How shelterin solves the end-protection problem

Lab members investigating How shelterin solves the end-protection problem:


Nanda Sasi and

Audrey Goldfarb: Molecular mechanisms of t-loop formation and ATM suppression by TRF2


John Zinder and Logan Myler: Structural aspects of human and mouse shelterin

Overview

The multi-subunit shelterin complex is crucial for the protection of telomeres from the DNA damage response and regulates telomere maintenance by telomerase. Shelterin is composed of six proteins: TRF1, TRF2, Rap1, TIN2, TPP1, and POT1. TRF1 and TRF2 bind to the duplex telomeric repeat array and anchor shelterin on telomeres. POT1 binds to single-stranded TTAGGG repeats and is recruited to telomeres through its interaction with TPP1. TPP1 in turns binds to TIN2, which interacts with both TRF1 and TRF2. Due to its specificity for the sequence and structure of the telomeric DNA, shelterin accumulates at the ends of human and mouse chromosomes but does not bind to DNA ends elsewhere in the genome. Thus, shelterin constitutes a unique marker of telomeres that allows cells to distinguish natural chromosome ends from sites of DNA damage. Our main approach to understanding how shelterin solves the end-protection problem is to generate mouse cells from which individual shelterin proteins can be removed using inducible systems, e.g. Cre-mediated deletion. Mouse and human shelterin are nearly identical except for the presence of two POT1 proteins (POT1a and POT1b) in mouse shelterin.

Cre-mediated deletion of individual shelterin subunits showed that shelterin is highly compartmentalized such that distinct subunits are dedicated to different DDR pathways. TRF2 is critical for the repression of ATM signaling and prevents fusion of telomeres by the c-NHEJ and alt-NHEJ pathways. POT1 is needed to prevent the activation of the ATR kinase at telomeres. POT1a is primarily responsible for this function, whereas POT1b controls the formation of the 3’ overhang. TPP1 mediates the functions of POT1 by tethering POT1 via TIN2 to the rest of shelterin. The function of Rap1 is to repress Homology-Directed Repair (HDR) together with one of the two POT1 proteins. TRF1 has no direct role in protecting the chromosome end but is dedicated to promoting the replication of the telomeric DNA.

Repression of ATM signaling and NHEJ by TRF2: t-loops

Inappropriate NHEJ at telomeres can lead to unstable dicentric chromosomes and needs to be stringently repressed. NHEJ-mediated fusion of telomeres is rampant when TRF2 is deleted from mouse cells, resulting in long trains of joined chromosomes. The telomere fusions that occur in the absence of TRF2 are formed through the loading of the Ku70/80 heterodimer onto telomere ends and involve ligation by DNA ligase IV, indicating that they are due to c-NHEJ. Alternative-NHEJ, mediated by PARP1 and DNA ligase III can also take place at telomeres but only when shelterin is impaired in cells that lack the Ku70/80 heterodimer.

Similarly, the activation of ATM signaling at telomeres needs to be averted. When TRF2 is deleted, most telomeres are recognized by the ATM kinase pathway, leading to DNA damage foci at telomeres (called Telomere Dysfunction Induced Foci or TIFs) that contain γ-H2AX, MDC1, 53BP1 and other DDR factors. ATM kinase activation at telomeres involves recognition of the telomere end by the Mre11/Rad50/Nbs1 (MRN complex).

In collaboration with Jack Griffith (University of North Carolina, Chapel Hill) we found that telomeres can occur in a lariat conformation, referred to as the t-loop. T-loops are formed through the strand invasion of the 3’ telomeric overhang into the duplex part of the telomere. Since the discovery of t-loops in mammals, they have been found in many other eukaryotes, including protozoa, plants, and some fungi.

Given that the telomere terminus is sequestered in the t-loop configuration we proposed that this structure would protect telomeres. Specifically, the t-loop structure would render telomeres impervious to c-NHEJ, which requires the loading of the Ku70/80 complex on free DNA ends, and would prevent the activation of the ATM kinase, which involves binding of the MRN complex to DNA ends. TRF2, the only shelterin protein required for the repression of c-NHEJ and ATM signaling, has the ability to make t-loops in vitro. We tested the TRF2/t-loop model by using super-resolution STORM imaging to detect t-loops in relaxed chromatin from cells with and without TRF2. The results demonstrated that TRF2, but not the other components of shelterin, is required for the establishment and/or maintenance of t-loops.

Repression of ATR signaling by POT1

The constitutive ssDNA at telomeres can activate the ATR kinase. The ATR kinase signaling is activated through the binding of RPA to ssDNA and the Rad17-dependent loading of 9-1-1 on the neighboring 5’ ds/ss transition. ATR is recruited by ATRIP-dependent binding to RPA and is activated when TopBP1 interacts with the 9-1-1 complex. The 3’ overhang of mammalian telomeres is of sufficient length to bind RPA and ATR activation can occur if shelterin fails to protect the telomeres. The t-loop configuration does not protect telomeres from ATR signaling because all DNA structures needed for RPA binding and TopBP1-mediated ATR activation are present at the base of the t-loop.  

Shelterin uses POT1 to repress ATR signaling. In human shelterin, this task is delegated to the single POT1 protein, whereas mouse shelterin has two ATR repressors, POT1a and POT1b. When POT1a and POT1b are both deleted, ATR is activated at telomeres throughout the cell cycle. As expected, this activation is dependent on ATRIP and RPA. By binding to the ss telomeric DNA, POT1 blocks the accumulation of RPA at telomeres and thereby prevents ATR activation. As RPA is much more abundant than POT1 and since POT1 and RPA have the same affinity for telomeric sequences, the tethering of POT1 to the rest of shelterin is the critical aspect of its ability to exclude RPA from the ssDNA.

Publications since 2010:
L.R. Myler, C.G. Kinzig, N.K. Sasi, G. Zakusilo, S.W. Cai and T. de Lange (2021) The evolution of metazoan shelterin.  Genes Dev 35:1625-1641.

L.A. Timashev & T. de Lange (2020) Characterization of t-loop formation by TRF2. Nucleus 11: 164-177.

K. Kratz & T. de Lange (2018) Protection of telomeres 1 proteins POT1a and POT1b can repress ATR signaling by RPA exclusion, but binding to CST limits ATR repression by POT1b. J Biol Chem: 293: 14384-14392.

T. de Lange (2018) Shelterin-mediated telomere protection. Ann. Rev. Genetics 52:223-247.

T. de Lange (2018) What I got wrong about shelterin. J. Biol. Chem. 293: 10453-10456.

L.A. Timashev, H. Babcock, X. Zhuang & T. de Lange (2017) The DDR at telomeres lacking intact shelterin does not require substantial chromatin decompaction. Genes Dev 31: 578-589.

I. Schmutz, L. Timashev, W. Xie, D.J. Patel & T. de Lange (2017) TRF2 binds branched DNA to safeguard telomere integrity. Nat. Struct. Mol. Biol.: 24:734-742.

F. Erdel, K. Kratz, S. Willcox, J.D. Griffith, E.C. Greene & T. de Lange (2017) Telomere Recognition and Assembly Mechanism of Mammalian Shelterin. Cell Rep 18: 41-53.

T. Kibe, M. Zimmermann & T. de Lange (2016) TPP1 Blocks an ATR-Mediated Resection Mechanism at Telomeres. Mol Cell 61: 236-246

Y. Doksani & T.  de Lange (2016) Telomere-Internal Double-Strand Breaks Are Repaired by Homologous Recombination and PARP1/Lig3-Dependent End-Joining. Cell Rep. 17: 1646-1656.

T. de Lange (2015) A loopy view of telomere evolution. Front. Genet. 6:321.

S. Kabir, D. Hockemeyer, T. de Lange (2014). TALEN Gene Knockouts Reveal No Requirement for the Conserved Human Shelterin Protein Rap1 in Telomere Protection and Length Regulation. Cell Rep 9:1273-1280

D. Frescas, and T. de Lange (2014) Binding of TPP1 to TIN2 is required for POT1a,b-mediated telomere protection. J Biol Chem 289: 24180-24187.

D. Frescas, and T. de Lange (2014) TRF2-Tethered TIN2 Can Mediate Telomere Protection by TPP1/POT1. Mol Cell Biol 34: 1349-1362.

D. Frescas and T. de Lange (2014) A TIN2 dyskeratosis congenita mutation causes telomerase-independent telomere shortening in mice. Genes Dev. 28: 153-166.

Y. Doksani*, J.Y. Wu*, T. de Lange, X. Zhuang (2013) Super-Resolution Fluorescence Imaging of Telomeres Reveals TRF2-Dependent T-Loop Formation. Cell 155: 345-356.  *equal contribution

A. Sfeir and T. de Lange (2012) Removal of shelterin reveals the telomere end-protection problem. Science 336: 593-597.

K. Takai, T. Kibe, J. Donigian, D. Frescas and T. de Lange (2011) Telomere Protection by TPP1/POT1 Requires Tethering to TIN2. Mol. Cell, 44: 647-659.

Y. Gong and T. de Lange (2010) A Shld1-controlled POT1a provides support for repression of ATR signaling at telomeres through RPA exclusion. Mol. Cell 40: 377-387.

A. Sfeir, S. Kabir, M. van Overbeek, G.B. Celli, T. de Lange (2010) Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal. Science 327: 1657-1661.

T. Kibe, G.A. Osawa, C.E. Keegan, T. de Lange (2010) Telomere protection by TPP1 is mediated by POT1a and POT1b. Mol. Cell. Biol. 30: 1059-1066.

K. Takai, S. Hooper, S. Blackwood, T. de Lange (2010) In Vivo stoichiometry of shelterin complex. J. Biol. Chem. 285: 1457-1467.