Genome-wide YFP Fluorescence Complementation Screen Identifies New Regulators for Telomere Signaling in Human Cells

Severance Hospital Integrative Research Institute for Cerebral and Cardiovascular Disease, Yonsei University Health System, Seoul, Korea.
Molecular &amp Cellular Proteomics (Impact Factor: 6.56). 11/2010; 10(2):M110.001628. DOI: 10.1074/mcp.M110.001628
Source: PubMed


Detection of low-affinity or transient interactions can be a bottleneck in our understanding of signaling networks. To address this problem, we developed an arrayed screening strategy based on protein complementation to systematically investigate protein-protein interactions in live human cells, and performed a large-scale screen for regulators of telomeres. Maintenance of vertebrate telomeres requires the concerted action of members of the Telomere Interactome, built upon the six core telomeric proteins TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. Of the ∼12,000 human proteins examined, we identified over 300 proteins that associated with the six core telomeric proteins. The majority of the identified proteins have not been previously linked to telomere biology, including regulators of post-translational modifications such as protein kinases and ubiquitin E3 ligases. Results from this study shed light on the molecular niche that is fundamental to telomere regulation in humans, and provide a valuable tool to investigate signaling pathways in mammalian cells.

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Available from: Dong Yang, May 14, 2014
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    • "Akt regulates telomere protection, X. Han et al. 1097 ª 2013 the Anatomical Society and John Wiley & Sons Ltd Cells expressing protein pairs respectively tagged with YFPc (residues 156–239 of YFP) and YFPn (residues 1–155 of Venus YFP) were analyzed by flow cytometry as previously described (Lee et al., 2011). 293T cells were used for transient expression and retroviral packaging. "
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    ABSTRACT: Telomeres are specialized structures at the ends of eukaryotic chromosomes that are important for maintaining genome stability and integrity. Telomere dysfunction has been linked to aging and cancer development. In mammalian cells, extensive studies have been carried out to illustrate how core telomeric proteins assemble on telomeres to recruit the telomerase and additional factors for telomere maintenance and protection. In comparison, how changes in growth signaling pathways impact telomeres and telomere-binding proteins remains largely unexplored. The phosphatidylinositol 3-kinase (PI3-K)/Akt (also known as PKB) pathway, one of the best characterized growth signaling cascades, regulates a variety of cellular function including cell proliferation, survival, metabolism, and DNA repair, and dysregulation of PI3-K/Akt signaling has been linked to aging and diseases such as cancer and diabetes. In this study, we provide evidence that the Akt signaling pathway plays an important role in telomere protection. Akt inhibition either by chemical inhibitors or small interfering RNAs induced telomere dysfunction. Furthermore, we found that TPP1 could homodimerize through its OB fold, a process that was dependent on the Akt kinase. Telomere damage and reduced TPP1 dimerization as a result of Akt inhibition was also accompanied by diminished recruitment of TPP1 and POT1 to the telomeres. Our findings highlight a previously unknown link between Akt signaling and telomere protection. This article is protected by copyright. All rights reserved.
    Aging cell 07/2013; 12(6). DOI:10.1111/acel.12137 · 6.34 Impact Factor
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    • "A major challenge in such studies is that many signaling regulators are low in abundance or might only transiently or weakly associate with telomeres. Conventional immunoprecipitation and mass-spectrometry might therefore have to be complemented with more sensitive techniques, such as bimolecular fluorescent complementation that enables analysis of protein interactions in living cells and has recently been applied to identify proteins interacting with shelterin (Lee et al., 2011). "
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    ABSTRACT: Telomeres are repetitive sequences at the natural ends of linear eukaryotic chromosomes that protect these from recognition as chromosome breaks. Their ability to do so critically depends on the binding of sufficient quantities of functional shelterin, a six-unit protein complex with specific and crucial roles in telomere maintenance and function. Insufficient telomere length, leading to insufficient concentration of shelterin at chromosome ends, or otherwise crippled shelterin function, causes telomere deprotection. While contributing to aging-related pathologies, loss of telomere protection can act as a barrier to tumorigenesis, as dysfunctional telomeres activate DNA-damage-like checkpoint responses that halt cell proliferation or trigger cell death. In addition, dysfunctional telomeres affect cancer development and progression by being a source of genomic instability. Reviewed here are the different approaches that are being undertaken to investigate the mammalian cellular response to telomere dysfunction and its consequences for cancer. Furthermore, it is discussed how current and future knowledge about the mechanisms underlying telomere damage responses might be applied for diagnostic purposes or therapeutic intervention.
    Frontiers in Oncology 04/2013; 3:88. DOI:10.3389/fonc.2013.00088
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    ABSTRACT: The processes regulating telomere function have major impacts on fundamental issues in human cancer biology. First, active telomere maintenance is almost always required for full oncogenic transformation of human cells, through cellular immortalization by endowment of an infinite replicative potential. Second, the attrition that telomeres undergo upon replication is responsible for the finite replicative life span of cells in culture, a process called senescence, which is of paramount importance for tumor suppression in vivo. The process of telomere-based senescence is intimately coupled to the induction of a DNA damage response emanating from telomeres, which can be elicited by both the ATM and ATR dependent pathways. At telomeres, the shelterin complex is constituted by a group of six proteins which assembles quantitatively along the telomere tract, and imparts both telomere maintenance and telomere protection. Shelterin is known to regulate the action of telomerase, and to prevent inappropriate DNA damage responses at chromosome ends, mostly through inhibition of ATM and ATR. The roles of shelterin have increasingly been associated with transient interactions with downstream factors that are not associated quantitatively or stably with telomeres. Here, some of the important known interactions between shelterin and these associated factors and their interplay to mediate telomere functions are reviewed.
    Nucleus (Austin, Texas) 03/2011; 2(2):119-35. DOI:10.4161/nucl.2.2.15135 · 3.03 Impact Factor
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