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

Abstract

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 ϳ12,000 human proteins examined, we identified over 300 proteins that associated with the six core telomeric pro- During mammalian DNA replication, linear chromosomal ends will gradually erode because of the inability of the DNA replication machinery to replicate the extreme 5Ј terminus of a linear DNA sequence (1, 2). This inherent "end replication problem" is circumvented through specialized chromosomal end structures (telomeres) and the action of the RNA-containing DNA polymerase -telomerase (3-9). Telomere homeostasis is essential for genome stability, cell survival, and growth.Telomeres and telomerase help to ensure genome integrity in eukaryotes by enabling complete replication of the ends of linear DNA molecules, and preventing chromosomal rearrangements or fusion. For dividing cells such as stem cells and the majority of cancer cells, the telomerase is an essential positive regulator of their telomere length and ultimately determines the proliferative potential of these cells.Mammalian telomeres consist of a series of (TTAGGG)n sequence repeats and terminate in 3Ј single-stranded DNA overhangs that are extendable by the telomerase (10). Exposed linear chromosome ends or naturally occurring doublestranded breaks pose additional risks including activation of DNA damage responses. The ends of telomeres in mammalian cells appear to fold back in a T-loop structure, with the 3Ј G-rich single-stranded overhang invading into the doublestranded telomere regions to form the D-loop (11). The structure of the telomeres, coupled with the coordinated action of a collection of proteins that protect the ends of chromosomes (12-15), contributes to the maintenance of telomere integrity, genome stability, and proper cell cycle progression.In mammals, the most widely studied telomere-associated proteins include the double-stranded DNA binding proteins TRF1 and TRF2 (16,17), the single-stranded telomeric DNA binding protein POT1 (18), and three associated factors (RAP1, TIN2, and TPP1) (19 -23). Work from our lab and others suggest that TPP1, along with POT1, TIN2, TRF1, TRF2, and RAP1, form a higher order complex (the telosome/ shelterin) at the telomeres (24 -27). Information regarding the state of the telomere ends can be transmitted from TRF1 and TRF2 to POT1, through TPP1 and the other subunits (28). Furthermore, TRF1 and TRF2 function as bona fide protein hubs and interact with a diverse array of factors/complexes that are involved in cell cycle, DNA repair, and recombinat...

Essential role for mammalian apurinic/apyrimidinic (AP) endonuclease Ape1/Ref-1 in telomere maintenance

“…Hence, the presence of Ape1 at telomeres may reflect the amount of BER running in telomeres during routine cell metabolism. Supporting our finding that Ape1 associates with the members of the telomere complex such as TRF2 and POT1, Ape1 was identified as a protein interacting with the core telomere proteins including TRF2 (35). Furthermore, shelterin proteins TRF1, TRF2, and POT1 have been recently shown to stimulate Ape1 endonuclease activity on a telomeric abasic substrate in vitro (36).…”

Screening for Host Proteins Interacting with Escherichia Coli O157:H7 EspF using Bimolecular Fluorescence Complementation

“…They then tagged the 300 genes with FLAG tag and coexpressed them individually with their respective GST-tagged bait proteins, determining results by GST pull down. They found that about 72% of the interactions found using BiFC could be verified by GST pull down [27,68]. We conducted largescale, high-precision screening of the proteins interacting with EHEC O157:H7 EspF, using a BiFC platform and flow-sorting technology for the first time.…”

Shelterin complex and associated factors at human telomeres

“…A number of proteomics studies have confirmed the architecture and composition of the shelterin complex, and linked telomere function to as many as over three hundred activities believed to participate in telomere integrity. [50][51][52][53] Notably, pathways linking shelterin with the ubiquitin-proteasome pathway, the DNA damage response, RNA processing and other systems have been uncovered. In this review, we focus on some of these pathways, all confirmed by proteomic studies and analyzed by cell and biochemical methods for which an important functional and mechanistic picture has emerged.…”