Long noncoding RNAs (lncRNAs) are key regulators of chromatin state, yet the nature and sites of RNA-chromatin interaction are mostly unknown. Here we introduce Chromatin Isolation by RNA ...Purification (ChIRP), where tiling oligonucleotides retrieve specific lncRNAs with bound protein and DNA sequences, which are enumerated by deep sequencing. ChIRP-seq of three lncRNAs reveal that RNA occupancy sites in the genome are focal, sequence-specific, and numerous. Drosophila roX2 RNA occupies male X-linked gene bodies with increasing tendency toward the 3′ end, peaking at CES sites. Human telomerase RNA TERC occupies telomeres and Wnt pathway genes. HOTAIR lncRNA preferentially occupies a GA-rich DNA motif to nucleate broad domains of Polycomb occupancy and histone H3 lysine 27 trimethylation. HOTAIR occupancy occurs independently of EZH2, suggesting the order of RNA guidance of Polycomb occupancy. ChIRP-seq is generally applicable to illuminate the intersection of RNA and chromatin with newfound precision genome wide.
► ChIRP-seq maps the binding sites of specific RNAs on chromatin genome wide ► RNA-genome interactions are numerous, focal, and sequence-specific ► Telomerase RNA TERC binds telomeres and Wnt pathway genes ► HOTAIR lncRNA nucleates broader domains of Polycomb and H3K27me3 occupancy
Telomere synthesis in cancer cells and stem cells involves trafficking of telomerase to Cajal bodies, and telomerase is thought to be recruited to telomeres through interactions with telomere-binding ...proteins. Here, we show that the OB-fold domain of the telomere-binding protein TPP1 recruits telomerase to telomeres through an association with the telomerase reverse transcriptase TERT. When tethered away from telomeres and other telomere-binding proteins, the TPP1 OB-fold domain is sufficient to recruit telomerase to a heterologous chromatin locus. Expression of a minimal TPP1 OB-fold inhibits telomere maintenance by blocking access of telomerase to its cognate binding site at telomeres. We identify amino acids required for the TPP1-telomerase interaction, including specific loop residues within the TPP1 OB-fold domain and individual residues within TERT, some of which are mutated in a subset of pulmonary fibrosis patients. These data define a potential interface for telomerase-TPP1 interaction required for telomere maintenance and implicate defective telomerase recruitment in telomerase-related disease.
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► Enforced expression of telomerase forms neo-Cajal bodies at telomeres ► TPP1 OB-fold domain recruits telomerase to a heterologous chromatin locus ► TPP1-OB alone sequesters telomerase within Cajal bodies and causes telomere shortening ► OB-fold mutations and some disease mutations in TERT block telomerase recruitment
The shelterin component TPP1 recruits telomerase from storage in Cajal bodies via a direct interaction with the enzyme’s catalytic subunit TERT. Mutations in TERT found associated with an idiopathic lung disease disrupt the interaction and impair telomerase mobilization.
Hepatocytes are replenished gradually during homeostasis and robustly after liver injury
. In adults, new hepatocytes originate from the existing hepatocyte pool
, but the cellular source of renewing ...hepatocytes remains unclear. Telomerase is expressed in many stem cell populations, and mutations in telomerase pathway genes have been linked to liver diseases
. Here we identify a subset of hepatocytes that expresses high levels of telomerase and show that this hepatocyte subset repopulates the liver during homeostasis and injury. Using lineage tracing from the telomerase reverse transcriptase (Tert) locus in mice, we demonstrate that rare hepatocytes with high telomerase expression (TERT
hepatocytes) are distributed throughout the liver lobule. During homeostasis, these cells regenerate hepatocytes in all lobular zones, and both self-renew and differentiate to yield expanding hepatocyte clones that eventually dominate the liver. In response to injury, the repopulating activity of TERT
hepatocytes is accelerated and their progeny cross zonal boundaries. RNA sequencing shows that metabolic genes are downregulated in TERT
hepatocytes, indicating that metabolic activity and repopulating activity may be segregated within the hepatocyte lineage. Genetic ablation of TERT
hepatocytes combined with chemical injury causes a marked increase in stellate cell activation and fibrosis. These results provide support for a 'distributed model' of hepatocyte renewal in which a subset of hepatocytes dispersed throughout the lobule clonally expands to maintain liver mass.
Aging is a complex process that affects multiple organs. Modeling aging and age-related diseases in the lab is challenging because classical vertebrate models have relatively long lifespans. Here, we ...develop the first platform for rapid exploration of age-dependent traits and diseases in vertebrates, using the naturally short-lived African turquoise killifish. We provide an integrative genomic and genome-editing toolkit in this organism using our de-novo-assembled genome and the CRISPR/Cas9 technology. We mutate many genes encompassing the hallmarks of aging, and for a subset, we produce stable lines within 2–3 months. As a proof of principle, we show that fish deficient for the protein subunit of telomerase exhibit the fastest onset of telomere-related pathologies among vertebrates. We further demonstrate the feasibility of creating specific genetic variants. This genome-to-phenotype platform represents a unique resource for studying vertebrate aging and disease in a high-throughput manner and for investigating candidates arising from human genome-wide studies.
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•The turquoise killifish is the shortest-lived vertebrate species bred in the lab•A complete genome-to-phenotype platform for this emerging model•Telomerase-deficient fish display the fastest onset of related human pathologies•A resource of targeted aging- and disease-related genes and available fish lines
Aging is the number one risk factor for many human pathologies, yet it is challenging to study as existing vertebrate models are relatively long lived. The development of an integrative genome-to-phenotype platform in a naturally short-lived vertebrate, the African turquoise killifish, opens the door to high-throughput in vivo modeling of vertebrate aging and complex human diseases.
Telomere maintenance by telomerase is impaired in the stem cell disease dyskeratosis congenita and during human aging. Telomerase depends upon a complex pathway for enzyme assembly, localization in ...Cajal bodies, and association with telomeres. Here, we identify the chaperonin CCT/TRiC as a critical regulator of telomerase trafficking using a high-content genome-wide siRNA screen in human cells for factors required for Cajal body localization. We find that TRiC is required for folding the telomerase cofactor TCAB1, which controls trafficking of telomerase and small Cajal body RNAs (scaRNAs). Depletion of TRiC causes loss of TCAB1 protein, mislocalization of telomerase and scaRNAs to nucleoli, and failure of telomere elongation. DC patient-derived mutations in TCAB1 impair folding by TRiC, disrupting telomerase function and leading to severe disease. Our findings establish a critical role for TRiC-mediated protein folding in the telomerase pathway and link proteostasis, telomere maintenance, and human disease.
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•Telomere maintenance requires assembly and trafficking of telomerase•Proteostasis factor TRiC folds the telomerase cofactor TCAB1•Loss of TRiC causes telomerase mislocalization and telomere elongation failure•TCAB1 mutations in patients disrupt TRiC-mediated folding, leading to disease
The assembly and localization of telomerase and the consequent control of telomere elongation are found to be dependent upon the chaperonin TRiC. Mutations in the telomerase cofactor TCAB1 that impair its folding by TRiC cause dyskeratosis congenita, thus highlighting a proteostatic level of control of telomere function.
Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of ...cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function.
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•The Rb tumor suppressor inhibits reprogramming of fibroblasts to iPSCs•The effect of Rb on reprogramming is independent of cell-cycle regulation•Rb promotes assembly of repressive chromatin at pluripotency network genes•Deletion of Sox2 prevents cancer initiation upon loss of Rb in mice
To investigate how the Rb tumor suppressor inhibits cellular dedifferentiation, Kareta et al. utilized iPSC reprogramming as a cellular system and observed that Rb restricts reprogramming by silencing pluripotency genes and networks. Rb repression of one pluripotency factor, Sox2, in particular, is critical to block cancer initiation in mice.
Telomerase is a ribonucleoprotein (RNP) complex that synthesizes telomere repeats in tissue progenitor cells and cancer cells. Active human telomerase consists of at least three principal subunits, ...including the telomerase reverse transcriptase, the telomerase RNA (TERC), and dyskerin. Here, we identify a holoenzyme subunit, TCAB1 (telomerase Cajal body protein 1), that is notably enriched in Cajal bodies, nuclear sites of RNP processing that are important for telomerase function. TCAB1 associates with active telomerase enzyme, established telomerase components, and small Cajal body RNAs that are involved in modifying splicing RNAs. Depletion of TCAB1 by using RNA interference prevents TERC from associating with Cajal bodies, disrupts telomerase-telomere association, and abrogates telomere synthesis by telomerase. Thus, TCAB1 controls telomerase trafficking and is required for telomere synthesis in human cancer cells.
Telomeres, the nucleoprotein complex at the ends of eukaryotic chromosomes, perform an essential cellular role in part by preventing the chromosomal end from initiating a DNA-damage response. This ...function of telomeres can be compromised as telomeres erode either as a consequence of cell division in culture or as a normal part of cellular ageing in proliferative tissues. Telomere dysfunction in this context leads to DNA-damage signaling and activation of the tumor-suppressor protein p53, which then can prompt either cellular senescence or apoptosis. By culling cells with dysfunctional telomeres, p53 plays a critical role in protecting tissues against the effects of critically short telomeres. However, as telomere dysfunction worsens, p53 likely exacerbates short telomere-driven tissue failure diseases, including pulmonary fibrosis, aplastic anemia, and liver cirrhosis. In cells lacking p53, unchecked telomere shortening drives chromosomal end-to-end fusions and cycles of chromosome fusion-bridge-breakage. Incipient cancer cells confronting these telomere barriers must disable p53 signaling to avoid senescence and eventually up-regulate telomerase to achieve cellular immortality. The recent findings of highly recurrent activating mutations in the promoter for the telomerase reverse transcriptase (TERT) gene in diverse human cancers, together with the widespread mutations in p53 in cancer, provide support for the idea that circumvention of a telomere-p53 checkpoint is essential for malignant progression in human cancer.
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 (TERT(ci)) to study the mechanism of TERT action in mammalian skin, an ideal tissue for studying progenitor cell biology. We show that TERT(ci) 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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Dyskeratosis congenita (DC) is a genetic disorder of defective tissue maintenance and cancer predisposition caused by short telomeres and impaired stem cell function. Telomerase mutations are thought ...to precipitate DC by reducing either the catalytic activity or the overall levels of the telomerase complex. However, the underlying genetic mutations and the mechanisms of telomere shortening remain unknown for as many as 50% of DC patients, who lack mutations in genes controlling telomere homeostasis. Here, we show that disruption of telomerase trafficking accounts for unknown cases of DC. We identify DC patients with missense mutations in TCAB1, a telomerase holoenzyme protein that facilitates trafficking of telomerase to Cajal bodies. Compound heterozygous mutations in TCAB1 disrupt telomerase localization to Cajal bodies, resulting in misdirection of telomerase RNA to nucleoli, which prevents telomerase from elongating telomeres. Our findings establish telomerase mislocalization as a novel cause of DC, and suggest that telomerase trafficking defects may contribute more broadly to the pathogenesis of telomere-related disease.
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