Lastly, our meta-analysis uncovers previously uncharacterized discrete subtelomeric domains associated with specific chromatin features offering a new viewpoint on how to separate subtelomeres from the core chromosome.

genomics↗