Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients

Abstract

Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that several telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients.

Figure 1. Derivation and characterization of DKC1 mutant iPS cells

a, DKC1 del37L fibroblasts and iPS colony. b, RT-PCR for endogenous ("endo") Oct4, Sox2, TERT, and Actin transcripts in del37L fibroblasts (Fib) and iPS cells derived with and without exogenous Tert. c, Histology of del37L iPS1 teratomas. d, PCR RFLP verification of del37L mutation in iPS clones. e, Telomerase activity assay in DKC1 mutant iPS lines (dash = background). f, Telomere Southern blot in DKC1 del37L fibroblasts and early passage iPS clones. g, Telomere Southern blot in DKC1 A386T fibroblasts and early passage iPS clones.

Figure 2. Telomere elongation in DKC1 mutant iPS cells

a, Telomere Southern blot of del37L fibroblasts (Fib) and iPS clones 1 and 2 as a function of passage. b, Quantitative real-time PCR (Q-PCR) analysis of telomere length in del37L fibroblasts and iPS clones at indicated passages (p). Error bars represent s.e.m. c, Quantitative fluorescence in situ hybridization (Q-FISH) for telomere length in del37L cells. a.f.u. = arbitrary fluorescence units. Inset: mean relative length values shown +/− s.d.

Figure 3. Upregulation of TERC and DKC1 in iPS cells

a, Q-PCR measurement of TERC transcripts in DKC1 mutant, TERC^+/−, and wild-type (WT) fibroblasts (fib) and iPS cells, and human ES (hES) cells, normalized to GAPDH and relative to WT fibroblasts. b, Quantitative TRAP assay in iPS cells. c, ChIP of the human TERC locus in WT iPS cells. 821 bp deletion in TERC^+/− cells is indicated. d, Q-PCR measurement of DKC1 transcripts (as in a). e, Western blot of dyskerin and actin in WT and DC fibroblasts and iPS clones. f, ChIP of the DKC1 locus (see map) in WT iPS cells. All error bars represent s.e.m.

Figure 4. Transcriptional silencing is associated with a 3' deletion in the TERC locus

a, ChIP using TERC^+/− iPS cells with allele-specific primers (F / R1 for WT; F / R2 for mutant). Error bars represent s.e.m. b, DNAse I hypersensitivity (HS) analysis probing a TERC locus NdeI (N) fragment in WT iPS cells, showing two discrete HS sites. c, Allele-specific DNAse I HS analysis probing TERC locus Bsu36I (B) fragments in TERC^+/− iPS cells (probe 1 assesses WT parent allele and HS site I (I^WT); probe 2 assesses both parent alleles (WT and del) but HS I only on the deleted allele (I^del)). d, As in c, with PflMI (P) digestion and indicated probe for simultaneous assessment of both WT and deleted parent alleles and HS sites.