Biomedical subjects

Eric Gilson

Publications and source records attributed to Eric Gilson.

29 records · Page 2Linked to original sources

PubMed2003

The number of vertebrate repeats can be regulated at yeast telomeres by Rap1-independent mechanisms.

The number of telomeric DNA repeats at chromosome ends is maintained around a mean value by a dynamic balance between elongation and shortening. In particular, proteins binding along the duplex part of telomeric DNA set the number of repeats by progressively limiting telomere growth. The paradigm of this counting mechanism is the Rap1 protein in Saccharomyces cerevisiae. We demonstrate here that a Rap1-independent mechanism regulates the number of yeast telomeric repeats (TG(1-3)) and of vertebrate repeats (T(2)AG(3)) when TEL1, a yeast ortholog of the human gene encoding the ATM kinase, is inactivated. In addition, we show that a T(2)AG(3)-only telomere can be formed and maintained in humanized yeast cells carrying a template mutation of the gene encoding the telomerase RNA, which leads to the synthesis of vertebrate instead of yeast repeats. Genetic and biochemical evidences indicate that this telomere is regulated in a Rap1-independent manner, both in TEL1 and in tel1Delta humanized yeast cells. Altogether, these findings shed light on multiple repeat-counting mechanisms, which may share critical features between lower and higher eukaryotes.

Base Sequence↗

PubMed2003

A telomere-binding protein (TRF2/MTBP) from mouse nuclear matrix with motives of an intermediate filament-type rod domain.

In previous work, we identified a telomeric DNA-binding protein (termed telomere-membrane binding protein, MTBP) in the envelope of the frog oocyte nucleus and raised antibodies against it. Here we present immunological evidence which suggests strongly that MTBP is identical with the vertebrate telomeric DNA-binding protein TRF2 (telomere-repeat factor 2). MTBP/TRF2 possesses motif which resembles rod domain characteristic of intermediate filament (IF) proteins as shown by immunological cross-reactivity with characteristic antibodies, as well as amino acid sequence homology. Anti-MTBP antibodies recognised a protein of the same M, as TRF2 in extracts of mouse nuclei and nuclear matrix as shown by ion-exchange chromatography, gel shift assays, and Western blots. This mouse MTBP analogue forms more stable complexes with the vertebrate telomeric DNA fragment (T(2)AG(3))(135) than with the corresponding fragment from Tetrahymena (T(2)G(4))(130). Proteins in each of these complexes are recognised by anti-MTBP antibody. In situ hybridization with the vertebrate telomeric DNA sequence (T(2)AG(3))(135) and immunofluorescence with anti-MTBP antibody had shown earlier that these are co-localised in the nucleus of mouse cells, and here MTBP is shown to be associated with the residual membrane of hepatocyte nuclei using Western blotting and immunofluorescence. Some immunofluorescence signal from MTBP is localized at chromosome extremities on metaphase plates from mouse cell culture, but the main signal is seen in patches scattered around the chromosomes which were identified as remnants of the nuclear envelope by double labelling with antibodies against lamin B. These observations suggest that MTBP/TRF2 is a good candidate for the attachment of telomeres to the nuclear envelope in somatic cells.

Animals↗

PubMed2003

A methyltransferase targeting assay reveals silencer-telomere interactions in budding yeast.

We have designed a modified version of the Dam identification technique and used it to probe higher-order chromatin structure in Saccharomyces cerevisiae. We fused the bacterial DNA methyltransferase Dam to the DNA-binding domain of TetR and targeted the resulting chimera to Tet operators inserted in the yeast genome at the repressed locus HML. We then monitored the methylation status of HML and other sequences by a quantitative technique combining methylation-sensitive restriction and real-time PCR. As expected, we found that TetR-Dam efficiently methylated HML in cis. More strikingly, when TetR-Dam was present at HML, we observed increased methylation in the III-L subtelomeric region but not in intervening sequences. This effect was lost when the HML silencers were inactivated by mutations. When the HM silencers and the Tet operators were transferred to a plasmid, strong methylation was clearly observed not only in the III-L subtelomeric region but also at other telomeres. These data indicate that HM silencers can specifically associate with telomeres, even those located on different chromosomes.

Cell Nucleus↗

PubMed2003

Expression of mRNAs for telomeric repeat binding factor (TRF)-1 and TRF2 in atypical adenomatous hyperplasia and adenocarcinoma of the lung.

PURPOSE AND EXPERIMENTAL DESIGN: It has been suggested that atypical adenomatous hyperplasia (AAH) may be a precursor of peripheral adenocarcinoma of the lung. Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA onto chromosomal ends. Its activity is thought to participate in the development of most human cancers. Telomere-specific DNA-binding proteins, such as telomeric repeat binding factor 1 and telomeric repeat binding factor 2, also control telomere length in a complex interplay with telomerase. Here we investigated the expressions of the mRNAs encoded by the TERF1 and TERF2 genes using in situ hybridization in surgically resected specimens [28 AAHs (11 lesions were interpreted as low-grade AAH, and 17 were interpreted as high-grade AAH) and 40 peripherally located bronchioloalveolar carcinoma (BAC). RESULTS: A clear overexpression of these mRNAs was recognized in low- and high-grade AAH and BAC samples (as compared with normal tissues) using in situ hybridization and these mRNAs were detected in normal AAH and BAC samples using reverse transcription-PCR. The expressions of TERF1 and TERF2 mRNA detected by in situ hybridization were scored positive in 36% and 82% of low-grade AAH, 65% and 83% of high-grade AAH, and 88% and 88% of BAC, respectively. Statistically significant differences in TERF1 mRNA expression could be shown between low-grade AAH and BAC and between high-grade AAH and BAC. There was no statistical difference in the positive expressions of TERF2 mRNA among low-grade AAH, high-grade AAH, and BAC. CONCLUSIONS: These results are consistent with (but are not enough to confirm) the idea that high-grade AAH is closely related to BAC.

Adenocarcinoma↗

PubMed2002

The vertebrate protein CTCF functions as an insulator in Saccharomyces cerevisiae.

Insulators are elements that shelter genes from the effects of silencers or enhancers. CTCF is the only vertebrate protein that has a recognized role in transcriptional insulation, but how it exerts its effect is unknown. In an attempt to better understand how CTCF functions, we have used an insulation assay in Saccharomyces cerevisiae. We show that CTCF acts as an insulator in yeast, where it can efficiently block the spreading of repressive telomeric chromatin. We identify two domains of the protein that are responsible for this activity: a short and very potent N-terminal domain, as well as the C-terminus of the protein.

Amino Acid Sequence↗

PubMed2002

Human telomeric position effect is determined by chromosomal context and telomeric chromatin integrity.

We investigated the influence of telomere proximity and composition on the expression of an EGFP reporter gene in human cells. In transient transfection assays, telomeric DNA does not repress EGFP but rather slightly increases its expression. In contrast, in stable cell lines, the same reporter construct is repressed when inserted at a subtelomeric location. The telomeric repression is transiently alleviated by increasing the dosage of the TTAGGG repeat factor 1 (TRF1). Upon a prolongated treatment with trichostatin A, the derepression of the subtelomeric reporter gene correlates with the delocalization of HP1alpha and HP1beta. In contrast, treating the cells with 5 azacytidin, a demethylating agent, or with sirtinol, an inhibitor of the Sir2 family of deacetylase, has no apparent effect on telomeric repression. Overall, position effects at human chromosome ends are dependent on a specific higher-order organization of the telomeric chromatin. The possible involvement of HP1 isoforms is discussed.

Chromatin↗

PubMed2002

General regulatory factors (GRFs) as genome partitioners.

Insulators are sequences that uncouple adjacent chromosome domains. Here we have shown that Saccharomyces cerevisiae Rap1p and Abf1p proteins are endowed with a potent insulating capacity. Insulating domains in Rap1p coincide with previously described transcription activation domains, whereas four adjacent subdomains spanning the whole of the Abf1p C terminus (440-731) were found to display autonomous insulating capacity. That both Rap1p and Abf1p silencing domains either contain or largely overlap with an insulating domain suggests that insulation conveys some undefined chromosome organization capacity that also contributes a function in silencing. Together with Reb1p and Tbf1p, previously involved in the activity of Saccharomyces cerevisiae subtelomeric insulators, insulating potential emerges as a supplementary common property of General Regulatory Factors (GRFs). Thus GRFs, which bind to sites scattered throughout the genome within promoters, would not only play a key role in regulating gene expression but also partition the genome in functionally independent domains.

Chromosomes, Fungal↗

PubMed2002

Natural and pharmacological regulation of telomerase.

The extremities of eukaryotic chromosomes are called telomeres. They have a structure unlike the bulk of the chromosome, which allows the cell DNA repair machinery to distinguish them from 'broken' DNA ends. But these specialised structures present a problem when it comes to replicating the DNA. Indeed, telomeric DNA progressively erodes with each round of cell division in cells that do not express telomerase, a specialised reverse transcriptase necessary to fully duplicate the telomeric DNA. Telomerase is expressed in tumour cells but not in most somatic cells and thus telomeres and telomerase may be proposed as attractive targets for the discovery of new anticancer agents.

Animals↗

PubMed2002

Protosilencers as building blocks for heterochromatin.

DNA repetitions may provoke heterochromatinization. We explore here a model in which multiple cis-acting sequences that display no silencing activity on their own (protosilencers) may cooperate to establish and maintain a heterochromatin domain efficiently. Protosilencers, first defined in budding yeast, have now been found in a wide range of genomes where they appear to stabilize and to extend the propagation of heterochromatin domains. Strikingly, isolated or moderately repeated protosilencers can also be found in promoters where they participate in transcriptional activation and have insulation functions. This suggests that the proper juxtaposition of a threshold number of protosilencers converts them from neutral or transactivating elements into ones that nucleate heterochromatin. Interactions might be transient or permanent, and are likely to occur over distances by looping. This model provides a conceptual framework for as varied phenomena as telomere-driven silencing in Drosophila, X inactivation in mammals, and rDNA silencing in S. cerevisiae. It may also account for the silencing that occurs when multiple copies of a transgene are inserted in tandem.

Animals↗

PubMed2002

Nuclear architecture and spatial positioning help establish transcriptional states of telomeres in yeast.

Recent experiments have shown that gene repression can be correlated with relocation of genes to heterochromatin-rich silent domains. Here, we investigate whether nuclear architecture and spatial positioning can contribute directly to the transcriptional activity of a genetic locus in Saccharomyces cerevisiae. By disassembling telomeric silent domains without altering the chromatin-mediated silencing machinery, we show that the transcriptional activity of silencer--reporter constructs depends on intranuclear position. This demonstrates that telomeric silent domains are actively involved in transcriptional silencing. Employing fluorescent in situ hybridization (FISH) in combination with genetic assays, we demonstrate that telomeres control the establishment of transcriptional states by reversible partitioning with the perinuclear silencing domains. Anchoring telomeres interferes with their ability to assume an active state, whereas disassembly of silencing domains prevents telomeres from assuming a repressed state. Our data support a model in which domains of enriched transcriptional regulators allow genes to determine transcriptional states by spatial positioning.

Cell Nucleus↗

PubMed2002

Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2.

We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2. To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced. We demonstrate that their targeting leads to telomere shortening. This indicates that these proteins act in cis to repress telomere elongation. Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting and telomerase inhibition have additive effects. The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase. In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.

Bacterial Proteins↗