Targeted deletion reveals an essential function for the telomere length regulator Trf1

Abstract

The human telomeric DNA binding factor TRF1 (hTRF1) and its interacting proteins TIN2, tankyrase 1 and 2, and PINX1 have been implicated in the regulation of telomerase-dependent telomere length maintenance. Here we show that targeted deletion of exon 1 of the mouse gene encoding Trf1 causes early (day 5 to 6 postcoitus) embryonic lethality. The absence of telomerase did not alter the Terf1(ex1Delta/ex1Delta) lethality, indicating that the phenotype was not due to inappropriate telomere elongation by telomerase. Terf1(ex1Delta/ex1Delta) blastocysts had a severe growth defect of the inner cell mass that was accompanied by apoptosis. However, no evidence was found for telomere uncapping causing this cell death; chromosome spreads of Terf1(ex1Delta/ex1Delta) blastocysts did not reveal chromosome end-to-end fusions, and p53 deficiency only briefly delayed Terf1(ex1Delta/ex1Delta) lethality. These data suggest that murine Trf1 has an essential function that is independent of telomere length regulation.

FIG. 1.

Targeted deletion of exon 1 of the mouse Terf1 locus. (A) Schematics of the targeting construct (top line), wild-type locus (second line), and targeted locus (third line) are shown. The bottom line shows the XhoI/HindIII and XhoI fragments used for genotyping. X, XhoI; E, EcoRI; H, HindIII. Positions of PCR primers used for genotyping are indicated by half arrows. Primers are as follows: C, shared by the wild-type and the targeted allele; wt, wild-type allele-specific primer; Δ, primer specific for the targeted allele. (B) Southern blot of XhoI/HindIII-digested DNA from ES cells of the indicated genotypes. The probe is the 0.8-kb XhoI/EcoRI fragment shown in panel A. (C) PCR of wild-type and Terf1^ex1Δ/+ ES cells. (D) Northern blot of total RNA from Terf1^+/+ and Terf1^ex1Δ/+ MEFs. Total RNA was hybridized first with radiolabeled Terf1 cDNA and subsequently with two oligonucleotides complementary to 7SK RNAs. “% Terf1 signal” refers to the signal derived from hybridization with the Terf1 cDNA normalized to loading (7SK signal). (E) Growth curves of Terf1^+/+ and Terf1^ex1Δ/+ MEFs from E12.5 embryos. (F) Telomeric overhangs and telomeric restriction fragments from Terf1^+/+ and Terf1^ex1Δ/+ MEFs obtained from littermates from two different litters. Molecular size markers are indicated in kilobase pairs. Relative G-strand overhang intensities were determined by normalization of the native (G-strand) signal to the signal obtained after denaturation (representing duplex TTAGGG repeats). The ratio of G-strand to duplex signals was arbitrarily set to 100 for the DNA in the first lane, and the other ratios were normalized to this value.

FIG. 2.

Morphologies of Terf1^+/+, Terf1^ex1Δ/+, and Terf1^ex1Δ/ex1Δ embryos. (A) Hematoxylin-and-eosin-stained sections of E6 to E6.5 embryos (in utero) derived from intercrosses between Terf1^ex1Δ/+ mice. Extraembryonic (ex), embryonic (em), and ectoplacental-cone (epc) regions are indicated. Genotyping was performed by laser capture and PCR. (B) Hematoxylin-and-eosin-stained sections of E5 to E5.5 embryos (in utero) derived from intercrosses between Terf1^ex1Δ/+ mice. Note that the homozygous mutant embryos are grossly similar to their heterozygous and wild-type littermates.

FIG. 3.

p53 deficiency attenuates the embryonic lethality of the Terf1^ex1Δ/ex1Δ genotype. (A) Hematoxylin-and-eosin-stained E7 to E7.5 sections of embryos (in utero) from intercrosses between Terf1^ex1Δ/+ p53^−/− mice. Extraembryonic (ex) and embryonic (em) tissues are indicated. Genotypes were identified by laser capture and PCR. Note that the Terf1^ex1Δ/ex1Δ embryo was grossly normal, although smaller than its littermates. (B) E8 to E8.5 embryos derived from intercrosses between Terf1^ex1Δ/+ p53^−/− mice. Extraembryonic and embryonic regions are indicated.

FIG. 4.

Terf1^ex1Δ/ex1Δ blastocysts show ICM cell death without detectable telomere fusions. (A) Day-3.5-postcoitus blastocysts isolated from Terf1^ex1Δ/+ intercrosses cultured in 96-well plates for 5 days. ICM and trophoblastic giant cells (TGC) are indicated. (B) TUNEL staining of Terf1^+/+, Terf1^ex1Δ/+, and Terf1^ex1Δ/ex1Δ blastocysts. Blastocysts were cultured for 4 days and subjected to TUNEL staining. Green, TUNEL signal; blue, DNA stain (4′,6′-diamidino-2-phenylindole [DAPI]). (C) Telomeric FISH on metaphase spreads of Terf1^+/+, Terf1^ex1Δ/+, and Terf1^ex1Δ/ex1Δ blastocysts at day 5.5. Cells were cultured for 2 days, and metaphase spreads were processed for telomeric FISH (green). DNA is stained with DAPI (blue).