Biomedical subjects

M Cristina Cardoso

Publications and source records attributed to M Cristina Cardoso.

At least 19 recordsLinked to original sources

PubMed2025

Dynamic association of H3K36me3 with pericentromeric heterochromatin regulates its replication time.

The flexibility of the spatio-temporal genome replication program during development and disease highlights the regulatory role of plastic epigenetic mechanisms over genetic determinants. Histone post-translational modifications are broadly implicated in replication timing control, yet the specific mechanisms through which individual histone marks influence replication dynamics, particularly in heterochromatin, remain unclear. Here, we demonstrate that H3K36me3 dynamically enriches at pericentromeric heterochromatin, composed of major satellite DNA repeats, prior to replication during mid S phase in mouse embryonic stem cells. By knocking down lysine 36-specific methyltransferases or by targeting the H3K36M oncohistone to pericentromeric heterochromatin, we reduce global or local H3K36me3 levels, respectively, revealing its essential role in preserving the replication timing of constitutive heterochromatin. Loss of H3K36me3 accompanies increased RNA polymerase II serine-5 phosphorylation and lowered major satellite RNA levels, indicating transcriptional dysregulation. Notably, we identify a strand-specific contribution of major satellite forward transcripts in regulating the replication timing of constitutive heterochromatin and maintaining chromatin stability, highlighting the importance of non-coding RNAs as critical regulators of replication timing.

Heterochromatin↗

PubMed2007

Nucleolar marker for living cells.

In the recent molecular and cell biological research, there is an increasing need for labeling of subcellular structures in living cells. Here, we present the use of a fluorescently labeled cell penetrating peptide for fast labeling of nucleoli in living cells of different species and origin. We show that the short peptide with ten amino acids was able to cross cellular membranes and reach the nucleolar target sites, thereby marking this subnuclear structure in living cells. The treatment of cells with actinomycin D and labeling of B23 protein and fibrillarin provided evidence for a localization to the granular component of the nucleolus. The fluorescently conjugated nucleolar marker could be used in combination with different fluorophores like fluorescent proteins or DNA dyes, and nucleolar labeling was also preserved during fixation and staining of the cells. Furthermore, we observed a high stability of the label in long-term studies over 24 h as well as no effect on the cellular viability and proliferation and on rDNA transcription. The transducible nucleolar marker is therefore a valuable molecular tool for cell biology that allows a fast and easy labeling of this structure in living cells.

3T3 Cells↗

PubMed2006

Differential recruitment of DNA Ligase I and III to DNA repair sites.

DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Ligase I, whereas we could detect only a faint accumulation of DNA Ligase IV. Recruitment of DNA Ligase I and III to repair sites was cell cycle independent. Mutational analysis and binding studies revealed that DNA Ligase I was recruited to DNA repair sites by interaction with PCNA while DNA Ligase III was recruited via its BRCT domain mediated interaction with XRCC1. Selective recruitment of specialized DNA Ligases may have evolved to accommodate the particular requirements of different repair pathways and may thus enhance efficiency of DNA repair.

Animals↗

PubMed2006

Spatiotemporal dynamics of p21CDKN1A protein recruitment to DNA-damage sites and interaction with proliferating cell nuclear antigen.

The cyclin-dependent kinase inhibitor p21CDKN1A plays a fundamental role in the DNA-damage response by inducing cell-cycle arrest, and by inhibiting DNA replication through association with the proliferating cell nuclear antigen (PCNA). However, the role of such an interaction in DNA repair is poorly understood and controversial. Here, we provide evidence that a pool of p21 protein is rapidly recruited to UV-induced DNA-damage sites, where it colocalises with PCNA and PCNA-interacting proteins involved in nucleotide excision repair (NER), such as DNA polymerase delta, XPG and CAF-1. In vivo imaging and confocal fluorescence microscopy analysis of cells coexpressing p21 and PCNA fused to green or red fluorescent protein (p21-GFP, RFP-PCNA), showed a rapid relocation of both proteins at microirradiated nuclear spots, although dynamic measurements suggested that p21-GFP was recruited with slower kinetics. An exogenously expressed p21 mutant protein unable to bind PCNA neither colocalised, nor coimmunoprecipitated with PCNA after UV irradiation. In NER-deficient XP-A fibroblasts, p21 relocation was greatly delayed, concomitantly with that of PCNA. These results indicate that early recruitment of p21 protein to DNA-damage sites is a NER-related process dependent on interaction with PCNA, thus suggesting a direct involvement of p21 in DNA repair.

Animals↗

PubMed2006

Targeting and tracing antigens in live cells with fluorescent nanobodies.

We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.

Animals↗

PubMed2006

Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells.

Cell-penetrating peptides (CPPs) are capable of introducing a wide range of cargoes into living cells. Descriptions of the internalization process vary from energy-independent cell penetration of membranes to endocytic uptake. To elucidate whether the mechanism of entry of CPP constructs might be influenced by the properties of the cargo, we used time lapse confocal microscopy analysis of living mammalian cells to directly compare the uptake of the well-studied CPP TAT fused to a protein (>50 amino acids) or peptide (<50 amino acids) cargo. We also analyzed various constructs for their subcellular distribution and mobility after the internalization event. TAT fusion proteins were taken up largely into cytoplasmic vesicles whereas peptides fused to TAT entered the cell in a rapid manner that was dependent on membrane potential. Despite their accumulation in the nucleolus, photobleaching of TAT fusion peptides revealed their mobility. The bioavailability of internalized TAT peptides was tested and confirmed by the strong inhibitory effect on cell cycle progression of two TAT fusion peptides derived from the tumor suppressor p21(WAF/Cip) and DNA Ligase I measured in living cells.

Amino Acid Sequence↗

PubMed2005

Replication of centromeric heterochromatin in mouse fibroblasts takes place in early, middle, and late S phase.

The replication of eukaryotic chromosomes takes place throughout S phase, but little is known how this process is organized in space and time. Early and late replicating chromosomal domains appear to localize to distinct spatial compartments of the nucleus where DNA synthesis can take place at defined times during S phase. In general, transcriptionally active chromatin replicates early in S phase whereas transcriptionally inactive chromatin replicates later. Here we provide evidence for significant deviation from this dogma in mouse NIH3T3 cells. While the bulk pericentromeric heterochromatin replicates exclusively during mid to late S phase, centromeric DNA domains associated with constitutive kinetochore proteins are replicated throughout all stages of S phase. On an average, 12+/-4% of centromeres replicate in early S phase. Early replication of a subset of centromeres was also detected in living C2C12 murine cells. Thus, in contrast to expectation, late replication is not an obligatory feature of centromeric heterochromatin in murine cells and it does not determine their 'heterochromatic state'.

3T3 Cells↗

PubMed2005

CPT1alpha over-expression increases long-chain fatty acid oxidation and reduces cell viability with incremental palmitic acid concentration in 293T cells.

To test the cellular response to an increased fatty acid oxidation, we generated a vector for an inducible expression of the rate-limiting enzyme carnitine palmitoyl-transferase 1alpha (CPT1alpha). Human embryonic 293T kidney cells were transiently transfected and expression of the CPT1alpha transgene in the tet-on vector was activated with doxycycline. Fatty acid oxidation was measured by determining the conversion of supplemented, synthetic cis-10-heptadecenoic acid (C17:1n-7) to C15:ln-7. CPT1alpha over-expression increased mitochondrial long-chain fatty acid oxidation about 6-fold. Addition of palmitic acid (PA) decreased viability of CPT1alpha over-expressing cells in a concentration-dependent manner. Both, PA and CPT1alpha over-expression increased cell death. Interestingly, PA reduced total cell number only in cells over-expressing CPT1alpha, suggesting an effect on cell proliferation that requires PA translocation across the mitochondrial inner membrane. This inducible expression system should be well suited to study the roles of CPT1 and fatty acid oxidation in lipotoxicity and metabolism in vivo.

Carnitine O-Palmitoyltransferase↗

PubMed2005

Restricted mobility of Dnmt1 in preimplantation embryos: implications for epigenetic reprogramming.

BACKGROUND: Mouse preimplantation development is characterized by both active and passive genomic demethylation. A short isoform of the prevalent maintenance DNA methyltransferase (Dnmt1S) is found in the cytoplasm of preimplantation embryos and transiently enters the nucleus only at the 8-cell stage. RESULTS: Using GFP fusions we show that both the long and short isoforms of Dnmt1 localize to the nucleus of somatic cells and the cytoplasm of preimplantation embryos and that these subcellular localization properties are independent of phosphorylation. Importantly, photobleaching techniques and salt extraction revealed that Dnmt1S has a very restricted mobility in the cytoplasm, while it is highly mobile in the nucleus of preimplantation embryos. CONCLUSION: The restricted mobility of Dnmt1S limits its access to DNA and likely contributes to passive demethylation and epigenetic reprogramming during preimplantation development.

Animals↗

PubMed2005

PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins.

In DNA replication, the leading strand is synthesized continuously, but lagging strand synthesis requires the complex, discontinuous synthesis of Okazaki fragments, and their subsequent joining. We have used a combination of in situ extraction and dual color photobleaching to compare the dynamic properties of three proteins essential for lagging strand synthesis: the polymerase clamp proliferating cell nuclear antigen (PCNA) and two proteins that bind to it, DNA Ligase I and Fen1. All three proteins are localized at replication foci (RF), but in contrast to PCNA, Ligase and Fen1 were readily extracted. Dual photobleaching combined with time overlays revealed a rapid exchange of Ligase and Fen1 at RF, which is consistent with de novo loading at every Okazaki fragment, while the slow recovery of PCNA mostly occurred at adjacent, newly assembled RF. These data indicate that PCNA works as a stationary loading platform that is reused for multiple Okazaki fragments, while PCNA binding proteins only transiently associate and are not stable components of the replication machinery.

Animals↗

PubMed2005

Recruitment of DNA methyltransferase I to DNA repair sites.

In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair.

Animals↗

PubMed2005

Methyl CpG-binding proteins induce large-scale chromatin reorganization during terminal differentiation.

Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG-binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG-binding domain (MBD) only. Similar to MeCP2, another methyl CpG-binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation.

Animals↗

PubMed2005

Cell cycle markers for live cell analyses.

Many cellular processes are regulated by cell cycle dependent changes in protein dynamics and localization. Studying these changes in vivo requires methods to distinguish the different cell cycle stages. Here we demonstrate the use of DNA Ligase I fused to DsRed1 as an in situ marker to identify S phase and the subsequent transition to G2 in live cells. Using this marker, we observed changes in the nuclear distribution of Dnmt1 during cell cycle progression. Based on the different nuclear distribution of DNA Ligase I and Dnmt1 in G2 and G1, we demonstrate that the combination of both proteins allows the direct discrimination of all cell cycle phases using either immunostainings or fusions with fluorescent proteins. These markers are new tools to directly study cell cycle dependent processes in both, fixed and living cells.

Animals↗

PubMed2005

DNA labeling in living cells.

BACKGROUND: Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. METHODS: We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous imaging and photobleaching in widefield epifluorescence and confocal laser scanning microscopy. In addition, we compared the DNA stainings with fluorescent histones as an independent fluorescent label to mark chromatin. RESULTS: From the dyes tested, only Hoechst and DRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had several advantages, namely low photobleaching, labeling of the chromatin compartments comparable to that of H2B-GFP fusion proteins, and deep red excitation/emission compatible with available genetically encoded fluorescent proteins such as C/G/YFP or mRFP. CONCLUSIONS: The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and can easily be combined with other fluorophores with blue to orange emission.

Animals↗

PubMed2005

Trapped in action: direct visualization of DNA methyltransferase activity in living cells.

DNA methyltransferases have a central role in the complex regulatory network of epigenetic modifications controlling gene expression in mammalian cells. To study the regulation of DNA methylation in living cells, we developed a trapping assay using transiently expressed fluorescent DNA methyltransferase 1 (Dnmt1) fusions and mechanism-based inhibitors 5-azacytidine (5-aza-C) or 5-aza-2'-deoxycytidine (5-aza-dC). These nucleotide analogs are incorporated into the newly synthesized DNA at nuclear replication sites and cause irreversible immobilization, that is, trapping of Dnmt1 fusions at these sites. We measured trapping by either fluorescence bleaching assays or photoactivation of photoactivatable green fluorescent protein fused to Dnmt1 (paGFP-Dnmt1) in mouse and human cells; mutations affecting the catalytic center of Dnmt1 prevented trapping. This trapping assay monitors kinetic properties and activity-dependent immobilization of DNA methyltransferases in their native environment, and makes it possible to directly compare mutations and inhibitors that affect regulation and catalytic activity of DNA methyltransferases in single living cells.

Animals↗

PubMed2004

Stable chromosomal units determine the spatial and temporal organization of DNA replication.

DNA replication occurs in mammalian cells at so-called replication foci occupying defined nuclear sites at specific times during S phase. It is an unresolved problem how this specific spatiotemporal organization of replication foci is determined. Another unresolved question remains as to what extent DNA is redistributed during S phase. To investigate these problems, we visualized the replicating DNA and the replication machinery simultaneously in living HeLa cells. Time-lapse analyses revealed that DNA was not redistributed to other nuclear sites during S phase. Furthermore, the results showed that DNA is organized into stable aggregates equivalent to replication foci. These aggregates, which we call sub-chromosomal foci, stably maintained their replication timing from S phase to S phase. During S-phase progression, the replication machinery sequentially proceeded through spatially adjacent sets of sub-chromosomal foci. These findings imply that the specific nuclear substructure of chromosomes and the order of their stable subunits determine the spatiotemporal organization of DNA replication.

Animals↗

PubMed2004

Dynamic targeting of the replication machinery to sites of DNA damage.

Components of the DNA replication machinery localize into discrete subnuclear foci after DNA damage, where they play requisite functions in repair processes. Here, we find that the replication factors proliferating cell nuclear antigen (PCNA) and RPAp34 dynamically exchange at these repair foci with discrete kinetics, and this behavior is distinct from kinetics during DNA replication. Posttranslational modification is hypothesized to target specific proteins for repair, and we find that accumulation and stability of PCNA at sites of damage requires monoubiquitination. Contrary to the popular notion that phosphorylation on the NH2 terminus of RPAp34 directs the protein for repair, we demonstrate that phosphorylation by DNA-dependent protein kinase enhances RPAp34 turnover at repair foci. Together, these findings support a dynamic exchange model in which multiple repair factors regulated by specific modifications have access to and rapidly turn over at sites of DNA damage.

Animals↗

PubMed2004

Replication-independent chromatin loading of Dnmt1 during G2 and M phases.

The major DNA methyltransferase, Dnmt1, associates with DNA replication sites in S phase maintaining the methylation pattern in the newly synthesized strand. In view of the slow kinetics of Dnmt1 in vitro versus the fast progression of the replication fork, we have tested whether Dnmt1 associates with chromatin beyond S phase. Using time-lapse microscopy of mammalian cells expressing green-fluorescent-protein-tagged Dnmt1 and DsRed-tagged DNA Ligase I as a cell cycle progression marker, we have found that Dnmt1 associates with chromatin during G2 and M. This association is mediated by a specific targeting sequence, shows strong preference for constitutive but not facultative heterochromatin and is independent of heterochromatin-specific histone H3 Lys 9 trimethylation, SUV39H and HP1. Moreover, photobleaching analyses showed that Dnmt1 is continuously loaded onto chromatin throughout G2 and M, indicating a replication-independent role of Dnmt1 that could represent a novel and separate pathway to maintain DNA methylation.

Animals↗