Control of human telomere length by TRF1 and TRF2

Abstract

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.

FIG. 1

Overexpression of TRF2 results in telomere shortening. (A) Two TRF2-expressing HTC75 cell lines (P7 and P33) were grown for 124 PD in media with (non-induced) or without (induced) doxycycline, and genomic DNA was isolated at the indicated PD. Protein-free DNA was digested with HinfI and RsaI, was size fractionated on an agarose gel, and was blotted. A telomere-specific TTAGGG repeat probe was used to detect the telomeric restriction fragments. Molecular weight standards are indicated to the left of the gels. Periods of telomere shortening and growth are highlighted below the lanes. (B) Immunoblotting analysis of the TRF2 expression in P7 and P33 cells under induced and noninduced conditions. TRF2 was detected with antibody 508 in immunoblots of equal amounts (30 μg) of whole-cell-extract proteins. Endogenous TRF2 is below detection limit under these conditions.

FIG. 2

Long-term overexpression of full-length TRF1 and TRF1^66-439 result in progressive telomere shortening. (A) Stabilization of telomeres at a short-length setting due to overexpression of TRF1. D4, a HTC75 cell line overexpressing TRF1 in absence of doxycycline (44), was maintained under inducing conditions for 304 PD, and telomere lengths were determined by genomic blotting as shown in Fig. 1. The plot represents mean telomere length values during induced growth (filled circles). At PD 208, doxycycline was added to a parallel culture and telomere length changes were monitored under noninduced conditions (open circles). (B) TRF1^66-439 localizes to telomeres, where it displaces TRF1 but not TRF2. TRF1^66-439, bearing an N-terminal FLAG tag, was transfected into HeLal.2.11 cells, and its subcellular localization was determined by using indirect immunofluorescence with the anti-FLAG antibody M2 (left panels). The effect of this TRF1 allele on endogenous TRF1 was determined with an antibody directed against the N terminus of TRF1 (371C2; right top panel) that does not detect the TRF1^66-439 mutant. The subnuclear localization of TRF2 was assessed with antibody 508 (right bottom panel). Arrows denote transfected cells. (C) TRF1^66-439 induces progressive telomere shortening. J24, an HTC75 cell line expressing TRF1^66-439, was maintained under induced and noninduced conditions, and changes in telomere length were evaluated as described in the legend of Fig. 1A.

FIG. 3

TRF2 can bind to interstitial telomeric repeat-related sequences. (A) Immunoblot of whole-cell extracts from HeLall cells and three hamster cell lines (BHK-21, CHO-K1, and AHL-1) showing expression of TRF2. TRF2 was detected with antibody 508. Baculovirus-derived human TRF2 (Bac-hTRF2) protein is used as a positive control (4). (B) Interstitial binding of TRF2 in BHK-21 and CHO-K1 chromosomes. Left panels: metaphase chromosomes from the indicated hamster cell lines were analyzed by FISH by using a PNA probe for TTAGGG repeats. Arrows indicate some of the BHK and CHO chromosomes with prominent interstitial telomere-related sequences. Right panels: indirect immunofluorescence with the TRF2 antibody 508 or 647 (as indicated). Arrows highlight TRF2 at pericentric interstitial sites in BHK and CHO chromosomes. No interstitial TTAGGG signals or chromosome-internal binding of TRF2 is observed in the AHL cells. DNA was stained with DAPI.

FIG. 4

Greater relative abundance of TRF1 and TRF2 on long human telomeres. (A) Immunoblot showing expression of TRF1 and TRF2 in whole-cell extracts from two HeLa cell lines with different telomere lengths. TRF1 was detected with antibody 371; TRF2 was detected with antibody 647. Cyclin D is used as a loading control. In the first two lanes, proteins from equal numbers of cells were loaded. The second two lanes were normalized based on protein amounts (30 μg). (B) Indirect immunofluorescence signals for TRF1 and TRF2 in two HeLa cell lines with the indicated telomere lengths. Immunofluorescence staining of the two cell lines was executed in parallel and was processed identically, and images are presented without any adjustment. For each combination of cell line and antibody, nuclei are presented at two magnifications. DNA was stained with DAPI.

FIG. 5

Lack of direct effects of TRF1 and TRF2 on telomerase. (A) Overexpression of TRF2 does not affect the expression of hTERT mRNA or hTR RNA. Total RNA was prepared from the indicated HTC75 cell lines after 9 days of induction with doxycycline. RNase protection assays were performed to determine the relative abundance of hTERT mRNA and hTR RNA. β-actin served as a control (as described in Materials and Methods). (B) Schematic of the telomerase substrates used in panel C. SB2 contains an optimal binding site for TRF1 and TRF2. TS does not bind TRF1 or TRF2. (C) Lack of effects of TRF1 and TRF2 on the in vitro activity of telomerase. Partially purified human telomerase (see Materials and Methods) was assayed with TS (25, 35) or SB2 as a substrate in the presence of excess baculovirus-derived TRF1 and TRF2, or BSA as a control. The assay uses incorporation of [α-³²P]dGTP into telomerase products (conventional assay). The asterisk indicates a labeled form of the SB2 substrate that is only observed in the presence of the baculovirus-derived proteins.