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 ∼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.

Fig. 1.

PCA/BiFC reveals complex interactions among the six telomeric proteins. A, Principle of the PCA/BiFC assay. The N-terminal fragment (residues 1–155) of Venus YFP (YFPn) and C-terminal fragment (residues 156–239) of YFP (YFPc) were tethered to the N- or C-terminal ends of candidate proteins and expressed in human cells for fluorescence complementation. B, Schematic representation of the retroviral expression vectors for BiFC-based screening. LTR, long terminal repeat. Puro, puromycin selection marker. Neo, neomycin selection marker. YFPn and YFPc, N- or C-terminal fragment of YFP. C, Whole cell extracts from HTC75 cells expressing different Venus YFPn-tagged proteins were Western blotted with anti-GFP antibodies. Fusion proteins are indicated by *. Cells expressing YFPn-tagged SOX2 were used as a control for subsequent analysis. D, Flow cytometry analysis of the 6 × 6 PCA/BiFC assays of core telomeric protein pairs. YFPn-tagged SOX2 served as a negative control. E, Structural arrangement of the subunits within the six telomeric protein complex as determined by PCA/BiFC assays as in (D).

Fig. 2.

Identification of proteins that associate with the six telomeric proteins through our BiFC-based screens. A, Screening data for TRF2. On the basis of CytoArray analysis, a cutoff value of 0.090 (90% confidence level) for the weighted positive ratio (WPR) was applied (indicated by the red line). X axis, prey proteins scored in the screen. Y axis, WPR values. Arrows indicate known interacting prey proteins. B, A heat-map of the identified interaction partners of the core telomere associated proteins. C, Enriched biological processes identified by the screens.

Fig. 3.

Confirmation of protein-protein interactions identified by the screens. A, Fluorescent microscopy images of bait-prey protein pairs tagged with YFP fragments. HTC75 cells stably coexpressing YFPn-tagged TRF1 and YFPc alone (negative control), YFPc-tagged TIN2 (positive control), YFPc-MDH1, or YFPc-SET were visualized live under a fluorescence microscope. Hoechst33342 was added to visualize the nuclei. B, A flow chart of secondary coprecipitation screens that were used to confirm the identified protein-protein interactions. C, Examples of coprecipitation screens. Cell extracts from 293T cells coexpressing FLAG-tagged candidate proteins with GST alone or GST-tagged telomeric bait proteins were incubated with GSH-agarose beads. The proteins bound to GSH beads were then dot-blotted and probed with anti-FLAG antibodies.

Fig. 4.

Identification of signaling networks and regulatory components in the Telomere Interactome. A, Interaction of telomeric proteins with histone proteins. B, DNA repair and chromatin regulators that associate with the six telomeric proteins. C, Protein kinases, phosphatases, and their regulators are identified from the screen. D, Putative ubiquitin E3 ligases were found in the screen.