Dynamics of Protein Binding to Telomeres in Living Cells: Implications for Telomere Structure and Function

Mattern, Karin; Swiggers, Susan J. J.; Nigg, Alex L.; Löwenberg, Bob; Houtsmuller, Adriaan B.; Zijlmans, J. M. J. M.

doi:10.1128/mcb.24.12.5587-5594.2004

Cited by 86 publications

(76 citation statements)

References 40 publications

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“…In CLEM-FRAP and CLEM-FLIP experiments, we observed an ephemeral binding for TRF2. The dynamic binding of TRF2 has been assessed before in HeLa cells and it was proposed to allow for rapid adaptation in telomere-length regulatory processes (40). The volatility of TRF2 might also be explained by its versatility in function.…”

Section: Discussionmentioning

confidence: 99%

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Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle

et al. 2008

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Telomeres are complex end structures that confer functional integrity and positional stability to human chromosomes. Despite their critical importance, there is no clear view on telomere organization in cycling human cells and their dynamic behavior throughout the cell cycle. We investigated spatiotemporal organization of telomeres in living human ECV-304 cells stably expressing telomere binding proteins TRF1 and TRF2 fused to mCitrine using four dimensional microscopy. We thereby made use of controlled light exposure microscopy (CLEM), a novel technology that strongly reduces photodamage by limiting excitation in parts of the image where full exposure is not needed. We found that telomeres share small territories where they dynamically associate. These territories are preferentially positioned at the interface of chromatin domains. TRF1 and TRF2 are abundantly present in these territories but not firmly bound. At the onset of mitosis, the bulk of TRF protein dissociates from telomere regions, territories disintegrate and individual telomeres become faintly visible. The combination of stable cell lines, CLEM and cytometry proved essential in providing novel insights in compartment-based nuclear organization and may serve as a model approach for investigating telomere-driven genome-instability and studying long-term nuclear dynamics. ' 2008 International Society for Advancement of Cytometry Key termstelomere; TRF1; TRF2; nuclear organization; chromatin dynamics; CLEM; live cell imaging TELOMERES are the natural ends of linear chromosomes. A mammalian telomere consists of a double stranded array of simple TTAGGG repeats ending in a single stranded overhang that folds back to form a T-loop structure (1). In combination with sufficient telomere repeats, a complex of indirect and direct telomere binding proteins, dubbed shelterin, assures proper telomere structure and function. The specificity of shelterin for telomeric DNA is due to the recognition of TTAGGG repeats by three of its components: the homodimers telomeric repeat binding factor 1 and 2 (TRF1 and TRF2) that bind the duplex part of telomeres, and protection of telomeres 1 that binds to the single stranded TTAGGG repeats present at the three-overhang and in the D loop of the T-loop configuration (2-5).The nucleus has a meticulous organization with functional relevance to the cell assuring proper gene expression, replication and genome stability (6-8). Telomeres are considered to play an important role in spatial genome organization. Whereas yeast telomeres are known to form few large silencing clusters at the heterochromatic nuclear periphery (9,10), there is no consensus on how telomeres are organized in the mammalian nucleus. From an architectural point of view, human telomeres have been proposed to be anchored to a nuclear scaffold, thereby possibly providing struc-

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Section: Discussionmentioning

confidence: 99%

“…To estimate the mobility of TRF2 in telomere regions, we used both FRAP and FLIP techniques (39,40). One of the main technical problems in FRAP is photobleaching during monitoring of fluorescence recovery.…”

Section: Trf2 Dynamically Binds To the Telomeric Regionmentioning

confidence: 99%

Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle

et al. 2008

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“…(96) Interestingly, the residence time of unbound GFP-TRF1 is only 3 seconds which is five times lower, compared to the residence time of this protein at ITS. (96) Therefore, like telomeres (98) and constitutive heterochromatin, (97) ITS are dynamic structures maintained by continuous exchange and competition of specific proteins for the binding sites in these structures.…”

Section: Global Sine Clustering and Line Depletion In Gc-rich Gene-rimentioning

confidence: 99%

Regulation of mammalian gene expression by retroelements and non‐coding tandem repeats

Tomilin

2008

BioEssays

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Genomes of higher eukaryotes contain abundant non‐coding repeated sequences whose overall biological impact is unclear. They comprise two categories. The first consists of retrotransposon‐derived elements. These are three major families of retroelements (LINEs, SINEs and LTRs). SINEs are clustered in gene‐rich regions and are found in promoters of genes while LINEs are concentrated in gene‐poor regions and are depleted from promoters. The second class consists of non‐coding tandem repeats (satellite DNAs and TTAGGG arrays), which are associated with mammalian centromeres, heterochromatin and telomeres. Terminal TTAGGG arrays are involved in telomere capping and satellite DNAs are located in heterochromatin, which is implicated in transcription silencing by gene repositioning (relocalization). It is unknown whether interstitial TTAGGG sequences, which are present in many vertebrates, have a function. Here, evidence will be presented that retroelements and TTAGGG arrays are involved in regulation of gene expression. Retroelements can provide binding sites for transcription factors and protect promoter CpG islands from repressive chromatin modifications, and may be also involved in nuclear compartmentalization of transcriptionally active and inactive domains. Interstitial telomere‐like sequences can form dynamically maintained three‐dimensional nuclear networks of transcriptionally inactive domains, which may be involved in transcription silencing like classic heterochromatin. BioEssays 30:338–348, 2008. © 2008 Wiley Periodicals, Inc.

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“…35,36 Using these methods, it has become clear that many nuclear proteins, which have previously been thought of as components of a stable nuclear structure, now exhibit rapid movement in the nucleus and fast exchange at specific sites. [37][38][39] We previously reported the successful application of these techniques to the study of the dynamic behavior of DNAPKcs at DSBs in living cells. 12 To understand how XLF and XRCC4 are recruited to DSBs, we employed the same approach to monitor the dynamics of XLF and XRCC4 in various NHEJ deficient cells and their complemented cells.…”

Section: Dynamic Movement Of Nuclear Proteins In Living Cellsmentioning

confidence: 99%

Live cell imaging of XLF and XRCC4 reveals a novel view of protein assembly in the non-homologous end-joining pathway

Yano¹,

Chen

2008

Cell Cycle

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XLF, also known as Cernunnos, is a newly identified core factor of the non-homologous end-joining (NHEJ) pathway for DNA doublestrand breaks (DSBs) repair. XLF is known to stimulate DNA ligase IV in vitro through its interaction with XRCC4. Here, we outline the key findings on the dynamic behavior of XLF and XRCC4 at DSBs in living cells. XLF is quickly recruited to DSBs in the absence of XRCC4 or DNA-PKcs. The recruited XLF molecules constantly exchange at DSBs, and XRCC4 modulates the exchange rate of the recruited XLF. XRCC4 can be recruited to DSBs without DNA-PKcs, but DNA-PKcs stabilizes the recruited XRCC4. These observations are inconsistent with the prevailing concept that NHEJ proteins are sequentially recruited to DSBs, which is mainly supported by in vitro evidence. We propose a novel two-phase model for the assembly of NHEJ factors to DSBs in vivo. XLF, XRCC4 and DNA-PKcs are independently recruited to Ku-bound DSBs. The recruited factors are assembled into a large complex, in which the protein interactions observed in vitro define the stability of the recruited factors. This new view has broad implications for the mechanism of DSB sensing and functional protein assembly in the NHEJ pathway.

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Dynamics of Protein Binding to Telomeres in Living Cells: Implications for Telomere Structure and Function

Cited by 86 publications

References 40 publications

Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle

Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle

Regulation of mammalian gene expression by retroelements and non‐coding tandem repeats

Live cell imaging of XLF and XRCC4 reveals a novel view of protein assembly in the non-homologous end-joining pathway

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