Jinkuk Choi

Telomerase modulates Wnt signalling by association with target gene chromatin

Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/β-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/β-catenin signalling by serving as a cofactor in a β-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior–posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert-/- mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/β-catenin signalling pathway.

TERT Promotes Epithelial Proliferation through Transcriptional Control of a Myc- and Wnt-Related Developmental Program

Telomerase serves a critical role in stem cell function and tissue homeostasis. This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood. Here, we use a TERT mutant lacking RT function (TERTci) to study the mechanism of TERT action in mammalian skin, an ideal tissue for studying progenitor cell biology. We show that TERTci retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To understand the nature of this RT-independent function for TERT, we studied the genome-wide transcriptional response to acute changes in TERT levels in mouse skin. We find that TERT facilitates activation of progenitor cells in the skin and hair follicle by triggering a rapid change in gene expression that significantly overlaps the program controlling natural hair follicle cycling in wild-type mice. Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer. These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.

Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics.

IDOL Stimulates Clathrin-Independent Endocytosis and Multivesicular Body-Mediated Lysosomal Degradation of the Low-Density Lipoprotein Receptor

ABSTRACT The low-density lipoprotein receptor (LDLR) is a critical determinant of plasma cholesterol levels that internalizes lipoprotein cargo via clathrin-mediated endocytosis. Here, we show that the E3 ubiquitin ligase IDOL stimulates a previously unrecognized, clathrin-independent pathway for LDLR internalization. Real-time single-particle tracking and electron microscopy reveal that IDOL is recruited to the plasma membrane by LDLR, promotes LDLR internalization in the absence of clathrin or caveolae, and facilitates LDLR degradation by shuttling it into the multivesicular body (MVB) protein-sorting pathway. The IDOL-dependent degradation pathway is distinct from that mediated by PCSK9 as only IDOL employs ESCRT (endosomal-sorting complex required for transport) complexes to recognize and traffic LDLR to lysosomes. Small interfering RNA (siRNA)-mediated knockdown of ESCRT-0 (HGS) or ESCRT-I (TSG101) components prevents IDOL-mediated LDLR degradation. We further show that USP8 acts downstream of IDOL to deubiquitinate LDLR and that USP8 is required for LDLR entry into the MVB pathway. These results provide key mechanistic insights into an evolutionarily conserved pathway for the control of lipoprotein receptor expression and cellular lipid uptake.

Telomere protection and TRF2 expression are enhanced by the canonical Wnt signalling pathway

The DNA‐binding protein TRF2 is essential for telomere protection and chromosome stability in mammals. We show here that TRF2 expression is activated by the Wnt/β‐catenin signalling pathway in human cancer and normal cells as well as in mouse intestinal tissues. Furthermore, β‐catenin binds to TRF2 gene regulatory regions that are functional in a luciferase transactivating assay. Reduced β‐catenin expression in cancer cells triggers a marked increase in telomere dysfunction, which can be reversed by TRF2 overexpression. We conclude that the Wnt/β‐catenin signalling pathway maintains a level of TRF2 critical for telomere protection. This is expected to have an important role during development, adult stem cell function and oncogenesis.

Telomere uncapping in progenitor cells with critical telomere shortening is coupled to S-phase progression in vivo

Telomeres protect chromosome ends and serve as a substrate for telomerase, a reverse transcriptase that adds DNA repeats to the telomere terminus. In the absence of telomerase, telomeres progressively shorten, ultimately leading to telomere uncapping, a structural change at the telomere that activates DNA damage responses and leads to ligation of chromosome ends. Telomere uncapping has been implicated in aging and cancer, yet the precise mechanism of uncapping and its relationship to cell cycle remain to be defined. Here, we show that telomeres uncap in an S-phase-dependent manner in gastrointestinal progenitors of TERT−/− mice. We develop an in vivo assay that allows a quantitative kinetic assessment of telomere dysfunction-induced apoptosis and its relationship to cell cycle. By exploiting the mathematical relationship between rates of generation and clearance of apoptotic cells, we show that 86.2 ± 8.8% of apoptotic gastrointestinal cells undergo programmed cell death either late in S-phase or in G2. Apoptosis is primarily triggered via a signaling cascade from newly uncapped telomeres to the tumor suppressor p53, rather than by chromosome fusion-bridge breakage, because mitotic blockade did not alter the rate of newly generated apoptotic bodies. These data support a model in which rapidly dividing progenitor cells within a tissue with short telomeres are vulnerable to telomere uncapping during or shortly after telomere replication.

Marker‐free motion correction in weight‐bearing cone‐beam CT of the knee joint

PURPOSE To allow for a purely image-based motion estimation and compensation in weight-bearing cone-beam computed tomography of the knee joint. METHODS Weight-bearing imaging of the knee joint in a standing position poses additional requirements for the image reconstruction algorithm. In contrast to supine scans, patient motion needs to be estimated and compensated. The authors propose a method that is based on 2D/3D registration of left and right femur and tibia segmented from a prior, motion-free reconstruction acquired in supine position. Each segmented bone is first roughly aligned to the motion-corrupted reconstruction of a scan in standing or squatting position. Subsequently, a rigid 2D/3D registration is performed for each bone to each of K projection images, estimating 6 × 4 × K motion parameters. The motion of individual bones is combined into global motion fields using thin-plate-spline extrapolation. These can be incorporated into a motion-compensated reconstruction in the backprojection step. The authors performed visual and quantitative comparisons between a state-of-the-art marker-based (MB) method and two variants of the proposed method using gradient correlation (GC) and normalized gradient information (NGI) as similarity measure for the 2D/3D registration. RESULTS The authors evaluated their method on four acquisitions under different squatting positions of the same patient. All methods showed substantial improvement in image quality compared to the uncorrected reconstructions. Compared to NGI and MB, the GC method showed increased streaking artifacts due to misregistrations in lateral projection images. NGI and MB showed comparable image quality at the bone regions. Because the markers are attached to the skin, the MB method performed better at the surface of the legs where the authors observed slight streaking of the NGI and GC methods. For a quantitative evaluation, the authors computed the universal quality index (UQI) for all bone regions with respect to the motion-free reconstruction. The authors quantitative evaluation over regions around the bones yielded a mean UQI of 18.4 for no correction, 53.3 and 56.1 for the proposed method using GC and NGI, respectively, and 53.7 for the MB reference approach. In contrast to the authors registration-based corrections, the MB reference method caused slight nonrigid deformations at bone outlines when compared to a motion-free reference scan. CONCLUSIONS The authors showed that their method based on the NGI similarity measure yields reconstruction quality close to the MB reference method. In contrast to the MB method, the proposed method does not require any preparation prior to the examination which will improve the clinical workflow and patient comfort. Further, the authors found that the MB method causes small, nonrigid deformations at the bone outline which indicates that markers may not accurately reflect the internal motion close to the knee joint. Therefore, the authors believe that the proposed method is a promising alternative to MB motion management.

Regulation of Cellular Immortalization and Steady-State Levels of the Telomerase Reverse Transcriptase through Its Carboxy-Terminal Domain

ABSTRACT Telomerase maintains cell viability and chromosomal stability through the addition of telomere repeats to chromosome ends. The reactivation of telomerase through the upregulation of TERT, the telomerase protein subunit, is an important step during cancer development, yet TERT protein function and regulation remain incompletely understood. Despite its close sequence similarity to human TERT (hTERT), we find that mouse TERT (mTERT) does not immortalize primary human fibroblasts. Here we exploit these differences in activity to understand TERT protein function by creating chimeric mouse-human TERT proteins. Through the analysis of these chimeric TERT proteins, we find that sequences in the human carboxy-terminal domain are critical for telomere maintenance in human fibroblasts. The substitution of the human carboxy-terminal sequences into the mouse TERT protein is sufficient to confer immortalization and maintenance of telomere length and function. Strikingly, we find that hTERT protein accumulates to markedly higher levels than does mTERT protein and that the sequences governing this difference in protein regulation also reside in the carboxy-terminal domain. These elevated protein levels, which are characteristic of hTERT, are necessary but not sufficient for telomere maintenance because stabilized mTERT mutants cannot immortalize human cells. Thus, the TERT carboxy terminus contains sequences that regulate TERT protein levels and determinants that are required for productive action on telomere ends.

Stem Cell Aging and Aberrant Differentiation within the Niche

Stem cells age, but the underlying mechanisms remain unclear. In a recent issue of Cell, Inomata and colleagues (2009) show that DNA damage, a prime suspect in stem cell aging, causes graying and loss of melanocyte stem cells by inducing premature differentiation, without inducing apoptosis or senescence.

Interventional dual‐energy imaging—Feasibility of rapid kV‐switching on a C‐arm CT system

PURPOSE In the last years, dual-energy CT imaging has shown clinical value, thanks to its ability to differentiate materials based on their atomic number and to exploit different properties of images acquired at two different energies. C-arm CT systems are used to guide procedures in the interventional suite. Until now, there are no commercially available systems that employ dual-energy material decomposition. This paper explores the feasibility of implementing a fast kV-switching technique on a clinically available angiographic system for acquiring dual-energy C-arm CT images. METHODS As an initial proof of concept, a fast kV-switching approach was implemented on an angiographic C-arm system and the peak tube voltage during 3D rotational scans was measured. The tube voltage measurements during fast kV-switching scans were compared to corresponding measurements on kV-constant scans. Additionally, to prove stability of the requested exposure parameters, the accuracy of the delivered tube current and pulse width were also recorded and compared. In a first phantom experiment, the voxel intensity values of the individual tube voltage components of the fast kV-switching scans were compared to their corresponding kV-constant scans. The same phantom was used for a simple material decomposition between different iodine concentrations and pure water using a fast kV-switching protocol of 81 and 125 kV. In the last experiment, the same kV-switching protocol as in the phantom scan was used in an in vivo pig study to demonstrate the clinical feasibility. RESULTS During rapid kV-switching acquisitions, the measured tube voltage of the x-ray tube during fast switching scans has an absolute deviation of 0.23 ± 0.13 kV compared to the measured tube voltage produced during kV-constant acquisitions. The stability of the peak tube voltage over different scan requests was about 0.10 kV for the low and 0.46 for the high energy kV-switching scans and less than 0.1 kV for kV-constant scans, indicating slightly lower stability for kV-switching scans. The tube current resulted in a relative deviation of -1.6% for the low and 6.6% overestimation for the high tube voltage of the kV-switching scans compared to the kV-constant scans. The pulse width showed no deviation for the longer pulse width and only minor deviations (0.02 ± 0.02 ms) for the shorter pulse widths compared to the kV-constant scans. The phantom experiment using different iodine concentrations showed an accurate correlation (R2 > 0.99) between the extracted intensity values in the kV-switching and kV-constant reconstructed volumes, and allows for an automatic differentiation between contrast concentration down to 10% (350 mg/ml iodine) and pure water under low-noise conditions. Preliminary results of iodine and soft tissue separation showed also promising results in the first in vivo pig study. CONCLUSIONS The feasibility of dual-energy imaging using a fast kV-switching method on an angiographic C-arm CT system was investigated. Direct measurements of beam quality in the x-ray field demonstrate the stability of the kV-switching method. Phantom and in vivo experiments showed that images did not deviate from those of corresponding kV-constant scans. All performed experiments confirmed the capability of performing fast kV-switching scans on a clinically available C-arm CT system. More complex material decomposition tasks and postprocessing steps will be part of future investigations.