X-linked DC is caused by mutations in the dyskerin gene (DKC1)⁹ which encodes an RNA binding protein whose inactivation destabilizes levels of TERC, resulting in shortened telomeres and premature senescence in patient cell lines^10,11. We asked whether a DKC1 mutant fibroblast line (del37L^9–11) could be reprogrammed and propagated in a pluripotent state. Compared to normal cells, the reprogramming efficiency of del37L cells was poor, yielding only 2–5 colonies from 10⁵ input cells with a delayed latency (Supplementary Table 1). Nevertheless, DKC1 mutant iPS colonies showed all hallmarks of pluripotency, including characteristic morphology (Fig. 1a), gene expression (Fig. 1b; Supplementary Fig. 2a), and formation of teratomas comprised of all three embryonic germ layers (Fig. 1c). PCR restriction fragment length polymorphism (RFLP) analysis for the DKC1 mutation confirmed the del37L mutation in iPS lines, and karyotype analysis was normal (Fig. 1d; Supplementary Fig. 2b,c). These data show that the somatic cells from patients with a genetic impairment in telomere elongation can be reprogrammed to pluripotency.

Despite induction of endogenous TERT and telomerase activity (Figs. 1b,e), early passage del37L iPS cell lines displayed shorter telomeres relative to the starting fibroblast population (Fig. 1f). Addition of TERT to the reprogramming factors did not result in telomere elongation in del37L mutant cells (Fig. 1f), unlike in normal cells (Supplementary Fig. 3), but did increase reprogramming efficiency (Supplementary Table 1; Supplementary Text). We obtained similar results with reprogramming of an independent DKC1 mutant line (A386T¹¹) (Fig. 1g; Supplementary Fig. 4; Supplementary Table 1). Given the telomerase dysfunction and shortened telomeres, we expected to observe limited passage of DKC1 mutant iPS cells in culture. However, unlike the parental DKC1 mutant fibroblasts, which senesced after 3–4 passages, we were able to continuously culture the DKC1 mutant iPS cell lines. Compared to the early passage cells, we found by TRF analysis that telomere length in del37L iPS lines increased with continued passage (Fig. 2a). Consistent with this, despite numerous interval population doublings, late passage del37L iPS lines had telomere lengths comparable to the original fibroblast population by quantitative PCR¹² (Fig. 2b). In a blinded assessment by the complementary method of quantitative telomere fluorescence in situ hybridization, we confirmed that telomere length was shortened immediately after derivation but increased over time (Fig. 2c). Late passage del37L iPS cells maintained a characteristic morphology, normal karyotype, and the same clonal fingerprint as early passage cells, and reversion of the genetic mutation in DKC1 was excluded (Supplementary Fig. 5). These data show that even in cells carrying genetic lesions that reduce telomerase function, reprogramming restores telomere elongation and self-renewal.

Previous studies have shown that reduced TERC levels compromise telomerase activity in DKC1 mutant fibroblasts¹¹. Ectopic expression of TERT alone results in telomere elongation in wild-type fibroblasts^13,14, whereas expression of both TERC and TERT is required to restore telomere elongation in DKC1 mutant fibroblasts¹¹ (Supplementary Fig. 6). We therefore investigated TERC levels in DC fibroblasts and iPS cells. By quantitative RT-PCR, we found that TERC levels in DKC1 mutant fibroblasts were 10–15% of TERC levels found in wild-type fibroblasts, consistent with previous reports^10,11 (Fig. 3a). Relative to parental fibroblasts from two patients with different DKC1 mutations, we found TERC levels increased 6–8 fold in the reprogrammed derivatives, approaching levels in normal fibroblasts (Fig. 3a). In normal iPS cells, we found that TERC levels were approximately 3-fold higher than the fibroblasts from which they were derived (Fig. 3a). We next examined a cell line from a patient with autosomal dominant DC carrying a heterozygous 821 bp deletion in the 3' region of the TERC locus (DCHSF1^15,16). In these TERC^+/− fibroblasts TERC is limiting for telomere elongation, even in the presence of exogenous TERT¹⁶. We reprogrammed the TERC^+/− patient fibroblasts and obtained multiple independent iPS lines which all showed characteristics of pluripotency and maintained the mutant genotype (Supplementary Fig. 7). TERC^+/− iPS cells also showed a 3-fold induction of TERC relative to fibroblasts (Fig. 3a), and displayed continuous self-renewal in contrast to the early senescence seen in the parental fibroblasts¹⁶. These data demonstrate that reprogramming of somatic cells is accompanied by upregulation of endogenous TERC levels, and provide a mechanism for telomere elongation and indefinite self-renewal in DC iPS cell lines (Supplementary Fig. 6).

We found that human embryonic stem (ES) cells maintain elevated TERC levels similar to those found in WT iPS cells (Fig. 3a), and that TERC levels attained in iPS cell lines derived from DKC1 mutant, TERC^+/− and WT cells correlated with their respective telomerase activity (Fig. 3b). Furthermore, TERC knockdown in DC iPS lines compromised telomere maintenance (Supplementary Fig. 8a). We found that the TERC locus is not amplified in DC iPS cells, and that upon differentiation of DC iPS cells, TERC expression reverted to the pathologically low levels found in patient fibroblasts, and telomere length decreased (Supplementary Figs. 8b–d). Collectively, these results show that TERC levels are increased by reprogramming, and are a dynamically regulated and reversible property of the pluripotent state.

TERC abundance is tightly regulated by multiple transcriptional and post-transcriptional mechanisms^17,18. To investigate transcriptional mechanisms of TERC upregulation, we performed chromatin immunoprecipitation (ChIP) in human iPS cells, and detected enhanced binding of Oct4 and Nanog in the TERC locus (Fig. 3c). These data were corroborated in murine ES cells, which likewise showed enhanced binding of Oct4 and Nanog at the TERC locus (Supplementary Fig. 9). However, we were unable to detect increased levels of nascent TERC transcription by nuclear run-off assay in iPS cells versus fibroblasts (Supplementary Fig. 10), suggesting that Oct4 and Nanog may affect transcriptional competence rather than transcriptional rate of the TERC locus in pluripotent cells. Additional mechanisms are required to explain the increased steady-state levels of TERC. To investigate post-transcriptional mechanisms of TERC regulation, we determined DKC1 levels, which correlate with TERC levels in human cancer cells¹⁹. In all normal and DC iPS lines tested, we found an increase in DKC1 transcript levels and dyskerin protein relative to the fibroblasts (Figs. 3d and e). Moreover, we found that human ES cells, like iPS cells, maintain higher levels of DKC1 (Fig. 3d), that DKC1 levels decreased with differentiation of iPS cells into embryoid bodies (Supplementary Fig. 11a), and that DKC1 knockdown caused a reduction in TERC levels and compromised cellular viability (Supplementary Figs. 11b,c). ChIP showed binding of Oct4 and Nanog to the DKC1 promoter in pluripotent cells (Fig. 3f), and infection of fibroblasts with retroviruses encoding the four reprogramming factors increased DKC1 levels (Supplementary Fig. 11d). These data establish that multiple telomerase components (TERT, TERC, DKC1) are upregulated after reprogramming, thereby accounting for increased telomerase activity in the pluripotent state and explaining the restoration of telomere maintenance in DC iPS cells.

In an autosomal dominant DC patient carrying an 821 bp deletion in the 3' region of TERC, prior studies have concluded that the haploinsufficient phenotype is explained by impaired post-transcriptional accumulation of TERC RNA and reduced assembly of the truncated TERC RNA into telomerase holoenzyme^20–22 (Supplementary Fig. 12). Our ChIP data indicated Oct4 and Nanog binding in a region downstream of TERC that overlaps the 821 bp deletion (Fig. 3c). Moreover, we found that part of this region is predicted to have regulatory potential based on comparative genomic sequence alignment (Supplementary Fig. 13a). We hypothesized that loss of this region might compromise transcriptional activity of the deleted allele. We employed two distinct allele-specific methods to analyze the chromatin configuration in TERC^+/− iPS cells, and in both cases found the mutant TERC allele to be transcriptionally inactive (Figure 4). First, allele-specific ChIP showed a striking abrogation of H3K4me3 marks and RNA polymerase II binding on the deleted allele (Fig. 4a and Supplementary Figs. 13b and 13c). Second, allele-specific DNAse I hypersensitivity analysis showed pronounced nuclease accessibility at the TERC promoter in normal cells (Fig. 4b) and on the normal allele in TERC^+/− cells, but complete loss of promoter hypersensitivity on the mutant TERC allele (Fig. 4c and d). Collectively, these results identify a cis-element in the 3' region of the TERC locus that appears essential for formation of a transcriptionally active chromatin structure, and suggest that mutations in this region can cause haploinsufficiency in autosomal dominant DC by diminishing TERC transcription (Supplementary Fig. 12).

By reprogramming somatic cells from patients with the human disease dyskeratosis congenita, we have discovered novel mechanisms of regulation of telomerase activity in the pluripotent state, thus illustrating the value of disease-specific iPS cells for basic and translational discovery. Moreover, we have shown that the RNA component of telomerase is upregulated in the pluripotent state to a degree sufficient to overcome limitations to telomere maintenance in X-linked and autosomal dominant DC. Drugs that activate the TERC locus may favor telomere maintenance over premature attrition in stem cell compartments where TERT is present but telomerase activity is limiting, such as hematopoietic stem cells^23–27, and thus might serve as therapeutic agents for bone marrow failure. Moreover, we speculate that in DC patients certain cell types such as germ cells and cancer cells, whose transcriptional programs share similarities with pluripotent cells, may also upregulate TERC to permit germline propagation of mutations and malignant proliferation.

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