Biology subjects

Cardoso, M. C.

Publications and source records attributed to Cardoso, M. C..

10 recordsLinked to original sources

bioRxiv2026

Constitutive ATR Signaling Enforces Domain-Wide Origin Synchrony

Eukaryotic DNA replication relies on organized spatial-temporal domains, yet how neighboring origins are coordinated at the single-molecule level remains unresolved. Combining single-molecule DNA combing with super-resolution microscopy, we demonstrate that during unperturbed early S-phase, adjacent replication origins across diverse vertebrate species fire synchronously as a coordinated unit. This domain-wide temporal harmony is continuously enforced by a baseline, constitutive ATR CHK1 signaling axis that restrains local CDK2 and CDC7 kinase activities. Strikingly, replication stress dismantles this coordination, shifting origin activation toward an asynchronous, replicon-autonomous mode. Mechanistically, stress-induced phosphorylation of the cohesin subunit SMC1A promotes an SMC1A ATR complex that sequesters ATR at stalled replication forks. This localized sequestration creates a regional ATR loss-of-function phenotype, relieving CDK2/CDC7 suppression and driving premature, uncoordinated origin firing. Super-resolution imaging shows that ATR sequestration structurally reorganizes replication foci, converting coordinated clustered foci into fragmented, lower-density clusters. Together, our findings uncover a baseline role for constitutive ATR signaling in enforcing domain synchrony and reveal a molecular switch that dynamically decouples origin activation during genomic stress to safeguard chromosome duplication.

molecular biology↗

bioRxiv2026

Heterochromatin organization and liquid-liquid phase separation: it is not about if but about when

Heterochromatin is a membraneless compartment within the cell nucleus. In recent years, a controversy arose on whether heterochromatin organization is driven by liquid-liquid phase separation or not. While many heterochromatin proteins were shown to undergo liquid-liquid phase separation in vitro, other studies reported that this does not happen in cells. Here, we tested the ability of heterochromatin proteins to generate heterochromatin barrier compartments in cells. We found that, while several proteins (H1.0, H1.4, HP1alpha, HP1beta, Mbd1, Mbd2 and MeCP2) form barrier compartments in mouse and/or human cells this differs between cell types. In addition, not all compartments in the same cell form barriers. We established and experimentally validated a model that predicted the ability to form barrier compartments is dependent on the protein accumulation in heterochromatin followed by the competition between compartments for the nucleoplasm pool of the protein and resulted in larger size for the barrier compartments. These findings resolve the existing controversy and rationalize how in cells heterochromatin compartments form and compete to establish dynamic barriers to the entry and exit of its components. HighlightsHeterochromatin barrier formation differs between proteins, cell lines and heterochromatin compartments within the cell. Barrier formation depends on heterochromatin anchors, including ligands and other scaffolds. Barrier compartments are defined by their larger size and higher protein enrichment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/729812v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@a63021org.highwire.dtl.DTLVardef@a20362org.highwire.dtl.DTLVardef@8c2390org.highwire.dtl.DTLVardef@72dde3_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Dynamic anatomy of the replisome under stress

When exposed to replication stress, the factors involved in DNA replication display differing responses that in many cases remain unexplained. In this study, we generated a series of genomically engineered embryonic stem cell lines with fluorescently tagged replication factors to track their spatiotemporal dynamics in live-cell microscopy during and after treatment with the replication inhibitors aphidicolin and hydroxyurea. All 3 replicative polymerases were found to accumulate upon stress, with the primase subunits of polymerase alpha retaining catalytic activity. This accumulation was dependent on new origin firing and accompanied by 9-1-1 clamp loading in the vicinity of stalled forks. Pulse-stress-pulse experiments and spatial correlation analyses of replicons indicated that, after stress is removed, replication preferentially restarts at new sites surrounding previously stalled forks. These findings shed light on the replisomes immediate reaction to stalled replication forks and provide comprehensive kinetic maps of the replisome under stress. Highlights- Comprehensible atlas of the replisome under stress - DNA polymerases accumulate during stress, with clamp switch near stalled forks - In the absence of DNA synthesis, new adjacent origins fire with active priming - During stress recovery, replication restart predominantly at new fired origins

cell biology↗

bioRxiv2026

RepliCNN: High-resolution inference of the DNA replication program from strand-specific 3' DNA end sequencing

During S phase, the genome is replicated in a tightly regulated spatiotemporal order described as DNA replication timing (RT). Discontinuous lagging-strand synthesis produces Okazaki fragments whose strand-specific distribution reflects replication dynamics. Here, we present RepliCNN, a deep learning framework based on one-dimensional convolutional neural networks to predict RT from Okazaki fragment distributions obtained from strand-specific 3' DNA end sequencing methods such as GLOE-Seq, TrAEL-seq, or OK-Seq. RepliCNN also automatically annotates replication origins, termination zones, replication fork directionality, and origin efficiency genome-wide from a single dataset. Benchmarking on public and in-house human and yeast datasets using leave-one-chromosome-out cross-validation demonstrates high predictive accuracy in both wild-type and perturbation experiments, enabling comprehensive analyses of replication dynamics from strand-specific DNA 3' end sequencing data. HighlightsO_LIRepliCNN enables integrated analysis of replication timing, fork directionality and replication features from strand-specific 3' DNA end sequencing data. C_LIO_LIHigh-resolution replication dynamics can be inferred from a single experiment, bypassing complex multi-fraction labelling approaches. C_LIO_LIThe framework generalizes across experimental protocols, datasets, and species. C_LIO_LIThis enables cost-effective comparative analysis of replication programs across biological conditions. C_LI

bioinformatics↗

bioRxiv2025

Chromatin organization controls nuclear stiffness

Cellular differentiation is driven by epigenetic modifiers and readers, including the methyl CpG binding protein 2 (MeCP2), whose level and mutations cause the neurological disorder Rett syndrome. During differentiation, most of the genome gets densely packed into heterochromatin, whose function has been simplistically viewed as gene silencing. However, gene expression changes reported in mutations leading to Rett syndrome have failed to be a predictor of disease severity. Here, we show that MeCP2 increases nuclear stiffness in a concentration dependent manner and dependent on its ability to cluster heterochromatin during differentiation. MeCP2-dependent stiffness increase could not be explained by changes in the expression of mechanobiology-related genes, but we found it is disrupted by Rett syndrome mutations and correlated with disease severity. Our results highlight the impact of chromatin organization in the mechanical properties of the cell as an alternative or complementary mechanism to changes in cytoskeleton components. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/643219v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@cb077dorg.highwire.dtl.DTLVardef@158c0c6org.highwire.dtl.DTLVardef@1d830e3org.highwire.dtl.DTLVardef@719456_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

TET dioxygenases localize at splicing speckles and promote RNA splicing

The dynamic regulation of RNA metabolism plays a crucial part in cellular function, with emerging evidence suggesting an important role for RNA modifications in this process. This study explores the relationship between RNA splicing and the TET dioxygenase activity, shedding light on the role of hm5C (RNA 5-hydroxymethylcytosine), and TET proteins, in RNA metabolism. Integrating data from mass spectrometry, AlphaFold structural modeling, microscopic analysis, and different functional assays including in vitro splicing, TET proteins were found to regulate splicing. We show that TET1, TET2, and TET3 interact with the splicing factors U2AF1 and U2AF2. Interestingly, TET dioxygenases localize in splicing speckles in mammalian and Drosophila cells. TET speckles association is RNA dependent, as it is TET interaction with splicing factors. Furthermore, in vitro splicing assays revealed that all three TET proteins promote splicing efficiency, and the oxidation of m5C to hm5C can restore splicing efficiency in vitro. The latter highlights the regulatory role of cytosine modifications in RNA metabolism. These findings provide insights into the complex interplay between RNA modifications and splicing, suggesting a multifaceted role for TET proteins in RNA metabolism beyond its canonical DNA demethylation function. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/641893v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1a304bborg.highwire.dtl.DTLVardef@18b91d3org.highwire.dtl.DTLVardef@12efeforg.highwire.dtl.DTLVardef@1464355_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- TET1 localizes in splicing speckles in an RNA-dependent manner - TET proteins, especially TET1, interact with the splicing factors U2AF1 and U2AF2 - TET proteins increase splicing efficiency, independent of their catalytic activity - RNA 5-methylcytosine (m5C) oxidation to 5-hydroxymethylcytosine (hm5C) restores splicing efficiently in vitro

cell biology↗

bioRxiv2025

Super-resolution compatible DNA labeling technique reveals chromatin mobility and organization changes during differentiation

Chromatin dynamics play a crucial role in cellular differentiation, yet tools for studying global chromatin mobility in living cells remain limited. Here, we developed a novel probe for the metabolic labeling of chromatin and tracking its mobility during neural differentiation. The labeling system utilizes a newly developed silicon rhodamine-conjugated deoxycytidine triphosphate (dCSiRTP). We show that this dCTP is efficiently delivered into living human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) via a synthetic transporter (SNTT1). Using correlative confocal microscopy and stimulated emission depletion (STED) super-resolution microscopy, we quantified the sizes of labeled chromatin domains. Time lapse super-resolution microscopy combined with single particle tracking revealed that chromatin mobility decreases during the transition from iPSCs (pluripotent state) to NSCs and neurons (differentiated state). This reduction in mobility correlates with the differentiation state, suggesting a role for chromatin dynamics in cellular plasticity. Concomitant mechanistic insights obtained from MNase digestion assays, chromatin compaction and histone modification analyses revealed a decrease in chromatin accessibility during neuronal differentiation, indicating that chromatin adopts a more constrained and compacted structure. These findings provide new insights into chromatin regulation during neurogenesis.

cell biology↗

bioRxiv2023

Replisome loading reduces chromatin motion independent of DNA synthesis

Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interphase human cells expressing GFP-tagged PCNA, a cell cycle marker and core component of the DNA replication machinery. We detected decreased chromatin mobility during the S-phase compared to G1 and G2 phases in tumor as well as normal diploid cells using automated particle tracking. To gain insight into the dynamical organization of the genome during DNA replication, we determined labeled chromatin domain sizes and analyzed their motion in replicating cells. By correlating chromatin mobility proximal to the active sites of DNA synthesis, we showed that chromatin motion was locally constrained at the sites of DNA replication. Furthermore, inhibiting DNA synthesis led to increased loading of DNA polymerases. This was accompanied by accumulation of the single-stranded DNA binding protein on the chromatin and activation of DNA helicases further restricting local chromatin motion. We, therefore, propose that it is the loading of replisomes but not their catalytic activity that reduces the dynamics of replicating chromatin segments in the S-phase as well as their accessibility and probability of interactions with other genomic regions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/531331v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@ed9743org.highwire.dtl.DTLVardef@65c9d4org.highwire.dtl.DTLVardef@a583a7org.highwire.dtl.DTLVardef@6681b1_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Direct and sequence unbiased labeling of DNA genome-wide - DNA labeled chromatin is more mobile in G1/G2 relative to the S-phase - Restriction of chromatin motion occurs proximal to sites of DNA replication - Loading of replisomes, even in the absence of processive DNA synthesis, restricts chromatin motion

cell biology↗

bioRxiv2022

Probing protein ubiquitination in live cells

The reversible attachment of ubiquitin governs the interaction, activity and degradation of proteins whereby the type and target of this conjugation determine the biological response. The investigation of this complex and multi-faceted protein ubiquitination mostly relies on painstaking biochemical analyses. Here, we employ recombinant binding domains to probe the ubiquitination of proteins in living cells. We immobilize GFP-fused proteins of interest at a distinct cellular structure and detect their ubiquitination state with red fluorescent ubiquitin binders. With this ubiquitin fluorescent three-hybrid (ubiF3H) assay we identified HP1{beta} as a novel ubiquitination target of UHRF1. The use of linkage specific ubiquitin binding domains enabled the discrimination of K48 and K63 linked protein ubiquitination. To enhance signal-to-noise ratio, we implemented fluorescence complementation (ubiF3Hc) with split YFP. Using in addition a cell cycle marker we could show that HP1{beta} is mostly ubiquitinated by UHRF1 during S phase and deubiquitinated by the protease USP7. With this complementation assay we could also directly detect the ubiquitination of the tumor suppressor p53 and monitor its inhibition by the anti-cancer drug Nutlin-3. Altogether, we demonstrate the utility of the ubiF3H assay to probe the ubiquitination of specific proteins and to screen for ligases, proteases and small molecules controlling this posttranslational modification.

cell biology↗

bioRxiv2019

Cohesin depleted cells pass through mitosis and reconstitute a functional nuclear architecture

Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is debatable. Using live-cell and super-resolved 3D microscopy, we demonstrate that cohesin depleted cells pass through an endomitosis and rebuild a single multilobulated nucleus (MLN) with chromosome territories (CTs) pervaded by interchromatin channels. CTs contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. Splicing speckles are located nearby within the lining channel system. These clusters form microscopically defined, active and inactive compartments, which correspond to A/B compartments, detected with ensemble Hi-C. Functionality of MLN despite continuous absence of cohesin was demonstrated by their ability to pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity.

cell biology↗