Deregulated telomere transcription causes replication-dependent telomere shortening and promotes cellular senescence

Abstract

Telomeres are transcribed into non-coding TElomeric Repeat containing RNAs (TERRA). We have employed a transcriptionally inducible telomere to investigate how telomere transcription affects telomere function in Saccharomyces cerevisiae. We report that telomere shortening resulting from high levels of telomere transcription stems from a DNA replication-dependent loss of telomere tracts, which can occur independent of both telomerase inhibition and homologous recombination. We show that in order for telomere loss to occur, transcription must pass through the telomere tract itself producing a TERRA molecule. We demonstrate that increased telomere transcription of a single telomere leads to a premature cellular senescence in the absence of a telomere maintenance mechanism (telomerase and homology directed repair). Similar rapid senescence and telomere shortening are also seen in sir2Δ cells with compromised telomere maintenance, where TERRA levels are increased at natural telomeres. These data suggest that telomere transcription must be tightly controlled to prevent telomere loss and early onset senescence.

Figure 1.

Telomere transcription causes telomere shortening in cis. (A) The constructs used to modify telomere 7L (see text). The values in bp refer to the distances between the PCR primer/Gal UAS and the start of the telomeric tract. (B) Northern blotting analysis revealed that while PT transcribes into the telomere in the presence of galactose, construct 4 transcription stops short of the telomere due to the presence of the natural URA3 terminator, therefore the production of telomeric transcripts only occurs in the PT strain. (C) Quantification of telomere length for construct 4 modified telomeres, comparing glucose vs. galactose after approximately 110 generations (n = 5). Values from UT, XT and PT have been included from Supplemental Figure 1B as comparison. For all above experiments error bars represent ± SEM.

Figure 2.

Transcription-induced shortening is additive with telomerase loss. (A) PT est2Δ cells grown in galactose had shorter telomeres than the same cells grown in glucose as well as PT EST2 cells grown in galactose for approximately 10 PD. Control Y′ telomeres from the same cells did not vary in length after 10 generations in glucose and galactose (n: PT est2Δ = 8, PT = 6). (B) Transcription of modified telomere 7L in PT cells (n = 9) results in an increased rate of senescence as compared to non-transcribing UT (n = 9) cells in an est2Δ rad52Δ genetic background. (C) Telomere PCR performed on modified telomere 7L during the senescence assay shown in (B) which revealed that this telomere was shorter in PT cells as compared to UT cells (two representative cultures (a and b) from each genotype—UT/PT est2Δ rad52Δ). (D) There was no apparent difference in terms of length at Y′ telomeres when comparing UT and PT strains. PD = population doublings following the formation of a colony after tetrad dissection.

Figure 3.

DNA replication is responsible for transcription-induced shortening. (A) Schematic of experimental design to assay for telomere length changes upon induction of telomere transcription in PT est2Δ synchronized cells. RNA and DNA samples were taken at time points indicated with asterisks. (B) Two micrograms RNA [from (A)] was spotted onto a nylon membrane and hybridized with labeled probes to recognize both TERRA and SCR1 as a loading control. (C) DNA was extracted and subjected to telomere PCRs specific for the induced telomere 7L, telomeres were cloned and sequenced and lengths were determined and plotted. A two-tailed unpaired Student’s t-test revealed a significant difference between telomere lengths of the transcribing G1 7 L telomeres (n = 30) and transcribing G2 telomeres (n = 27) (P = 0.0084). No difference between lengths in non-transcribing (n = 35) and transcribing G1 arrested cells (P=0.6499) was detected. Median telomere length is indicated with a line across the data points.

Figure 4.

Telomere transcription leads to early senescence. (A) sir2Δ rad52Δ est2Δ (n = 5) cells lose viability more quickly than est2Δ rad52Δ cells (n = 4) when subjected to a senescence assay, whereas there is no difference in growth profiles when comparing rad52Δ (n = 6) cells with rad52Δ sir2Δ (n = 6), error bars represent ± SEM. PD values refer to PD after the spore colony had grown to saturation on the dissection plate (approximately 25 generations). (B) Telomere lengths were analyzed via telomere PCR at the indicated number of PD for the natural X-only telomere 7L (one representative culture from the curve in (A) (spore a) for the indicated genotypes). (C) Telomere length for natural telomere 1L was analyzed for a second independent culture from the curve in (A) (spore b) by telomere PCR. (D) Y′ telomeres were only slightly shorter upon loss of SIR2 (Y′ telomeres were analyzed for both spores (a) and (b) from panels B and C).

Figure 5.

Transcription-induced telomere shortening. Wild type telomeres transcribe TERRA at very low levels as the TERRA promoter (arrow) is negatively regulated by the Sir2/3/4 complex as well as the Rif1/Rif2 proteins, which allows for a fully functional and capped telomere (top diagram, arrow in subtelomere indicates approximate TERRA transcription start site). When TERRA transcription increases, either by galactose induction in the PT strain or in a sir2Δ, both the RNAPII occupancy at the telomere as well as the number of TERRA molecules increase. Telomere loss could result from a TERRA intermediate that leads to a replication fork stall and eventually a double strand break (lightning bolt). Increased transcription may also render telomeres more accessible to exonucleases, which would result in shortening. Telomerase is able to act at the transcribing telomere to compensate for the loss, although potentially at reduced capacity due to partial TERRA/transcription inhibition (dashed line). In the absence of telomerase critically short telomeres accumulate leading to an earlier activation of checkpoint-induced cellular senescence.