Genomic analysis of mice with endothelium-specific TERT knockout (RNA-Seq)
Ontology highlight
ABSTRACT: Health of endothelial cells (EC) is fundamental for function of vital organs. There is evidence accumulating that senescence of EC is associated with aging-related diseases. However, it has not been established whether EC senescence underlies the pathophysiology of aging. Here, we report a mouse model of EC senescence. Inactivation of telomerase (TERT) expression sets the stage for aging and predisposes cells to replicative senescence. TERT maintains telomere length and has telomere-independent effects on cell transcriptome and physiology. We generated mice with TERT gene knock-out (KO) specifically in EC. We analyzed EC from adipose tissues (AT) and skeletal muscle cells from Tert-EC-KO and control mice raised on either chow or high-calorie diet (HCD) and subjected them to RNA sequencing (RNAseq). Single cell RNAseq data were also obtained for subcutaneous and visceral AT. Preliminary analysis of data deposited online indicates that TERT KO induces premature cell senescence and dysfunction. Moreover, HCD feeding exacerbates the effect of TERT loss. We conclude that the Tert-EC-KO mice are a useful model to study EC senescence and its effects on aging-associated organ dysfunction.
Project description:Health of endothelial cells (EC) is fundamental for function of vital organs. However, it has not been established whether EC senescence underlies the pathophysiology of aging. Here, we report a mouse model of EC senescence. Inactivation of telomerase (TERT) expression sets the stage for aging and predisposes cells to replicative senescence. TERT maintains telomere length and has telomere-independent effects on cell transcriptome and physiology. We generated mice with TERT gene knock-out (KO) specifically in EC. We analyzed EC from adipose tissues (AT) and skeletal muscle cells from Tert-EC-KO and control mice raised on either chow or high-calorie diet (HCD) and subjected them to RNA sequencing (RNAseq). Single cell RNAseq data were also obtained for subcutaneous and visceral AT. Preliminary analysis of data deposited online indicates that TERT KO induces premature cell senescence and dysfunction. Moreover, HCD feeding exacerbates the effect of TERT loss. We conclude that the Tert-EC-KO mice are a useful model to study EC senescence and its effects on aging-associated organ dysfunction.
Project description:Aging is the consequence of intra- and extracellular events that promote cellular senescence. Dyskeratosis congenita (DC) is an example of a premature aging disorder caused by underlying telomere/telomerase-related mutations. Cells from these patients offer an opportunity to study telomere-related aging and senescence. Our previous work has found that telomere shortening stimulates DNA damage responses (DDR) and increases reactive oxygen species (ROS), thereby promoting entry into senescence. This work also found that telomere elongation via TERT expression, the catalytic component of the telomere-elongating enzyme telomerase, or p53 shRNA could decrease ROS by disrupting this telomere-DDR-ROS pathway. To further characterize this pathway, we performed a CRISPR/Cas9 knockout screen to identify genes that extend lifespan in DC cells. Of the cellular clones isolated due to increased lifespan, 34% had a guide RNA (gRNA) targeting CEBPB while gRNAs targeting WSB1, MED28 and p73 were observed multiple times. CEBPB is a transcription factor associated with activation of proinflammatory response genes suggesting that inflammation may be present in DC cells. The inflammatory response was investigated using RNA-Seq to compare DC and control cells. Expression of inflammatory genes were found to be significantly elevated (p<0.0001) in addition to a key subset of these inflammation-related genes (IL1B, IL6, IL8, IL12A, CXCL1 (GROa), CXCL2 (GROb), and CXCL5) which are regulated by CEBPB. Exogenous TERT expression led to downregulation of RNA/protein CEBPB expression and the inflammatory response genes suggesting a telomere-length dependent mechanism to regulate CEBPB. Furthermore, unlike exogenous TERT and p53 shRNA, CEBPB shRNA did not significantly decrease ROS suggesting that CEBPB’s contribution in DC cells’ senescence is ROS-independent. Our findings demonstrate a key role for CEBPB in engaging senescence by mobilizing an inflammatory response within DC cells.
Project description:We generated mice with telomerase (TERT) gene knock-out (KO) that undergo premature telomere shortening in adipose stromal cells expressing Pdgfra or Pdgfrb. Stromal vascular cells were isolated from inguinal adipose tissue of Pdgfra-Cre; TERT fl/fl mice (Sample name A_SAT), Pdgfrb-Cre; TERT fl/fl mice (Sample name KO_SAT), and of control TERT WT mice (Sample name PBS_SAT).
Project description:Telomere erosion in cells with insufficient levels of the telomerase reverse transcriptase, TERT, contributes to age-associated tissue dysfunction and senescence, and p53 plays a crucial role in this response. We undertook a genome-wide screen to identify gene deletions that sensitized p53-positive human cells to telomerase inhibition. We uncovered a previously unannotated gene, C16ORF72, which we term Telomere Attrition and p53 Response 1, TAPR1, that exhibited a synthetic-sick relationship with TERT loss. A genome-wide screen in TAPR1-disrupted cells also identified TERT as a sensitizing gene deletion. Additional genetic and transcriptome analysis of TAPR1-disrupted cells revealed that TAPR1 tapered p53 activation in response to eroded telomeres, p53 stabilization with nutlin-3a, or DNA damage. Importantly, deletion of TP53 rescued the fitness defect in TAPR1-disrupted cells. These findings identify C16ORF72/TAPR1 as new regulator at the nexus between telomere integrity and p53 regulation.
Project description:Telomere erosion in cells with insufficient levels of the telomerase reverse transcriptase, TERT, contributes to age-associated tissue dysfunction and senescence, and p53 plays a crucial role in this response. We undertook a genome-wide screen to identify gene deletions that sensitized p53-positive human cells to telomerase inhibition. We uncovered a previously unannotated gene, C16ORF72, which we term Telomere Attrition and p53 Response 1, TAPR1, that exhibited a synthetic-sick relationship with TERT loss. A genome-wide screen in TAPR1-disrupted cells also identified TERT as a sensitizing gene deletion. Additional genetic and transcriptome analysis of TAPR1-disrupted cells revealed that TAPR1 tapered p53 activation in response to eroded telomeres, p53 stabilization with nutlin-3a, or DNA damage. Importantly, deletion of TP53 rescued the fitness defect in TAPR1-disrupted cells. These findings identify C16ORF72/TAPR1 as new regulator at the nexus between telomere integrity and p53 regulation.
Project description:Telomere erosion in cells with insufficient levels of the telomerase reverse transcriptase, TERT, contributes to age-associated tissue dysfunction and senescence, and p53 plays a crucial role in this response. We undertook a genome-wide screen to identify gene deletions that sensitized p53-positive human cells to telomerase inhibition. We uncovered a previously unannotated gene, C16ORF72, which we term Telomere Attrition and p53 Response 1, TAPR1, that exhibited a synthetic-sick relationship with TERT loss. A genome-wide screen in TAPR1-disrupted cells also identified TERT as a sensitizing gene deletion. Additional genetic and transcriptome analysis of TAPR1-disrupted cells revealed that TAPR1 tapered p53 activation in response to eroded telomeres, p53 stabilization with nutlin-3a, or DNA damage. Importantly, deletion of TP53 rescued the fitness defect in TAPR1-disrupted cells. These findings identify C16ORF72/TAPR1 as new regulator at the nexus between telomere integrity and p53 regulation.
Project description:Telomere dysfunction induces two types of cellular responses: cellular senescence and apoptosis. Here we analyzed the influence of the cellular level of telomere dysfunction and the role of p53 on induction of apoptosis and senescence in mouse liver using the experimental system of adenoviral mediated, transient expression of a dominant negative version of TRF2 (TRF2DBDM). Gene-profiling experiments identified p53-dependent and p53-independent changes in gene expression in response to telomere deprotection and transcription factors potentially regulating these genes.
Project description:Cellular senescence due to telomere dysfunction has been hypothesized to play a role in age-associated diseases including idiopathic pulmonary fibrosis (IPF). It has been postulated that paracrine mediators originating from senescent alveolar epithelia signal to surrounding mesenchymal cells and contribute to disease pathogenesis. However, murine models of telomere-induced alveolar epithelial senescence fail to display the canonical senescence-associated secretory phenotype (SASP) that is observed in senescent human cells. In an effort to understand human-specific responses to telomere dysfunction, we modelled telomere dysfunction-induced senescence in a human alveolar epithelial cell line. We hypothesized that this system would enable us to probe for differences in transcriptional and proteomic senescence pathways in vitro and to identify novel secreted protein (secretome) changes that potentially contribute to the pathogenesis of IPF. Following induction of telomere dysfunction, a robust senescence phenotype was observed. RNA-Seq analysis of the senescent cells revealed the SASP and comparisons to previous murine data highlighted species-specific responses to telomere dysfunction. We then conducted a proteomic analysis of the senescent cells using a novel biotin ligase capable of labeling secreted proteins. Candidate biomarkers selected from our transcriptional and secretome data were then evaluated in IPF and control patient plasma. Four novel proteins were found to be differentially expressed between the patient groups: stanniocalcin-1, contactin-1, tenascin C, and total inhibin. Our data show that human telomere-induced, alveolar epithelial senescence results in a transcriptional SASP that is distinct from that seen in analogous murine cells. Our findings suggest that studies in animal models should be carefully validated given the species-specific responses to telomere dysfunction. We also describe a pragmatic approach for the study of the consequences of telomere-induced alveolar epithelial cell senescence in humans.
Project description:Telomere erosion contributes to age-associated tissue dysfunction and senescence, and p53 plays a crucial role in this response. We undertook a genome-wide screen to identify gene deletions that sensitized p53-positive human cells to loss of telomere integrity, and uncovered a previously unannotated gene, C16ORF72, which we term Telomere Attrition and p53 Response 1: TAPR1. CRISPR-Cas9 mediated deletion of TAPR1 led to elevated p53 and induction of p53 transcriptional targets. TAPR1-disrupted cells exhibited a synthetic-sick relationship with the loss of telomerase, or treatment with the topoisomerase II inhibitor doxorubicin. Stabilization of p53 with nutlin-3a further decreased cell fitness in cells lacking TAPR1 or telomerase, whereas deletion of TP53 rescued the decreased fitness of TAPR1-deleted cells. We propose that TAPR1 regulates p53 turnover, thereby tapering the p53-dependent response to telomere erosion. We discuss the possible implications of such a mechanism in the preservation of genome integrity during senescence or aging.
Project description:Telomerase reverse transcriptase (TERT) plays a crucial role in maintaining telomere length, which are specialised protective caps at the end of chromosomes. The lack of in vitro aging models, particularly for the central nervous system (CNS), has impeded progress in understanding aging and age-associated neurodegenerative diseases. In this study, we aimed to explore the possibility of accelerating aging in vitro using hiPSC (human induced pluripotent stem cell) technology. To achieve this, we utilised CRISPR/Cas9 to generate TERT loss-of-function hiPSCs, resulting in a loss of telomerase function and shortened telomeres. Through directed differentiation, we generated motor neurons and astrocytes to investigate whether telomere shortening could lead to age-associated phenotypes in CNS cell types.