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 & 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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    • "All tested BiFC-positive TFs were found by co-ip (Supplementary file 3), highlighting that the S2 cell environment is appropriate for revealing interactions with tissue-specific TFs. Thus, observations from BiFC could be reproduced by co-ip, as previously noticed (Lee et al., 2011). Co-ip was also performed with the three positive competitors that did not produce BiFC with AbdA (Kr, Lmd, Pan). "
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    ABSTRACT: eLife digest In all animals, it is important that cells are correctly organised into tissues and organs. This organisation starts in the embryo, and cells are instructed to perform different roles depending on their position within the body. A family of proteins called the Hox proteins coordinates the organisation of the cells in the animal embryo by binding to and controlling the expression of specific genes. To properly control their target genes, Hox proteins need to interact with other proteins called transcription factors that can also bind to the genes. However, only a few of these transcription factors have been identified so far, and it is not clear how Hox proteins are able to interact with them. Here, Baëza, Viala, Heim et al. identified several more transcription factors that can bind to the Hox proteins in fruit fly embryos. The experiments show that Hox proteins are able to bind to many transcription factors that are very different from each other. Baëza, Viala, Heim et al. also show that two short sections within the Hox proteins known as short linear motifs are important for controlling these interactions. A fly Hox protein that was missing these motifs was able to interact with new transcription factors. This inhibitory role was found in Hox proteins from mice and sea anemones, suggesting that these motifs may play the same role in all animals. Baëza, Viala, Heim et al.'s findings challenge the traditional view of the role of the short linear motifs in interactions between proteins. Also, the findings provide an alternative explanation for how the Hox proteins are only able to interact with particular transcription factors in animal embryos. The next step will be to find out whether the inhibitory role of short linear motifs could more generally apply to many other protein families. DOI:
    Full-text · Article · Apr 2015 · eLife Sciences
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    • "Bi - molecular fluorescence complementation assay Pair - wise examination of protein – protein interactions by BiFC was carried out as previously described ( Lee et al . , 2011 ) . HTC75 cells stably co - expressing two proteins respectively tagged with YFP fragments ( YFPn and YFPc ) were generated for fluorescence complementation assessment in live cells by flow cytometry ."
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    ABSTRACT: Most human cancers depend on the telomerase to maintain telomeres; however, about 10% of cancers are telomerase negative and utilize the Alternative Lengthening of Telomeres (ALT) mechanism. Mutations in the DAXX gene have been found frequently in both telomerase-positive and ALT cells, and how DAXX mutations contribute to cancers remains unclear. We report here that endogenous DAXX can localize to Cajal bodies, associate with the telomerase, and regulate telomerase targeting to telomeres. Furthermore, disease mutations that are located in different regions of DAXX differentially impacted its ability to interact with its binding partners, and its targeting to Cajal bodies and telomeres. In addition, DAXX inhibition by RNAi led to reduced telomerase targeting to telomeres and telomere shortening. These findings collectively support a DAXX-centric pathway for telomere maintenance, where DAXX interaction with the telomerase regulates telomerase assembly in Cajal bodies and telomerase targeting to telomeres.
    Preview · Article · Nov 2014 · Journal of Cell Science
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    • "It is interesting to note that our BIFC screen of genome-wide binding partners of TPP1 identified many candidates with no apparent function on telomeres (Lee et al., 2011). And recent findings of TIN2 are prime examples of how telomere proteins may have important roles in diseases and in areas outside of telomeres. "
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    ABSTRACT: Telomeres, the ends of linear eukaryotic chromosomes, are tandem DNA repeats and capped by various telomeric proteins. These nucleoprotein complexes protect telomeres from DNA damage response (DDR), recombination, and end-to-end fusions, ensuring genome stability. The human telosome/shelterin complex is one of the best-studied telomere-associated protein complexes, made up of six core telomeric proteins TRF1, TRF2, TIN2, RAP1, POT1, and TPP1. TPP1, also known as adrenocortical dysplasia protein homolog (ACD), is a putative mammalian homolog of TEBP-β and belongs to the oligonucleotide binding (OB)-fold-containing protein family. Three functional domains have been identified within TPP1, the N-terminal OB fold, the POT1 binding recruitment domain (RD), and the carboxyl-terminal TIN2-interacting domain (TID). TPP1 can interact with both POT1 and TIN2 to maintain telomere structure, and mediate telomerase recruitment for telomere elongation. These features have indicated TPP1 play an essential role in telomere maintenance. Here, we will review important findings that highlight the functional significance of TPP1, with a focus on its interaction with other telosome components and the telomerase. We will also discuss potential implications in disease therapies.
    Full-text · Article · Jun 2014
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