Bai-Wei Gu

A pathogenic dyskerin mutation impairs proliferation and activates a DNA damage response independent of telomere length in mice.

Telomeres are nucleoprotein structures that cap the ends of chromosomes, protecting them from exonucleases and distinguishing them from double-stranded breaks. Their integrity is maintained by telomerase, an enzyme consisting of a reverse transcriptase, TERT and an RNA template, TERC, and other components, including the pseudouridine synthase, dyskerin, the product of the DKC1 gene. When telomeres become critically short, a p53-dependent pathway causing cell cycle arrest is induced that can lead to senescence, apoptosis, or, rarely to genomic instability and transformation. The same pathway is induced in response to DNA damage. DKC1 mutations in the disease dyskeratosis congenita are thought to act via this mechanism, causing growth defects in proliferative tissues through telomere shortening. Here, we show that pathogenic mutations in mouse Dkc1 cause a growth disadvantage and an enhanced DNA damage response in the context of telomeres of normal length. We show by genetic experiments that the growth disadvantage, detected by disparities in X-inactivation patterns in female heterozygotes, depends on telomerase. Hemizygous male mutant cells showed a strikingly enhanced DNA damage response via the ATM/p53 pathway after treatment with etoposide with a significant number of DNA damage foci colocalizing with telomeres in cytological preparations. We conclude that dyskerin mutations cause slow growth independently of telomere shortening and that this slow growth is the result of the induction of DNA damage. Thus, dyskerin interacts with telomerase and affects telomere maintenance independently of telomere length.

Dyskerin, telomerase and the DNA damage response

The bone marrow failure syndrome Dyskeratosis congenita (DC), though rare, has attracted a great deal of attention in the last few years because it is caused by mutations in genes whose products are involved in telomere maintenance. The disease presents with a variety of features that can all be due to failure of tissues that require constant renewal via stem cell activity. It is thought this is caused by defects in telomere maintenance leading eventually to cell cycle arrest or cell death caused by critically short telomeres. The most common form of DC is the X-linked form caused by mutations in DKC1 encoding the nucleolar protein, dyskerin. We recently reported a mouse model of the X-linked form of the disease in which females heterozygous for a mutation that copies a human pathogenic mutation showed a growth disadvantage in cells expressing the mutant dyskerin. This growth disadvantage, which was associated with an enhanced DNA damage response, was dependent on telomerase but appeared to be independent of telomere shortening. Here we discuss these results in terms of the role of dyskerin in telomere maintenance and the possible role that the DNA damage response plays in the pathogenesis of DC.

Accelerated hematopoietic stem cell aging in a mouse model of dyskeratosis congenita responds to antioxidant treatment

Mutations in DKC1, encoding telomerase associated protein dyskerin, cause X‐linked dyskeratosis congenita (DC), a bone marrow (BM) failure, and cancer susceptibility syndrome. Decreased accumulation of telomerase RNA resulting in excessive telomere shortening and premature cellular senescence is thought to be the primary cause of disease in X‐linked DC. Affected tissues are those that require constant renewal by stem cell activity. We previously showed that in Dkc1Δ15 mice, which contain a mutation that is a copy of a human mutation causing DC, mutant cells have a telomerase‐dependent proliferative defect and increased accumulation of DNA damage in the first generation before the telomeres are short. We now demonstrate the presence of the growth defect in Dkc1Δ15 mouse embryonic fibroblasts in vitro and show that accumulation of DNA damage and levels of reactive oxygen species increase with increasing population doublings. Treatment with the antioxidant, N‐acetyl cysteine (NAC), partially rescued the growth disadvantage of mutant cells in vitro and in vivo. Competitive BM repopulation experiments showed that the Dkc1Δ15 mutation is associated with a functional stem cell defect that becomes more severe with increasing age, consistent with accelerated senescence, a hallmark of DC hematopoiesis. This stem cell phenotype was partially corrected by NAC treatment. These results suggest that a pathogenic Dkc1 mutation accelerates stem cell aging, that increased oxidative stress might play a role in the pathogenesis of X‐linked DC, and that some manifestations of DC may be prevented or delayed by antioxidant treatment.

Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients.

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1 mutations Q31E, A353V and ΔL37. In addition we constructed “corrected” lines with a copy of the wild type dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1 mutations telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin, is variable. The recurrent mutation A353V shows the most severe effect on telomere maintenance. A353V cells but not Q31E or ΔL37 cells, are refractory to correction by expression of wild type DKC1 cDNA. Because dyskerin is involved in both telomere maintenance and ribosome biogenesis it has been postulated that defective ribosome biogenesis and translation may contribute to the disease phenotype. Evidence from mouse and zebra fish models has supported the involvement of ribosome biogenesis but primary cells from human patients have so far not shown defects in pseudouridylation or ribosomal RNA processing. None of the mutant iPS cells presented here show decreased pseudouridine levels in rRNA or defective rRNA processing suggesting telomere maintenance defects account for most of the phenotype of X-linked DC. Finally gene expression analysis of the iPS cells shows that WNT signaling is significantly decreased in all mutant cells, raising the possibility that defective WNT signaling may contribute to disease pathogenesis.

Slow growth and unstable ribosomal RNA lacking pseudouridine in mouse embryonic fibroblast cells expressing catalytically inactive dyskerin

Pseudouridine is the most abundant modified nucleotide in ribosomal RNA throughout eukaryotes and archaea but its role is not known. Here we produced mouse embryonic fibroblast cells expressing only catalytically inactive dyskerin, the pseudouridine synthase that converts uridine to pseudouridine in ribosomal RNA. The mutant dyskerin protein, D125A, was extremely unstable but cells were able to divide and grow very slowly. Abnormalities in ribosome RNA synthesis were apparent but mature cytoplasmic RNAs lacking pseudouridine were produced and were very unstable. We conclude that pseudouridine is required to stabilize the secondary structure of ribosomal RNA that is essential for its function.

Anomalous electrophoretic migration of newly synthesized ribosomal RNAs and their precursors from cells with DKC1 mutations

Mutations in the X‐linked gene, DKC1, encoding dyskerin, cause dyskeratosis congenita by leading to decreased telomerase activity and causing short telomeres. Dyskerin is also a pseudouridine synthase that modifies nascent ribosomal and other RNAs and it is not known if this function is affected by the mutations. Here we show that newly synthesized ribosomal RNA, extracted from human and mouse cells with pathogenic mutations, shows anomalous mobility in agarose gels under certain denaturation conditions. The anomalously migrating RNA is turned over rapidly. Analysis of ribosomal RNA in these cells suggests the altered mobility is due to inefficient pseudouridylation.

Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop a Defect in Erythropoiesis

MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the Mdm2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA) and in 5q- myelodysplastic syndrome (MDS). DBA and 5q- MDS are associated with inherited (DBA) or acquired (5q- MDS) haploinsufficiency of ribosomal proteins. A mutation in Mdm2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (Mdm2C305F), retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we noticed that Mdm2C305F homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM), these mice showed a significant decrease in Ter119hi cells compared to wild type (WT) littermates, while no decrease in the number of mature erythroid cells (Ter119hiCD71low) was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (p < 0.01). This erythropoietic defect was abrogated by concomitant p53 deficiency (Trp53ko/ko). Further investigation revealed that in Mdm2C305F animals, there was a decrease in Lin-Sca-1+c-Kit+ (LSK) cells, accompanied by significant decreases in multipotent progenitor (MPP) cells (p < 0.01). Competitive BM repopulation experiments showed that donor BM harboring the Mdm2C305F mutation possessed decreased repopulation capacity compared to WT BM, suggesting a functional stem cell deficit. These results suggest that there is a fine tuned balance in the interaction of ribosomal proteins with the MDM2/p53 axis which is important in normal hematopoiesis.

Defects in mTR stability and telomerase activity produced by the Dkc1 A353V mutation in dyskeratosis congenita are rescued by a peptide from the dyskerin TruB domain

BackgroundThe predominant X-linked form of dyskeratosis congenita results from mutations in dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. Materials and MethodsHere, we have generated F9 mouse cell lines expressing the most frequent mutation found in X-DC patients, A353V and study the effect of expressing the GSE24.2 cDNA or GSE24.2 peptide on telomerase activity by TRAP assay, and mTERT and mTR expression by Q-PCR. Point mutation in GSE24.2 residues were generated by site-directed mutagenesis.ResultsExpression of GSE24.2 increases mTR and to a lesser extent mTERT RNA levels, and leads to recovery of telomerase activity. Point mutations in GSE24.2 residues known to be highly conserved and crucial for the pseudouridine-synthase activity of dyskerin abolished the effect of the peptide. Recovery of telomerase activity and increase in mTERT levels were found when the GSE24.2 peptide purified from bacteria was introduced into the cells. Moreover, mTR stability was also rescued by transfection of the peptide GSE24.2.Discussion These data indicate that supplying GSE24.2, either from a cDNA vector, or as a peptide, can reduces the pathogenic effects of Dkc1 mutations and could form the basis of a novel therapeutic approach.

Variable expression of DKC1 mutations in mice

In humans mutations in DKC1, cause the rare bone marrow failure syndrome dyskeratosis congenita. We have used gene targeting to produce mouse ES cells with Dkc1 mutations that cause DC when in humans. The mutation A353V, the most common human mutation, causes typical DC to very severe DC in humans. Male chimeric mice carrying this mutation do not pass the mutated allele to their offspring. The mutation G402E accounts for a single typical case of DC in a human family. The allele carrying this mutation was transmitted to the offspring with high efficiency. Expression of RNA and protein was reduced compared to wild type animals, but no abnormalities of growth and development or in blood values were found in mutant mice. Thus Dkc1 mutations have variable expression inmice, as in humans. genesis 47:366–373, 2009.

Variations in reactive oxygen species between mouse strains.

Fig. 1. Variation in reactive oxygen species (ROS) generation in bone marrow, peripheral blood, and spleen fromWT andΔ15 C57/BL6, CAST, and/or DBAmice. (A) Total ROS accrual was measured using the general oxidative stress fluorescent indicator, 5-(and -6)carboxy-2′,7′-dichlorodihydrofluorescein diacetate (CMH2DCFDA; Invitrogen C6827, Grand Island, NY, USA). Conversion of CMH2DCFDA to its fluorescent product was monitored using flow cytometry 488 nm FL1 (CFP) channel. Asterisks represent significant differences between Δ15 and WT C57/BL6 mice or WT C57/BL6 and WT CAST as determined by Students t-test with P b 0.05. (B) Total ROS accrual wasmeasured using CellRox oxidative stress reagent (Molecular Probes by Life Biosciences, Eugene, Oregon, USA). Fluorescencewasmonitored using BD FACSCanto IIflowcytometry 488 nm laser: Fluorescence laser FL1 (CFP) channel for CellROX Green. Asterisks represent significant differences between WT C57/BL6, DBA, and CAST mice as determined by Students t-test with P b 0.05. FlowJo was utilized to analyze all flow cytometry data, and mean peak values are shown. To the Editor,