The length of mammalian telomeres is governed by a homeostasis mechanism. Telomeres have a species-specific length setting (26
) which is constant over the generations, despite high levels of telomerase activity in the germline (47
). For instance, the telomeres of Mus musculus
are maintained at 20 to 50 kb, while the closely related mouse species Mus spretus
has telomeric tracts closer in length to human telomeres, usually ranging from 5 to 15 kb. Similarly, despite high levels of telomerase, the telomeres of many human tumor cell lines do not grow but are stably maintained at a setting characteristic for each individual cell line (12
). Telomere length homeostasis is also evident when new telomeres are generated by transfection of short stretches of telomeric DNA into cultured cells. The transfected telomeric tracts are elongated, presumably by telomerase, until their length matches the other telomeres in the transfected cells (3
). The new telomere presumably recruits telomere binding proteins and so attains all functions of telomeres, including the length regulation characteristic of a given cell. Regulated growth of a single telomere, as observed in this context, suggests that cells can measure and modulate the length of the telomeric repeat array at individual chromosome ends, implying a cis
-acting regulatory mechanism.
Human telomeres contain two related TTAGGG repeat binding factors, TRF1 and TRF2 (7
). Both TRF proteins have a Myb-like helix-turn-helix domain in their carboxy terminus and a central conserved domain that includes sequences responsible for the formation of homodimers. The two proteins do not heterodimerize, and they differ substantially at the N terminus, which is acidic in TRF1 but basic in TRF2 (10
). TRF1 and -2 are most closely related in their Myb-type DNA binding domains (56% identity), and both proteins can bind duplex telomeric DNA in vitro. The DNA binding site of TRF1, as determined by systematic evolution of ligands by exponential enrichment (SELEX), is composed of two identical YTAGGGTTR half sites which each engage one Myb domain in the TRF1 homodimer (6
). There is no constraint on the distance between the two half sites, and the sites can be bound in direct or inverted orientation. TRF1 has architectural features, including the ability to loop the sequence between two half sites (6
) and the ability to pair two telomeric tracts (20
). Although TRF2 displays a similar preference for duplex TTAGGG repeats in vitro (4
), its DNA binding features have not been established in detail.
In human cells, both TRF1 and TRF2 are predominantly located at chromosome ends where they contribute to the protection and maintenance of telomeric DNA. TRF1 has been demonstrated to regulate telomere length (44
). Overexpression of TRF1 in the tetracycline-responsive human fibrosarcoma cell line HTC75 results in a gradual decline in telomere length at a rate of ~10 bp/population doubling (PD). Conversely, the expression of a dominant negative allele of TRF1, which removes endogenous TRF1 from telomeres, leads to telomere elongation. In this system, TRF1 did not affect the activity of telomerase detectable in cell extracts, suggesting that TRF1 does not affect telomerase activity globally in the cell. Instead, we have proposed that TRF1 acts in cis
as a negative length regulator at each individual telomere. According to the current model, an inappropriately long telomere would recruit a large amount of TRF1 protein, blocking telomerase-mediated elongation of that particular chromosome end and thus leading to a resetting of the telomere length in cis
. A similar protein-counting model was proposed for telomere length homeostasis in yeast (31
An important question is how TRF1, which binds along the length of the telomere, modulates telomerase, an enzyme that acts at telomere termini. One mechanism that could be considered in this context is that accessibility of a DNA end to telomerase is diminished by the presence of TRF1 on or near the telomere terminus. An alternative proposal has recently emerged from the finding that telomeres fold back, forming a large duplex lariat called the t loop (21
). In the t loop, the 3′ single-stranded telomeric overhand of TTAGGG repeats is tucked into the duplex part of the telomeric repeat tract. The t loop is proposed to sequester telomeres from activities that might act on chromosome ends, including telomerase. In vitro studies suggest that telomerase requires an accessible 3′ overhang (28
), a structure predicted to be absent from t loops. Therefore, t loops could control the action of telomerase at individual chromosome ends. Based on biochemical studies, the formation of t loops was proposed to involve both TRF1 and TRF2. TRF1 has the ability to induce bending, looping, and pairing of duplex telomeric DNA (5
), activities that could facilitate the folding back of the telomere. TRF2 was found to induce the invasion of the 3′ single-stranded TTAGGG repeat tail into duplex telomeric DNA, forming t loops in vitro (21
; R. Stansel, T. de Lange, and J. D. Griffith, unpublished data). Thus, a t-loop-based mechanism for telomere length regulation would predict that both TRF1 and TRF2 are required for the length homeostasis of human telomeres. Specifically, the model predicts that, like TRF1, TRF2 acts as a negative regulator of telomere length.
The role of TRF2 in telomere length regulation cannot easily be examined through the inhibition of its function. Interference with the activity of TRF2 results in immediately deleterious phenotypes, possibly due to inappropriate exposure of unfolded telomere termini to DNA damage checkpoints and repair activities. For instance, cells forced to express a dominant negative allele of TRF2 rapidly initiate an apoptotic pathway (24
), and telomeres lacking TRF2 lose their 3′ overhangs and undergo covalent fusions (45
; A. Smogorzewska and T. de Lange, unpublished data). Therefore, we have addressed the role of TRF2 in telomere length regulation by overexpression of the full-length protein. The results demonstrate that TRF2 is a second negative regulator of telomere length in mammalian cells and are consistent with a t-loop-based mechanism for telomere length homeostasis.