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The role of DNA sequence in telomeric chromatin features
No full textThe structure of human telomeres is not yet completely characterized. Several proteins involved in the formation of the human telomeric complex have been identified, such as hTRF1 and hTRF2 which bind to GGGTAA duplex telomeric repeats. Notwithstanding most human telomeric DNA is organized into tightly spaced nucleosomes, the role of nucleosomes in telomeric chromatin is largely unknown. We previously found that telomeric nucleosomes are less stable than bulk nucleosomes and occupy multiple isoenergetic positions spaced every telomere repeat, due to the peculiar features of telomeric repeated sequences. We will present data on some important sequence-dependent features of telomeric nucleosomes, such as intrinsic nucleosome mobility and the assembly in the 30 nm fiber, by means of an in vitro model system, AFM visualization and theoretical calculations. Data on the interaction of telomeric nucleosomes with the proteins hTRF1 and hTRF2 will be also presented
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
Structural and dynamic features of telomeric chromatin
No full textTelomeres are the specialized nucleoprotein structures that protect the ends of eukaryotic chromosomes. Vertebrate telomeres consist of several kb of double-stranded TTAGGG repeats, ending in 100-200 bases of single-stranded TTAGGG, named 3’-overhang. Protection of chromosome termini is achieved through the establishment of specialized structures, whose precise organization is not known yet. Telomeres are organized in nucleosomal arrays, with an unusually shorter spacing than bulk chromatin. Little is known about their role in the higher order telomeric chromatin structure.
In order to investigate structural features of telomeric chromatin, in this thesis a model system has been developed and studied at single molecule level by Atomic Force Microscopy (AFM) imaging. Human telomeric nucleosomal arrays, reconstituted on a 1500 bp long telomeric DNA have been analyzed. The same nucleosomal arrays have also been studied by a theoretical method, which derives nucleosomal positioning from the sequence-dependent DNA mechanical properties (curvature and flexibility). A satisfactory correlation between the theoretical and experimental results has been obtained, suggesting a relevant role of DNA sequence in telomeric chromatin basic organization. Both the experimental and theoretical telomeric nucleosome positioning appear characterized by nucleosome dyad axis multiple positioning with the six bp periodicity of human telomeric DNA. Moreover, the barriers between adjacent free energy minima of nucleosome formation are so low that it is possible to consider the nucleosomal organization on human telomeric DNA as almost continuous and the nucleosomes as free moving along DNA. These features suggest a higher mobility of telomeric nucleosomes with respect to that of bulk nucleosomes.
Since the intrinsic mobility of telomeric nucleosomes could be a relevant feature in telomeres dynamics, a model system has been set up to study nucleosome mobility. A construct has been realized, in which nucleosomes, initially located at the end of an 800 bp long DNA fragment, are adjacent to a strong nucleosome positioning sequence that should act as a nucleosome trap. By a restriction enzyme assay and AFM imaging it has been found a significant higher mobility of telomeric nucleosomes than average sequence nucleosomes. In fact, the mobility of telomeric nucleosome increases rising the temperature and the ionic strength. Moreover, the telomeric nucleosome mobility is influenced by specific telomeric protein, hTRF1, since hTRF1 binding to telomeric nucleosome gives rise to significant nucleosome repositioning
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
Synthetic oligonucleotides as models of superstructural features of regulative DNA sequences
No full textSELECTIVE BINDING OF ACTINOMYCIN-D INDUCES A REVERSIBLE CONFORMATIONAL TRANSITION OF NUCLEOSOMES
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