Haploinsufficiency of mTR results in defects in telomere elongation

Abstract

Telomeres are usually maintained about an equilibrium length, and the set point for this equilibrium differs between species and between strains of a given species. To examine the requirement for telomerase in mediating establishment of a new telomere length equilibrium, we generated interspecies crosses with telomerase mTR knockout mice. In crosses between C57BL/6J (B6) and either of two unrelated mouse species, CAST/Ei and SPRET/Ei, telomerase mediated establishment of a new telomere length equilibrium in wild-type mTR(+/+) mice. This new equilibrium was characterized by elongation of the short telomeres of CAST/Ei or SPRET/Ei origin. In contrast, mTR(-/-) offspring of interspecies crosses failed to elongate telomeres. Unexpectedly, haploinsufficiency was observed in mTR(+/-) heterozygous interspecies mice, which had an impaired ability to elongate short SPRET/Ei or CAST/Ei telomeres to the new equilibrium set point that was achieved in wild-type mTR(+/+) mice. These results demonstrate that elongation of telomeres to a new telomere set point requires telomerase and indicate that telomerase RNA may be limiting in vivo.

Figure 1

CAST/Ei telomeres are elongated in offspring of a CAST/Ei × B6 cross. Telomere restriction fragments (TRF) from a B6, a CAST/Ei, and a (B6 × CAST/Ei) F1 mouse are shown. (A) End-labeled (CCCTAA)₃ probe was used in in-gel hybridization to DpnII digests of agarose-embedded genomic DNA. The boxed region represents the shortest telomeres where differences in the distributions are most evident. (B) Q-FISH analysis of B6, CAST/Ei, and (B6 × CAST/Ei) F1 telomeres. Frequency distributions of 800 telomere signals from each mouse are shown.

Figure 2

Elongation of short telomeres is impaired in mTR^+/− offspring of B6 mTR^+/− × SPRET/Ei and B6 mTR^+/− × CAST/Ei crosses. (A) TRF distributions from the B6 mTR^+/− and SPRET/Ei parents and mTR^+/+ and mTR^+/− offspring of a B6 mTR^+/− × SPRET/Ei cross. End-labeled (CCCTAA)₃ probe was used in in-gel hybridization to DpnII digests of agarose-embedded genomic DNA. The boxed region represents the shortest telomeres where differences in the distributions are most evident. (B) TRF distributions from the B6 mTR^+/− and CAST/Ei parents and mTR^+/+ and mTR^+/− offspring of a B6 mTR^+/− × CAST/Ei cross. (C) Q-FISH analysis of B6 mTR^+/−, SPRET/Ei, and (B6 × SPRET/Ei) mTR^+/+ and mTR^+/− telomeres. Frequency distributions of 1,600 telomere signals from two mice of each genotype are shown.

Figure 3

Telomeres shorten in mTR^−/− offspring of crosses between B6 mTR^+/− and mTR^+/− mice with short telomeres. (A) TRF distributions from B6 mTR^+/− and (B6 mTR^+/− × SPRET/Ei) mTR^+/− parents and mTR^+/+, mTR^+/−, and mTR^−/− offspring of a (B6 mTR^+/− × SPRET/Ei) mTR^+/− × B6 mTR^+/− cross. End-labeled (CCCTAA)₃ probe was used in in-gel hybridization to DpnII digests of agarose-embedded genomic DNA. The boxed region represents the shortest telomeres where differences in the distributions are most evident. (B) TRF distributions from B6 mTR^+/− and CAST/Ei mTR^+/− parents and mTR^+/+, mTR^+/−, and mTR^−/− offspring of a B6 mTR^+/− × CAST/Ei mTR^+/− cross. CAST/Ei mTR^+/− mice used here were bred by backcrossing mTR^+/− (B6 × CAST/Ei) F1 mice three times to CAST/Ei. The boxed region represents the shortest telomeres where difference in the distributions are most evident. (C) Q-FISH analysis of telomeres from mTR^+/+, mTR^+/−, and mTR^−/− offspring of a [B6 mTR^+/− × (B6 mTR^+/− × SPRET/Ei) mTR^+/−] cross. Frequency distributions of 2,400 telomere signals from three mice of each genotype are shown.

Figure 4

Similar levels of telomerase activity are expressed in mTR^+/+ and mTR^+/− mice. Lysates were prepared from in vitro activated spleen cells derived from age-matched [(B6 mTR^+/− × SPRET/Ei) × B6 mTR^+/−] mice that were genotypically mTR^+/+, mTR^+/−, or mTR^−/− and serially diluted before analyzing telomerase activity. Telomerase activity is shown for 4,000, 2,000, 1,000, and 500 cell equivalents. As a control for telomerase activity, a sample (4,000 cell equivalents) was heated to 85°C for 10 min. IS, the band produced by the internal standard included in the TRAP reaction.

Figure 5

mTR RNA levels are reduced in mTR heterozygous mice. Relative RNA concentrations (ng) were calculated from a standard curve generated from wild-type CAST/Ei mice (see Materials and Methods) and are indicated for each genotype. mTR levels were 365 in mTR^+/+ and 118 in mTR^+/− mice, and GAPDH levels were 320 in mTR^+/+ and 280 in mTR^+/− mice. Closed circles represent 10 independent threshold cycle values from mTR^+/− mice, and the open circle represents the average. Closed squares represent 12 independent threshold cycle values from mTR^+/+ mice, and the open square represents the average. (A) mTR. (B) GAPDH.