Shelterin Genes Research Articles

Abstract 941: Exome sequencing identified POT1, a telomere shelterin gene, as a major susceptibility gene for familial cutaneous malignant melanoma

Abstract Approximately 10% of cutaneous malignant melanoma (CMM) cases occur in a familial setting and known high-penetrance melanoma susceptibility genes (CDKN2A, CDK4, BAP1, and TERT) account for melanoma susceptibility in a small proportion of melanoma-prone families. To identify additional high-penetrance susceptibility genes for familial CMM, we performed whole exome sequencing in 101 CMM cases/obligate carriers in 56 unrelated melanoma-prone families recruited from the Romagna area in Italy. We identified a germline founder mutation in Protection Of Telomeres 1(POT1) (g.7:124493086 C>T, p.S270N) in five unrelated melanoma-prone families. The mutation was also found in a single sporadic CMM case from Romagna out of 1,824 Italian cases examined, but it was not seen in public databases, our internal exome databases of over 1000 subjects, and 878 Spanish CMM cases and 3,489 controls (2,038 Italian and 1,451Spanish) genotyped. POT1 is a component of the telomeric shelterin complex that plays a critical role in maintaining telomere integrity and regulating telomere length. The p.S270N mutation is highly conserved among vertebrates and predicted to be deleterious by most computational programs we evaluated. Carriers of the p.S270N mutation had increased length and heterogeneity as well as increased average number of fragile telomeres in peripheral blood mononuclear cell (PBMC) DNA compared to age-matched CMM cases (controls) without the mutation. Exome sequencing analysis of Italian families also identified two other rare missense substitutions in POT1 (g.7:124464052 C>G [p.Q623H] and g.7:124503540 C>T [p.R137H]) that were found in all cases sequenced in two families. Both missense mutations are absent from public databases and 3,489 genotyped controls, and are predicted to be deleterious by most algorithms. The carriers of these two mutations showed slightly but significantly increased telomere intensity signals and telomere fragility in PBMCs compared to age-matched controls. We further sequenced POT1 exons in 768 CMM cases and 768 controls collected from Italy and we found that CMM cases showed a significant increase in the burden of rare exonic variants compared to controls (OR=5.4, 95% CI=1.5-29.2, P=0.0021). Subsequently, we identified two novel recurrent germline missense mutations in POT1 (g.7:124499043 C>T, p.D224N and g.7:124469308 C>G, p.A532P) in American and French familial melanoma cases. Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations and that genes involved in telomere maintenance may play important roles in melanoma development. Citation Format: Jianxin Shi, Xiaohong R. Yang, Bari Ballew, Melissa Rotunno, Donato Calista, Maria C. Fargnoli, Paola Ghiorzo, Brigitte Bressac-de Paillerets, Eduardo Nagore, NCI DCEG Cancer Sequencing Working Group, Xing Hua, Paula Hyland, Jinhu Yin, Haritha Vallabhaneni, Weihang Chai, Sarangan Ravichandran, Alexander Eggermont, Mark Lathrop, Ketty Peris, Giovanna Bianchi-Scarra, Giorgio Landi, Sharon Savage, Joshua Sampson, Ji He, Meredith Yeager, Lynn Goldin, Florence Demenais, Stephen Chanock, Margaret Tucker, Alisa Goldstein, Yie Liu, Maria T. Landi. Exome sequencing identified POT1, a telomere shelterin gene, as a major susceptibility gene for familial cutaneous malignant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 941. doi:10.1158/1538-7445.AM2014-941

The effect of chemotherapeutic agents on telomere length maintenance in breast cancer cell lines.

Mammalian telomeric DNA consists of tandem repeats of the sequence TTAGGG associated with a specialized set of proteins, known collectively as Shelterin. These telosomal proteins protect the ends of chromosomes against end-to-end fusion and degradation. Short telomeres in breast cancer cells confer telomere dysfunction and this can be related to Shelterin proteins and their level of expression in breast cancer cell lines. This study investigates whether expression of Shelterin and Shelterin-associated proteins are altered, and influence the protection and maintenance of telomeres, in breast cancer cells. 5-aza-2′-deoxycytidine (5-aza-CdR) and trichostatin A (TSA) were used in an attempt to reactivate the expression of silenced genes. Our studies have shown that Shelterin and Shelterin-associated genes were down-regulated in breast cancer cell lines; this may be due to epigenetic modification of DNA as the promoter region of POT1 was found to be partially methylated. Shelterin genes expression was up-regulated upon treatment of 21NT breast cancer cells with 5-aza-CdR and TSA. The telomere length of treated 21NT cells was measured by q-PCR showed an increase in telomere length at different time points. Our studies have shown that down-regulation of Shelterin genes is partially due to methylation in some epithelial breast cancer cell lines. Removal of epigenetic silencing results in up-regulation of Shelterin and Shelterin-associated genes which can then lead to telomere length elongation and stability.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-014-2975-x) contains supplementary material, which is available to authorized users.

Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma.

Although CDKN2A is the most frequent high-risk melanoma susceptibility gene, the underlying genetic factors for most melanoma-prone families remain unknown. Using whole exome sequencing, we identified a rare variant that arose as a founder mutation in the telomere shelterin POT1 gene (g.7:124493086 C>T, Ser270Asn) in five unrelated melanoma-prone families from Romagna, Italy. Carriers of this variant had increased telomere length and elevated fragile telomeres suggesting that this variant perturbs telomere maintenance. Two additional rare POT1 variants were identified in all cases sequenced in two other Italian families, yielding a frequency of POT1 variants comparable to that of CDKN2A mutations in this population. These variants were not found in public databases or in 2,038 genotyped Italian controls. We also identified two rare recurrent POT1 variants in American and French familial melanoma cases. Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations.

Hematopoietic stem cells are acutely sensitive to Acd shelterin gene inactivation

The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene Acd, encoding TPP1. Homozygosity for a hypomorphic acd allele preserved the emergence and expansion of fetal HSCs but led to profoundly defective function in transplantation assays. Upon complete Acd inactivation, HSCs expressed p53 target genes, underwent cell cycle arrest, and were severely depleted within days, leading to hematopoietic failure. TPP1 loss induced increased telomeric fusion events in bone marrow progenitors. However, unlike in epidermal stem cells, p53 deficiency did not rescue TPP1-deficient HSCs, indicating that shelterin dysfunction has unique effects in different stem cell populations. Because the consequences of telomere shortening are progressive and unsynchronized, acute loss of shelterin function represents an attractive alternative for studying telomere crisis in hematopoietic progenitors.

TP53 Disruption and Telomere Instability In Chronic Lymphocytic Leukemia

BackgroundDisruptions of the TP53 tumor suppressor pathway by TP53 gene mutations and/or by17p deletions resulting in loss of the TP53 locus are clearly associated with poor survival and chromosomal instability in chronic lymphocytic leukemia (CLL). Chromosomal instability is promoted by telomere dysfunction, and the TP53 pathway is involved in the monitoring of telomere integrity. AimThe purpose of the present study was to describe the changes in main telomeric parameters, which can affect telomere integrity, associated with TP53 mutations and 17p deletions and to evaluate their potential prognostic value. Patients and MethodsWe performed a comparison between a group of TP53 disrupted CLL patients (n= 24) and a group of TP53 wild-type CLL patients (n= 61). Median age was 70 [39-89] years, M/F ratio was 2.4/1. At the time of sampling, 35 patients were in Binet stage A, 19 patients in stage B and 31 patients in stage C. A total of 50 patients were subsequently treated by immunochemotherapy (FCR n= 30, other n= 8), alemtuzumab (n= 8) or alkylating agent-based regimens (n= 4). TP53 gene mutation screening was performed using Sanger sequencing of the entire coding region (exons 2–11). Cytogenetic analysis (karyotype and FISH) were performed to evaluate the chromosomal instability and detect the marks of telomeric fragility. Quantitative PCR approaches were used to measure the relative telomere length and to evaluate expression levels of shelterin genes (TRF1, TRF2, POT1, RAP1, TPP1, and TIN2) and telomerase (hTERT). ResultsThe patients showing a TP53 mutation without a 17p deletion and the patients carrying a 17p deletion with or without TP53 mutation appear to display the same level of telomeric instability. These cases are characterized by significant telomeric erosion (P<10-7), hTERT over expression (P<10-4) and decreased TRF1 (P<0.04) and TPP1 (P<0.016) expression levels. The impact of TP53 status on telomeric shortening is independent on Binet stages and classical markers of CLL progression (IgVH status, CD38 expression, lymphocyte doubling time). Moreover, TP53 disruption is strongly associated with telomere deletions, end-to-end fusions and chromosomal aberrations. Multivariate analysis on time to progression (TTP) including TP53 status and telomere characteristics revealed that two shelterin genes TTP1 (HR 7.1; 95% CI 2.0, 25.0; P= 0.0026) and RAP1 (HR 3.7; 95% CI 1.4, 9.6; P= 0.0088) have a significant prognostic impact which was independent on that of TP53 status (HR 6.9; 95% CI 2.3, 20.5; P= 0.0053). Low TPP1 and high RAP1 expression identify subsets of TP53 wild type patients with significantly shorter TTP as illustrated in Figure 1 and 2. [Display omitted] [Display omitted] ConclusionThese results suggest that TP53 disruption may play a major role in telomeric and chromosomal instability in CLL. Shelterin gene expression may help to identify a subset of TP53 wild-type patients with adverse prognosis. Disclosures:No relevant conflicts of interest to declare.

Cause-specific telomere factors deregulation in hepatocellular carcinoma

BackgroundAmong the numerous genetic defects associated with hepatocarcinogenesis, telomere abnormalities appear to play a role both in tumor promotion and maintenance. Telomeres, the chromosome extremities, are protected by specific proteins, the shelterin complex and by additional factors. Besides telomerase dysregulation, expression changes of these telomere factors have been observed in cancers.MethodsHere, we tested the hypothesis that such dysregulation might occur in hepatocellular carcinoma (HCC) with specific patterns depending on the cause of HCC. We compared telomere length, telomerase activity (TA), hTERT and telomere genes expression using PCR and Western-blot analyses between non-cirrhotic liver, peritumoral cirrhotic tissue (40 samples) and cancerous tissue (40 samples) derived from 40 patients with HBV-, HCV-, or alcohol-related HCC.ResultsAlterations in TA, hTERT expression and telomere length between non-cirrhotic, cirrhotic, and tumor samples were not significantly influenced by the cause of HCC. In contrast, the expression pattern of hTR, shelterin, and non-shelterin telomere protective factors clearly distinguished the 3 causes of cirrhosis and HCC. For patients with HBV diseased liver, when compared with non-cirrhotic liver, the cirrhotic tissue underexpressed all shelterin and all but HMRE11A and RAD50 non-shelterin telomere factors. For HCV the expression level of POT1, RAP1, Ku80, and RAD50 was higher in cirrhotic than in non-cirrhotic liver samples without evidence for significant transcriptional change for the remaining genes. For alcohol-related liver diseases, the expression level of POT1, RAP1, TIN2, hMRE11A, hMRE11B, Ku70, Ku80, RAD50, TANK1, and PINX1 was higher in cirrhotic than in non-cirrhotic liver samples. For the 3 causes of HCC, there was no significant change in shelterin and non-shelterin gene expression between cirrhosis and HCC samples.ConclusionsThese results validate our hypotheses and demonstrate that cirrhosis and HCC add-up numerous telomere dysfunctions including numerous cause-specific changes that appear to occur early during the course of the disease.

Telomere length and gene expression of shelterin in CD3(+) T cell of severe aplastic anemia patients

To explore the changes in telomere length and gene expression of complex shelterin (composed of 6 core components: TRF1, TRF2, POT1, TIN2, TPP1 and RAP1) in severe aplastic anemia (SAA). Bone marrow samples were obtained from 20 SAA patients and 10 normal controls. CD3(+)T cells were sorted by immunomagnetic separation. Telomere length was tested by Southern blot and the gene expressions of TRF1, TRF2, POT1, TIN2, TPP1 and RAP1 were detected by reverse transcription-PCR(RT-PCR). Telomeres of CD3(+)T cells were found significantly shorter in SAA untreated ((4.4 ± 1.1) kb, n = 9) and recovering groups((5.8 ± 1.0) kb, n = 11) than control group ((9.2 ± 3.3) kb, P < 0.05). Telomere length of CD3(+)T cells shortened with TH/S decreasing (r = 0.564, P = 0.029). The mRNA expression of POT1 decreased in untreated SAA patients (0.16(0.02-0.29)) and over-expressed in recovering patients (1.17(0.82-1.86), P < 0.05). The mRNA expression of RAP1 was significantly higher in untreated patients (4.14 (1.93-6.92)) than that in recovering group (0.87 (0.30-1.73) ) and controls (0.62 (0.45-4.07) , both P < 0.05). Changes in telomere length and shelterin gene expression occur in CD3(+)T cells of SAA patients and may be correlated with disease severity.

Telomere Dynamics and Homeostasis in a Transmissible Cancer

BackgroundDevil Facial Tumour Disease (DFTD) is a unique clonal cancer that threatens the world's largest carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii) with extinction. This transmissible cancer is passed between individual devils by cell implantation during social interactions. The tumour arose in a Schwann cell of a single devil over 15 years ago and since then has expanded clonally, without showing signs of replicative senescence; in stark contrast to a somatic cell that displays a finite capacity for replication, known as the “Hayflick limit”.Methodology/Principal FindingsIn the present study we investigate the role of telomere length, measured as Telomere Copy Number (TCN), and telomerase and shelterin gene expression, as well as telomerase activity in maintaining hyperproliferation of Devil Facial Tumour (DFT) cells. Our results show that DFT cells have short telomeres. DFTD TCN does not differ between geographic regions or between strains. However, TCN has increased over time. Unlimited cell proliferation is likely to have been achieved through the observed up-regulation of the catalytic subunit of telomerase (TERT) and concomitant activation of telomerase. Up-regulation of the central component of shelterin, the TRF1-intercating nuclear factor 2 (TINF2) provides DFT a mechanism for telomere length homeostasis. The higher expression of both TERT and TINF2 may also protect DFT cells from genomic instability and enhance tumour proliferation.Conclusions/SignificanceDFT cells appear to monitor and regulate the length of individual telomeres: i.e. shorter telomeres are elongated by up-regulation of telomerase-related genes; longer telomeres are protected from further elongation by members of the shelterin complex, which may explain the lack of spatial and strain variation in DFT telomere copy number. The observed longitudinal increase in gene expression in DFT tissue samples and telomerase activity in DFT cell lines might indicate a selection for more stable tumours with higher proliferative potential.

Lack of Mutations of the Telomerase RNA Component in Familial Papillary Thyroid Cancer with Short Telomeres

The occurrence of familial papillary thyroid cancer (FPTC) is well established but no susceptibility genes for this disease have been discovered. Our group has recently demonstrated that patients with FPTC have shorter telomeres, not associated with mutations in telomerase reverse transcriptase, gene than patients with sporadic papillary thyroid cancer (SPTC), healthy subjects (HS), and unaffected family members (UFMs). Several diseases, however, have short telomeres associated with mutations in the telomerase RNA component (TERC) gene or in the shelterin complex (POT1, RAP1, TIN2, TPP1, TRF1, and TRF2) genes. The objective of the present study was to verify whether short telomeres observed in FPTC patients were related to mutations in TERC or shelterin genes. Sixty-six patients with FPTC, 46 UFMs, 111 patients with SPTC, and 153 HS were analyzed by polymerase chain reaction followed by denaturing high performance liquid chromatography analysis and direct sequencing for the presence of TERC or shelterin gene mutations. When present, single-nucleotide polymorphisms were tested by χ(2) analysis at the genotypic, allelic, and haplotypic levels. The entire sequence of the TERC gene was analyzed with particular attention to known mutations known to be associated with short telomeres. All samples appeared to be homozygous wild type for A-771G, C-99G, G305A, G322A, C323T, C408G, G450A, T467C, G508A, A514G, G623A, and C727G substitutions and for the 378Δ→3' deletion in the TERC gene. In addition, upon analysis of all samples for shelterin proteins, we observed a significant decrease in POT1 and RAP1 protein expression in the blood of FPTC patients compared with SPTC subjects. However, no mutations or polymorphisms were found when in the coding sequences of both genes. To our knowledge this is the first study of TERC mutations or alterations in the shelterin complex in relation to FPTC. Shorter telomeres observed in FPTC are not linked to mutations or polymorphisms in TERC, POT1, or RAP1 genes.

Telomeric damage in early stage of chronic lymphocytic leukemia correlates with shelterin dysregulation

AbstractCells of B-cell chronic lymphocytic leukemia (B-CLL) are characterized by short telomeres despite a low proliferative index. Because telomere length has been reported to be a valuable prognosis criteria, there is a great interest in a deep understanding of the origin and consequences of telomere dysfunction in this pathology. Cases of chromosome fusion involving extremely short telomeres have been reported at advanced stage. In the present study, we address the question of the existence of early telomere dysfunction during the B-CLL time course. In a series restricted to 23 newly diagnosed Binet stage A CLL patients compared with 12 healthy donors, we found a significant increase in recruitment of DNA-damage factors to telomeres showing telomere dysfunction in the early stage of the disease. Remarkably, the presence of dysfunctional telomeres did not correlate with telomere shortening or chromatin marks deregulation but with a down-regulation of 2 shelterin genes: ACD (coding for TPP1; P = .0464) and TINF2 (coding for TIN2; P = .0177). We propose that telomeric deprotection in the early step of CLL is not merely the consequence of telomere shortening but also of shelterin alteration.

Abstract 2996: DNA-damaging agents disrupt telomere structure

Abstract Telomeres are found at chromosome ends. When these ends are not protected by telomeric capping, the telomeres are recognized as double-strand breaks and the consequent recruitment of DNA repair machinery may affect cellular fate. Furthermore, telomeric capping proteins have been shown to interact with some of the proteins involved in DNA repair. A direct link thus exists between DNA repair and telomeres. However, few data are available on the changes in telomere structure associated with the different modes of DNA repair. To study the effects of three DNA-damaging agents on telomere structure, we exposed normal human primary fibroblasts to H202, gamma radiation, or UVB rays. The cells were then harvested at different times over a period of 24 hours. First the telomere nuclear architecture was studied by quantitative fluorescence in situ hybridization with a telomere-specific peptide nucleic acid probe followed by 3D telomere imaging. H202 treatment induced a significant reduction of telomere signals and a disruption of the nuclear volume that became normal after 24 hours of recovery. Also, telomere positioning was positively correlated to nuclear volume that disrupted over time. For gamma irradiation, the nuclear volume was positively correlated to the number of telomere signals and negatively correlated to telomere average intensity for each time point. UVB lead to a significant formation of telomeric aggregates and disruption of telomere nuclear shape by forming ellipsoid instead of circular shape. Next, we studied the effects of these different DNA damage agents on the expression profile of telomere shelterin genes. The most affected genes were TRF1, POT1, TIN2 and Rap1. For instance, while the three agents showed a high induction of TIN2, gamma and H202 treatments presented high induction for POT1 and only H202 treatment induced significant increase of TRF1. In conclusion, certain DNA-damaging events alter telomere structure by acting on nuclear telomere architecture and the expression profile of some telomere shelterin genes. The disruption of telomere structure due to acute insults might have an influence on cell homeostasis and the pathways involved can shed light for a better understanding of some pathological and therapeutic processes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2996. doi:10.1158/1538-7445.AM2011-2996

Developmental studies ofXenopusshelterin complexes: the message to reset telomere length is already present in the egg

The 6-protein complex shelterin protects the telomeres of human chromosomes. The recent discovery that telomeres are important for epigenetic gene regulation and vertebrate embryonic development calls for the establishment of model organisms to study shelterin and telomere function under normal developmental conditions. Here, we report the sequences of the shelterin-encoding genes in Xenopus laevis and its close relation Xenopus tropicalis. In vitro expression and biochemical characterization of the Xenopus shelterin proteins TRF1, TRF2, POT1, TIN2, RAP1, TPP1, and the shelterin accessory factor PINX1 indicate that all main functions of their human orthologs are conserved in Xenopus. The XlTRF1 and XtTRF1 proteins bind double-stranded telomeric DNA sequence specifically and interact with XlTIN2 and XtTIN2, respectively. Similarly, the XlTRF2 and XtTRF2 proteins bind double-stranded telomeric DNA and interact with XlRAP1 and XtRAP1, respectively, whereas the XlPOT1 and XtPOT1 proteins bind single-stranded telomeric DNA. Real-time PCR further reveals the gene expression profiles for telomerase and the shelterin genes during embryogenesis. Notably, the composition of shelterin and the formation of its subcomplexes appear to be temporally regulated during embryonic development. Moreover, unexpectedly high telomerase and shelterin gene expression during early embryogenesis may reflect a telomere length-resetting mechanism, similar to that reported for induced pluripotent stem cells and for animals cloned through somatic nuclear transfer.

Telomere uncapping during in vitro T‐lymphocyte senescence

Normal lymphocytes represent examples of somatic cells that are able to induce telomerase activity when stimulated. As previously reported, we showed that, during lymphocyte long-term culture and repeated stimulations, the appearance of senescent cells is associated with telomere shortening and a progressive drop in telomerase activity. We further showed that this shortening preferentially occured at long telomeres and was interrupted at each stimulation by a transitory increase in telomere length. In agreement with the fact that telomere uncapping triggers lymphocyte senescence, we observed an increase in gamma-H2AX and 53BP1 foci as well as in the percentage of cells exhibiting DNA damage foci in telomeres. Such a DNA damage response may be related to the continuous increase of p16(ink4a) upon cell stimulation and cell aging. Remarkably, at each stimulation, the expression of shelterin genes, such as hTRF1, hTANK1, hTIN2, hPOT1 and hRAP1, was decreased. We propose that telomere dysfunction during lymphocyte senescence caused by iterative stimulations does not only result from an excessive telomere shortening, but also from a decrease in shelterin content. These observations may be relevant for T-cell biology and aging.

HTERT Transcriptional Repression in Acute Myeloid Leukemias

Telomeres consist of repetitive DNA sequences and specific proteins, creating a specialized structure called the telosome. Unraveling the specific telomerase and telosome changes in leukemia is thought for providing new knowledge about oncogenesis, together with useful clinical markers and specific therapeutic targets. Here we measured telomerase activity (TA) by a quantitative TRAP assay in 57 human acute leukemia samples including 20 acute lymphoblastic leukemias (ALL) and 37 acute myeloid leukemias (AML). AML, TALL (n=7) and BALL (n=13) displayed significantly higher levels of TA than their normal counterparts [normal bone marrow CD34+ cells deriving from donors (BMCs, 17 samples) for AML, B-lymphocytes (n=20) for BALL and T-lymphocytes (n=24) for TALL; p<0.005 for all]. hTERT encodes telomerase reverse transcriptase which is the rate-limiting factor for TA. In BALL and TALL, hTERT expression was respectively 53 (p=0.0005) and 12 (p=0.05) times higher than in control cells. In contrast AML blasts displayed 4.5 times lower hTERT expression levels than BMCs (p=0.0008). hTERT transcripts include the full-length functional hTERT mRNA and non-functional sequences. AML blasts displayed a global decrease of all hTERT transcripts (p<0.0006) without any shift in the splicing pattern. c-Myc is a transcriptional activator of hTERT; we evidenced a positive correlation between c-Myc and hTERT transcription in BMCs (p=0.005) but not in myeloblasts. WT1, which represses hTERT transcription ex vivo in certain cell lines, can act as a transcriptional regulator or repressor depending on the cellular context. WT1 was significantly overexpressed in all leukemic subtypes (p<0.05 for all). In AML, WT1 expression negatively correlated hTERT expression (p=0.022) whereas both transcripts were positively correlated in BALL (p=0.029). We investigated the transcriptional control of hTERT by comparing the hTERT promoter occupancy between AML blasts and BMCs. Nuclear extracts deriving from 9 AML and 3 normal BMC samples were incubated with the hTERT core promoter (hCP). The protein complexes interacting with hCP were purified and components were identified by mass spectrometry. The specific AML hCP peptidome included 19 proteins of which 4, 5, 9, and 1 are involved in transcriptional regulation (including 3 factors possessing negative transcriptional effects), chromatin remodeling (including the DEK protein), mRNA maturation, and DNA methylation [DNA methyl transferase 1 (DNMT1)], respectively. With the exception of DNMT1, which is involved in hTERT transcriptional repression via CpG methylation, these factors have not been previously found negatively correlated with hTERT transcription. DEK is a chromatin remodeling protein frequently overexpressed in AML. Using CHIP, RNAi, and transfection experiments we validated and characterized the transcriptional repression of hTERT by DEK. To investigate the paradoxical association of hTERT transcriptional repression with elevated TA in AML, we measured the transcription of 36 genes involved in the post-translational regulation of hTERT and/or encoding shelterin and non-shelterin telosome genes. AML blasts overexpressed AKT1 (p=0.01) and underexpressed Ku70 (p=0.039). AKT1 activates hTERT through phosphorylation while Ku70 may act as a negative regulator of hTERT by reducing the ability of telomerase to access the exposed 3′ overhang. One can propose that these gene deregulations help compensate hTERT transcriptional repression towards an increased TA in AML. In B and TALL, telomeric gene deregulation involved shelterin and non-shelterin genes with a transcriptome pattern consistent with telomere deprotection. This is the first report of a hTERT transcriptional repression in de novo AML resulting from a global repression of all hTERT transcripts, depending on WT1 expression and involving an acquired defect in the transcriptional control of hTERT by c-Myc. DEK and DNMT1 participate to this process in vivo; our proteomic survey evidenced additional promising candidates. hTERT transcriptional repression is known to promote telomere dysfunctions and thereby genetic instability. We propose that it helps initiate the AML phenotype, which is subsequently stabilized by additional deregulations including AKT1 and Ku70-dependent hTERT post-transcriptional activation. Finally these results support important telosome modifications in acute leukemias with specific features depending on the disease phenotype.

A balanced transcription between telomerase and the telomeric DNA-binding proteins TRF1, TRF2 and Pot1 in resting, activated, HTLV-1-transformed and Tax-expressing human T lymphocytes

BackgroundThe functional state of human telomeres is controlled by telomerase and by a protein complex named shelterin, including the telomeric DNA-binding proteins TRF1, TRF2 and Pot1 involved in telomere capping functions. The expression of hTERT, encoding the catalytic subunit of telomerase, plays a crucial role in the control of lymphocyte proliferation by maintaining telomere homeostasis. It has been previously found that hTERT activity is down-regulated by the human T cell leukaemia virus type 1 (HTLV-1) Tax protein in HTLV-1 transformed T lymphocytes. In this study, we have examined the effects of Tax expression on the transcriptional profile of telomerase and of shelterin in human T lymphocytes.ResultsWe first provide evidence that the up-regulation of hTERT transcription in activated CD4+ T lymphocytes is associated with a down-regulation of that of TERF1, TERF2 and POT1 genes. Next, the down-regulation of hTERT transcription by Tax in HTLV-1 transformed or in Tax-expressing T lymphocytes is found to correlate with a significant increase of TRF2 and/or Pot1 mRNAs. Finally, ectopic expression of hTERT in one HTLV-1 T cell line induces a marked decrease in the transcription of the POT1 gene. Collectively, these observations predict that the increased transcriptional expression of shelterin genes is minimizing the impact on telomere instability induced by the down-regulation of hTERT by Tax.ConclusionThese findings support the notion that Tax, telomerase and shelterin play a critical role in the proliferation of HTLV-1 transformed T lymphocytes.