1,720,979 research outputs found
Structural study of the human telomeric chromatin
No full textTelomeres are the special nucleoprotein structures that protect chromosome ends from both recombination and degradation. In most organisms, telomeric DNA consists of short sequences repeated in tandem ending in single stranded G-rich-overhangs.
About 80% of telomeric DNA is organized in tightly packed nucleosomes separated by 10-20 bp of linker DNA. Several specific proteins contribute to telomeric structure. The protein hPOT1 binds to single stranded G-rich-overhangs, whereas double stranded telomeric DNA is specifically recognized by hTRF1 and hTRF2. hTRF1, hTRF2, and hPOT1 associate with hRap1, Tin2 and TPP1 to form the complex named shelterin, essential for telomere protection. In addition, several other factors, many of which are involved in DNA repair and recombination, are also recruited to telomeres. At the moment, a satisfactory description of telomere organization is still lacking. The emerging view is that there is an interconversion among different structures along with the cell cycle and development.
While the relevance of telomeric proteins has been widely investigated, the role played by nucleosomes and histone post-translational modifications in the protection of chromosome ends represents a research field almost completely unexplored.
This thesis addresses a relevant question in telomere biology, that is whether the human telomeric proteins TRF1 and TRF2 interplay with telomeric nucleosomes and whether they affect the organization and the epigenetic status of telomeric chromatin.
In particular, the relation of TRF2, the protein essential for telomere protection, with nucleosomes has been studied by overexpressing TRF2, by transient expression of wild-type and dominant-negative genes. Cancer cells (C33A) and immortalized fibroblasts (HT1080) have been transfected with plasmids encoding hTRF2, hTRF2ΔBΔC. The effect of altered concentrations of TRF2 has been analyzed by chromatin immunoprecipitation assay (ChIP). We found that the density of nucleosomes at human telomere depends on TRF2 expression. Moreover, in order to establish if the association of telomeric proteins and histones at telomeres are cell-cycle regulated we performed ChIP experiments after cell synchronization. Cell synchronization has been checked by flow cytometry and ChIP assay has been performed at various times after cell synchronization. We observed that the remodeling effect of TRF2 occurs outside replication. In parallel with ChIP assay, we also evaluated the organization of telomeric chromatin by MNase mapping. Nuclei of control C33A cells and TRF2-overexpressing cells were isolated and then digested with increasing amounts of micrococcal nuclease (MNase). We found that telomeric chromatin shows a higher sensitivity to MNase in TRF2-overexpressing cells. In addition, using Drosophila extracts to assemble nucleosomal arrays in vitro, we showed that the spacing between telomeric nucleosomes is increased by the presence of TRF2. Finally, the epigenetic status of C33A telomeres as a function of TRF2 expression has been characterized by ChIP, using specific antibodies for heterochromatic marks (H3K9me3, H4K20me3), euchromatic marks (H3K4me2), histone variants (H2AX). We observed a decreased density of epigenetic marks in TRF2-overexpressing cells and an enrichment of H2AX at telomeres. All together, these results indicate an impact of TRF2 on nucleosomal organization at mammalian telomeres, further highlighting the importance of TRF2 in telomere protection
Telomeric nucleosomes: Forgotten players at chromosome ends
No full textTelomeres are the special nucleoprotein structures that protect chromosome ends from both recombination and degradation. In most organisms, telomeric DNA consists of short sequences repeated in tandem ending in single-stranded G-rich overhangs. In higher eukaryotes, about 80%of telomeric DNAis organized in tightly packed nucleosomes separated by 10–20 bp of linker DNA. Several specific proteins contribute to telomeric structure. At the moment, a satisfactory description of telomere organization is still lacking. Whereas the role played by telomeric proteins in telomere function and regulation has been widely investigated, little is known about the contribution of nucleosomes to the protection of chromosome ends. In this review we present an overview on the chromatin organization in lower and higher eukaryotes, and discuss the recent results on the peculiar features of telomeric nucleosomes and on the epigenetic status of mammalian telomeres
Telomere maintenance in the dynamic nuclear architecture
No full textTelomeres, the protective structures at the end of eukaryotic chromosomes, play a pivotal role in several regulatory pathways that determine the cell fate. Human telomeres consist of thousands of TTAGGG repeats organized in a peculiar compact chromatin and bound by the six-protein complex shelterin. In germinal and embryonic stem cells telomere length is maintained by the activity of telomerase that adds TTAGGG repeats at the 3’ ends of chromosomes. On the contrary, in somatic cells telomerase is inactive and consequently telomeres shorten at each replication, till they reach a critical length that triggers a DNA damage response pathway leading to cell growth arrest, a state known as replicative senescence.
In this chapter, we review what is known about telomere structure and telomeric chromatin organization. We will discuss the dynamic changes of telomeres and the epigenetic and structural modifications linked to telomere shortening and the entry in replicative senescence
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
TRF2 NEGATIVELY AFFECTS NUCLEOSOME ASSEMBLY ON TELOMERIC SEQUENCES
No full textAn unresolved question in the organization of mammalian telomeres is whether specific telomeric proteins compete with the histone octamer for binding to telomeric sequences or whether they cooperate to form a telomeric protective structure. By means of in vitro model systems we previously demonstrated that TRF1 is able to recognize nucleosomal binding sites and to alter nucleosome structure. We show that, unlike TRF1, TRF2 is unable to bind to nucleosomal binding sites, suggesting that TRF2 and the histone octamer compete for binding to telomeres. To investigate how TRF2 interplays with telomeric nucleosomes, we set up a model system to assemble telomeric nucleosomal arrays by using Drosophila embryonic extracts. We found that telomeric nucleosomes are spaced every 155±5 bp, indicating that the short nucleosomal spacing found in vivo is a sequence-dependent feature. Remarkably, when added to the Drosophila extracts TRF2 increases nucleosomal repeat length to about 195±5 bp, indicating that TRF2 may preferentially position between nucleosomes
INTERPLAY BETWEEN NUCLEOSOMES AND TELOMERIC PROTEINS. DO THEY INTERACT OR COMPETE FOR TELOMERIC SEQUENCES?
No full textIn higher eukaryotes, both specific proteins and the histone octamer bind to telomeric repeats. Whether histones and specific telomeric proteins compete for telomeric DNA binding (and hence occupy different telomere domains) or whether they cooperate in the formation of the telomeric complex is a relevant issue in order to understand telomeric structure and its dynamics.
In humans, TRF1 and TRF2 bind as preformed homodimers recognizing two telomeric TTAGGG repeats. Since most of telomeric DNA is packed in nucleosomes, a possible hypothesis is that TRF proteins, after saturating the short DNA linkers between nucleosomes, could interact with nucleosomal binding sites. We have previously shown that hTRF1 forms stable ternary complexes with telomeric nucleosomes.
By means of in vitro model systems we show that also hTRF2 recognize nucleosomal binding sites, but with a lower affinity with respect to hTRF1. This difference does not depend on DNA binding domains (DBDs), since the DBDs of hTRF1 and hTRF2 have the same affinity for nucleosomal binding sites. Binding of hTRF1 to naked DNA, but not of hTRF2, is favored by the presence of an adjacent nucleosome. Importantly, hTRF1 binding induces nucleosome sliding, enhancing the intrinsic mobility of telomeric nucleosomes, whereas hTRF2 does not apparently influence nucleosome mobility. These results suggest that the different interactions of hTRF1 and hTRF2 with nucleosomes may play a major role in the architecture of telomeric chromatin
Targeting hTERT core promoter with specific G-quadruplex ligands
No full textIn mammalian somatic cells telomeres shorten at each replication till they reach a critical length that leads to cell replicative senescence. Cancer cells overcome this limit by acquiring the capability to maintain functional telomeres. In most cases, this is due to upregulation of the expression of hTERT, encoding the proteic moiety of telomerase. Therefore, several anti-cancer strategies that target telomerase activity and hTERT expression are being developed. One of these strategies is based on ligands that induce the formation of a G-quadruplex structure at the ends of chromosomes, thus preventing telomerase elongation.
Recently, it has been reported that the hTERT core promoter contains a G-rich region able to form multiple G-quadruplex structures, representing a possible target for G-quadruplex ligands. By means of circular dichroism (CD) spectroscopy and DNA polymerase stop assay we report that a series of perylene derivatives with different hydrophilic side chains (HPDIs, hydrosoluble perylene diimides) are able to specifically stabilize G-quadruplex structures and superstructures on the hTERT promoter and to inhibit DNA replication. These data suggest that ligand-induced G-quadruplex formation on hTERT promoter could affect hTERT expression. An analysis of HPDIs effect on hTERT promoter activity by luciferase reporter assay is in progress.
An attractive hypothesis is that G-quadruplex ligands could inhibit telomerase activity not only by targeting the terminal telomeric G-tails but also by inducing G-quadruplex structures on the hTERT promoter
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