Telomere dysfunction in Tert knockout mice delays BrafV600E -induced melanoma development.

Telomerase activation is a crucial step in melanomagenesis, often occurring because of ultraviolet radiation (UVR)-induced mutations at the telomerase gene (TERT) promoter and rendering TERT transcription in response to the activated Raf-MAP kinase pathway by BRAFV600E mutation. Due to the excessively long telomeres in mice, this process does not occur during melanomagenesis in mouse models. To investigate the impact of telomere dysfunction on melanomagenesis, BrafV600E was induced in generations 1 and 4 (G1 and G4) of Tert-/- mice. Our findings revealed that, regardless of UVR exposure, melanoma development was delayed in G4 mice, which had shorter telomeres compared to G1 and wild-type C57BL/6J (G0) mice. Moreover, many G4 tumors displayed an accumulation of excessive DNA damage, as evidenced by increased γH2A.X staining. Tumors from UVR-exposed mice exhibited elevated p53 protein expression. Cultured tumor cells isolated from G4 mice displayed abundant chromosomal fusions and rearrangements, indicative of telomere dysfunction in these cells. Additionally, tumor cells derived from UVB-exposed mice exhibited constitutively elevated expression of mutant p53 proteins, suggesting that p53 was a target of UVB-induced mutagenesis. Taken together, our findings suggest that telomere dysfunction hampers melanomagenesis, and targeting telomere crisis-mediated genomic instability may hold promise for the prevention and treatment of melanoma.

Polymorphic tandem DNA repeats activate the human telomerase reverse transcriptase gene.

Multiple independent sequence variants of the hTERT locus have been associated with telomere length and cancer risks in genome-wide association studies. Here, we identified an intronic variable number tandem repeat, VNTR2-1, as an enhancer-like element, which activated hTERT transcription in a cell in a chromatin-dependent manner. VNTR2-1, consisting of 42-bp repeats with an array of enhancer boxes, cooperated with the proximal promoter in the regulation of hTERT transcription by basic helix-loop-helix transcription factors and maintained hTERT expression during embryonic stem-cell differentiation. Genomic deletion of VNTR2-1 in MelJuSo melanoma cells markedly reduced hTERT transcription, leading to telomere shortening, cellular senescence, and impairment of xenograft tumor growth. Interestingly, VNTR2-1 lengths varied widely in human populations; hTERT alleles with shorter VNTR2-1 were underrepresented in African American centenarians, indicating its role in human aging. Therefore, this polymorphic element is likely a missing link in the telomerase regulatory network and a molecular basis for genetic diversities of telomere homeostasis and age-related disease susceptibilities.

ETS variant transcription factor 5 and c-Myc cooperate in derepressing the human telomerase gene promoter via composite ETS/E-box motifs.

The human telomerase gene (hTERT) is repressed in most somatic cells. How transcription factors activate the hTERT promoter in its repressive chromatin environment is unknown. Here, we report that the ETS family protein ETS variant transcription factor 5 (ETV5) mediates epidermal growth factor (EGF)-induced hTERT expression in MCF10A cells. This activation required MYC proto-oncogene bHLH transcription factor (c-Myc) and depended on the chromatin state of the hTERT promoter. Using chromatinized bacterial artificial chromosome (BAC) reporters in human fibroblasts, we found that ETV5 and c-Myc/MYC-associated factor X (MAX) synergistically activate the hTERT promoter via two identical, but inverted, composite Ets/E-box motifs enclosing the core promoter. Mutations of Ets or E-box sites in either DNA motif abolished the activation and reduced or eliminated the synergism. ETV5 and c-Myc facilitated each other's binding to the hTERT promoter. ETV5 bound to the hTERT promoter in both telomerase-negative and -positive cells, but it activated the repressed hTERT promoter and altered histone modifications only in telomerase-negative cells. The synergistic ETV5/c-Myc activation disappeared when hTERT promoter repression became relieved because of the loss of distal regulatory elements in chimeric human/mouse BAC reporters. Our results suggest that the binding of c-Myc and ETS family proteins to the Ets/E-box motifs derepresses the hTERT promoter by inducing an active promoter configuration, providing a mechanistic insight into hTERT activation during tumorigenesis.

Photon transport model for dense polydisperse colloidal suspensions using the radiative transfer equation combined with the dependent scattering theory.

In near-infrared imaging and spectroscopy, high-fidelity modeling of photon transport for dense polydisperse colloidal suspensions is crucial. We developed photon transport models using the radiative transfer equation (RTE) with the dependent scattering theory (DST) at volume fractions up to 20%. The polydispersity and interference effects strongly influence results of the scattering properties and the RTE in cases of small mean diameter and large variance of the particle size distribution. We compared the RTE-results for the Henyey-Greenstein (conventional) function with those for the phase function using the DST. The RTE-results differ between both functions at low volume fractions for forward scattering media, suggesting the limitation of the conventional function.

Crispr/Cas9-mediated cleavages facilitate homologous recombination during genetic engineering of a large chromosomal region.

Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1-kb genomic region around the mTert gene with a recombinant fragment of over 45.5 kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with DNA double-strand breaks (DSBs) generated by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and three clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at single-guide RNA target sites. Our results indicated that the Crispr/Cas9 system significantly increased the efficiency of homologous recombination-mediated gene editing over a large genomic region in mammalian cells, and also caused frequent mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions.

Repression of telomerase gene promoter requires human-specific genomic context and is mediated by multiple HDAC1-containing corepressor complexes.

The human telomerase reverse transcriptase (hTERT) gene is repressed in most somatic cells, whereas the expression of the mouse mTert gene is widely detected. To understand the mechanisms of this human-specific repression, we constructed bacterial artificial chromosome (BAC) reporters using human and mouse genomic DNAs encompassing the TERT genes and neighboring loci. Upon chromosomal integration, the hTERT, but not the mTert, reporter was stringently repressed in telomerase-negative human cells in a histone deacetylase (HDAC)-dependent manner, replicating the expression of their respective endogenous genes. In chimeric BACs, the mTert promoter became strongly repressed in the human genomic context, but the hTERT promoter was highly active in the mouse genomic context. Furthermore, an unrelated herpes simplex virus-thymidine kinase (HSV-TK) promoter was strongly repressed in the human, but not in the mouse, genomic context. These results demonstrated that the repression of hTERT gene was dictated by distal elements and its chromatin environment. This repression depended on class I HDACs and involved multiple corepressor complexes, including HDAC1/2-containing Sin3B, nucleosome remodeling and histone deacetylase (NuRD), and corepressor of RE1 silencing transcription factor (CoREST) complexes. Together, our data indicate that the lack of telomerase expression in most human somatic cells results from its repressive genomic environment, providing new insight into the mechanism of long-recognized differential telomerase regulation in mammalian species.-Cheng, D., Zhao, Y., Wang, S., Zhang, F., Russo, M., McMahon, S. B., Zhu, J. Repression of telomerase gene promoter requires human-specific genomic context and is mediated by multiple HDAC1-containing corepressor complexes.

Therapeutic targeting of replicative immortality.

One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed "senescence," can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells' heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy.

Human Telomerase Reverse Transcriptase (hTERT) Transcription Requires Sp1/Sp3 Binding to the Promoter and a Permissive Chromatin Environment.

The transcription of human telomerase gene hTERT is regulated by transcription factors (TFs), including Sp1 family proteins, and its chromatin environment. To understand its regulation in a relevant chromatin context, we employed bacterial artificial chromosome reporters containing 160 kb of human genomic sequence containing the hTERT gene. Upon chromosomal integration, the bacterial artificial chromosomes recapitulated endogenous hTERT expression, contrary to transient reporters. Sp1/Sp3 expression did not correlate with hTERT promoter activity, and these TFs bound to the hTERT promoters in both telomerase-positive and telomerase-negative cells. Mutation of the proximal GC-box resulted in a dramatic decrease of hTERT promoter activity, and mutations of all five GC-boxes eliminated its transcriptional activity. Neither mutations of GC-boxes nor knockdown of endogenous Sp1 impacted promoter binding by other TFs, including E-box-binding proteins, and histone acetylation and trimethylation of histone H3K9 at the hTERT promoter in telomerase-positive and -negative cells. The result indicated that promoter binding by Sp1/Sp3 was essential, but not a limiting step, for hTERT transcription. hTERT transcription required a permissive chromatin environment. Importantly, our data also revealed different functions of GC-boxes and E-boxes in hTERT regulation; although GC-boxes were essential for promoter activity, factors bound to the E-boxes functioned to de-repress hTERT promoter.

Dual roles of c-Myc in the regulation of hTERT gene.

Human telomerase gene hTERT is important for cancer and aging. hTERT promoter is regulated by multiple transcription factors (TFs) and its activity is dependent on the chromatin environment. However, it remains unsolved how the interplay between TFs and chromatin environment controls hTERT transcription. In this study, we employed the recombinase-mediated BAC targeting and BAC recombineering techniques to dissect the functions of two proximal E-box sites at -165 and +44 nt in regulating the hTERT promoter in the native genomic contexts. Our data showed that mutations of these sites abolished promoter binding by c-Myc/Max, USF1 and USF2, decreased hTERT promoter activity, and prevented its activation by overexpressed c-Myc. Upon inhibition of histone deacetylases, mutant and wildtype promoters were induced to the same level, indicating that the E-boxes functioned to de-repress the hTERT promoter and allowed its transcription in a repressive chromatin environment. Unexpectedly, knockdown of endogenous c-Myc/Max proteins activated hTERT promoter. This activation did not require the proximal E-boxes but was accompanied by increased promoter accessibility, as indicated by augmented active histone marks and binding of multiple TFs at the promoter. Our studies demonstrated that c-Myc/Max functioned in maintaining chromatin-dependent repression of the hTERT gene in addition to activating its promoter.

ANLN Promotes the Proliferation and Migration of Gallbladder Cancer Cells via STRA6-Mediated Activation of PI3K/AKT Signaling.

The ANLN gene encodes anillin, a protein that binds to actin. Recent research has identified ANLN's function in the initiation and advancement of different cancers. However, its impact on gallbladder cancer (GBC) remains unexplored. This study aimed to elucidate its possible molecular mechanisms in GBC. ANLN expression was assessed using quantitative real-time polymerase chain reaction (QRT-PCR), Western blotting (WB), and immunohistochemistry (IHC), revealing elevated levels in GBC tissues. ANLN knockdown resulted in the inhibition of cell proliferation and migration, leading to apoptosis and cell cycle arrest. Conversely, ANLN overexpression had the opposite effects on GBC cells. In vivo experiments confirmed that ANLN knockdown inhibited GBC cell growth. RNA-seq and bioinformatics analysis revealed ANLN's function in activating the PI3K/AKT signaling pathway. We further confirmed that ANLN could upregulate STRA6 expression, which activated PI3K/AKT signaling to enhance the growth and movement of GBC cells. These findings demonstrate ANLN's involvement in GBC initiation and progression, suggesting its potential as a novel target for GBC.

Humanization of the mouse Tert gene reset telomeres to human length.

Telomeres undergo shortening with each cell division, serving as biomarkers of human aging, which is characterized by short telomeres and restricted telomerase expression in adult tissues. Contrarily, mice, featuring their longer telomeres and widespread telomerase activity, present limitations as models for understanding telomere-related human biology and diseases. To bridge this gap, we engineered a mouse strain with a humanized mTert gene, hmTert, wherein specific non-coding sequences were replaced with their human counterparts. The hmTert gene, encoding the wildtype mTert protein, was repressed in adult tissues beyond the gonads and thymus, closely resembling the regulatory pattern of the human TERT gene. Remarkably, the hmTert gene rescued telomere dysfunction in late generations of mTert-knockout mice. Through successive intercrosses of Terth/- mice, telomere length progressively declined, stabilizing below 10-kb. Terth/h mice achieved a human-like average telomere length of 10-12 kb, contrasting with the 50-kb length in wildtype C57BL/6J mice. Despite shortened telomeres, Terth/h mice maintained normal body weight and cell homeostasis in highly proliferative tissues. Notably, colonocyte proliferation decreased significantly in Terth/h mice during dextran sodium sulfate-induced ulcerative colitis-like pathology, suggesting limitations on cellular renewal due to short telomeres. Our findings underscore the genetic determination of telomere homeostasis in mice by the Tert gene. These mice, exhibiting humanized telomere homeostasis, serve as a valuable model for exploring fundamental questions related to human aging and cancer.

Association Between Nonthyroidal Illness Syndrome and Disseminated Intravascular Coagulation in Sepsis.

Purpose: Both nonthyroidal illness syndrome (NTIS) and disseminated intravascular coagulation (DIC) are commonly occurred in sepsis. The objective of this study is to evaluate the association between NTIS and DIC, as well as their impacts on the mortality in adults with sepsis. Patients and methods: A total of 1219 septic patients in two Chinese academic centers from October 2012 and October 2022 were enrolled in analysis. We conduct logistic regression models to analyze the independent risk factors for DIC. Modified Poisson regression models are used to estimate the relative risk (RR) of NTIS on the 28 days mortality in septic patients with DIC. Correlation analysis between thyroid function parameters and coagulation parameters is performed with Pearson coefficient be reported. Results: DIC is diagnosed on 388 (31.8%) of all the 1219 enrolled septic patients within 72 hours after admission. In multivariate logistic regression models, NTIS (OR 3.19; CI 2.31-4.46; p<0.001) is a statistically significant independent risk factor for DIC after adjustment for potential confounders. Twenty-eight days mortality is significantly higher in DIC patients complicated with NTIS compared with the other DIC patients (23.2% vs 14.0%, p=0.024). This result is also robust in different modified Poisson regression models (Model 1: RR 1.46; CI 1.25-1.70; p<0.001; Model 2: RR 1.35; CI 1.14-1.60; p<0.001; Model 3: RR 1.18; CI 1.02-1.37; p=0.026). Correlation analysis reveals that the thyroid function parameters of FT3, FT4 and TSH only have weak correlations with coagulation parameters of platelet count, fibrinogen, FDP, D-dimers, PT, APTT and INR in sepsis. Conclusion: NTIS is an independent risk factor for DIC in adults with sepsis. DIC patients complicated with NTIS have significantly higher severity and higher rate of mortality.

Different subtypes of nonthyroidal illness syndrome on the prognosis of septic patients: a two-centered retrospective cohort study.

Background: Nonthyroidal illness syndrome (NTIS) is a common endocrine dysfunction predicting unfavorable outcomes in critical illness. The objective of the study is to evaluate the association between different NTIS subtypes with outcomes in septic patients. Methods: Septic patients in two Chinese academic centers from October 2012 and October 2022 are enrolled in analysis. Multivariable regressions are used to assess associations between NTIS and outcomes. Outcomes include in-hospital mortality, length of stay in hospital (LOS), non-invasive ventilation failure and weaning failure. Patients with NTIS are categorized into 4 types according to the different levels of FT4 and TSH. The association between different NTIS subtypes and mortality are further analyzed. Survival curve is plotted using the Kaplan-Meier method. Results: After screening, a total of 1226 septic patients with complete thyroid hormones result are eventually enrolled. Among them, 520 (42.4%) patients are diagnosed as NTIS. In multivariable regression analysis, NTIS is independently associated with increased 30-days mortality (OR=1.759, CI 1.009-3.104, p=0.047), but has no association with 60-days mortality (OR=1.524, CI 0.893-2.618, p=0.123), 90-days mortality (OR=1.411, CI 0.831-2.408, p=0.203), LOS, non-invasive ventilation failure or weaning failure. In NTIS subtypes, NTIS patients with low FT3 and TSH levels, regardless of the FT4 values, have significantly higher mortality than euthyroid patients (30-days mortality, OR= 6.488, CI 1.546-27.808, p=0.01; 60-days mortality, OR=3.973, CI 1.006-15.579, p=0.046; 90-days mortality, OR=3.849, CI 0.977-15.088, p=0.051). This result is consistent in patients with low FT3 and FT4 levels, regardless of the TSH values (30-days mortality, OR=3.349, CI 1.402-7.957, p=0.006; 60-days mortality, OR= 2.594, CI 1.122-5.930, p=0.024; 90-days mortality, OR=2.55, CI 1.110-5.804, p=0.025). There is no survival difference between NTIS patients with low FT3 only and euthyroid patients. Survival plot shows the worst prognosis is in NTIS patients with low FT3, FT4 and TSH level. Conclusions: NTIS is frequent in sepsis. A reduction of FT3 together with FT4 or TSH, but not FT3 only, is associated with an increased risk of mortality.

A BAC transgenic reporter recapitulates in vivo regulation of human telomerase reverse transcriptase in development and tumorigenesis.

Telomerase is tightly regulated in humans relative to mice, owing to the differential regulation of TERT genes. To explore hTERT regulation in vivo, we engineered mice with a 160-kb transgenic bacterial artificial chromosome (BAC) spanning the hTERT locus with a Renilla luciferase (Rluc) cassette downstream of its promoter. Analysis of multiple founder lines revealed that the Rluc expression profile from the transgenic hTERT reporter locus reproduced that of the native hTERT gene in all tissues and organs examined, demonstrating that genetic sequence determined the species-specific developmental regulation of the hTERT gene and that mouse epigenetic and transcription machineries faithfully regulated hTERT transcription. Thus, these mice allowed detailed analyses of developmental hTERT regulation. Both the transgenic hTERT reporter and the endogenous mTERT locus were expressed in early embryonic stages, and their mRNA levels progressively decreased throughout embryonic and postnatal development. Whereas hTERT transcription was much lower than mTERT expression in most organs, it increased significantly during postnatal development of thymus, testis, and ovary. In testis, the Rluc mRNA was enriched in elongating spermatids of seminiferous tubules. In addition, the transcription of transgenic hTERT reporter, but surprisingly not the endogenous mTERT gene, was activated during Wnt1-induced mammary tumorigenesis, allowing the monitoring of tumor development via noninvasive bioluminescent imaging. Collectively, our results demonstrate that the hTERT transgenic reporter system recapitulates the developmental regulation of the hTERT gene in a chromosomal position-independent manner and serves as a legitimate model to explore telomerase regulation in the development of normal and neoplastic tissues in vivo.

Robust activation of the human but not mouse telomerase gene during the induction of pluripotency.

Pluripotent stem cells (PSCs) express telomerase and have unlimited proliferative potential. To study telomerase activation during reprogramming, 3 classes of embryonic stem cell (ESC)-like clones were isolated from mouse fibroblasts containing a transgenic hTERT reporter. Class I expressed few pluripotency markers, whereas class II contained many, but not Oct4, Nanog, and Sox2. Neither class of cells differentiated efficiently. Class III cells, the fully reprogrammed induced PSCs (iPSCs), expressed all pluripotency markers, formed teratomas indistinguishable from those of mESCs, and underwent efficient osteogenic differentiation in vitro. Interestingly, whereas the endogenous mTERT gene expression was only moderately increased during reprogramming, the hTERT promoter was strongly activated in class II cells and was further elevated in class III cells. Treatment of class II cells with chemical inhibitors of MEKs and glycogen synthase kinase 3 resulted in their further reprogramming into class III cells, accompanied by a strong activation of hTERT promoter. In reprogrammed human cells, the endogenous telomerase level, although variable among different clones, was dramatically elevated. Only in cells with the highest telomerase were telomeres restored to the lengths in hESCs. Our data, for the first time, demonstrated that the hTERT promoter was strongly activated in discrete steps, revealing a critical difference in human and mouse cell reprogramming. Because telomere elongation is crucial for self-renewal of hPSCs and replicative aging of their differentiated progeny, these findings have important implications in the generation and applications of iPSCs.

Differential repression of human and mouse TERT genes during cell differentiation.

Differential regulation of telomerase reverse transcriptase (TERT) contributes to the distinct aging and tumorigenic processes in humans and mice. Here, we report that the hTERT gene was strongly repressed during differentiation of human cells, whereas modest mTERT expression was detected in terminally differentiated and post-mitotic cells. The stringent hTERT repression depended on the native chromatin environment because transiently transfected hTERT promoters were not repressed in differentiated cells. Conversely, the transiently transfected mTERT core promoter was repressed during cell differentiation, suggesting that the repression of mTERT promoter did not require its endogenous chromatin structures. To understand the mechanisms of this differential regulation, we examined chromatin structures of the endogenous TERT loci during cell differentiation. In both human and mouse cells, repression was accompanied by the loss of multiple DNase I hypersensitive sites at the TERT promoters and their upstream regions, revealing positions of potential regulatory elements. Interestingly, the hTERT locus was located within a nuclease-resistant chromatin domain in human cells, whereas a corresponding chromatin domain was not detected for the mTERT locus. Taken together, our study indicated that, unlike the repression of mTERT gene, the condensed native chromatin environment of hTERT locus was central to its silencing during cell differentiation.

Studying human telomerase gene transcription by a chromatinized reporter generated by recombinase-mediated targeting of a bacterial artificial chromosome.

The endogenous human telomerase reverse transcriptase (hTERT) gene is repressed in somatic cells. To study the mechanisms of its repression, we developed a strategy of retrovirus-directed Cre recombinase-mediated BAC targeting, or RMBT, to generate single-copy integrations of BAC at pre-engineered chromosomal sites. This technique involved retroviral transduction of acceptor loci, containing an HSV thymidine kinase marker, and subsequent integration of BAC constructs into the acceptor sites, utilizing the loxP and lox511 sites present in the vector backbones. The BAC reporter, with a Renilla luciferase cassette inserted downstream of the hTERT promoter, was retrofitted with a puromycin marker. Through puromycin selection and ganciclovir counter-selection, a targeting efficiency of over 50% was achieved. We demonstrated that the activity and chromatin structures of the hTERT promoter in chromosomally integrated BAC reporter recapitulated its endogenous counterpart of the host cells. Therefore, we have established a genetically amendable platform to study chromatin and epigenetic regulation of the hTERT gene. The highly efficient and versatile RMBT technique has general applicability for studying largely unexplored chromatin-dependent mechanisms of promoter regulation of various genes.

The regulatory function of tandem repeat VNTR2-1 in hTERT gene involves basic Helix-loop-helix family transcription factors.

Telomerase is a ribonucleoprotein enzyme that synthesizes telomere end sequence. The expression of hTERT gene, encoding the catalytic subunit of human telomerase, is restricted to highly proliferative tissues and is undetectable in most somatic cells. Abnormal activation of hTERT gene is found in 90% of human tumors. Previously, we identified tandem repeat of 42-bp/unit, VNTR2-1, in intron 2 of the hTERT gene, as a novel regulatory element important for hTERT transcription in cancer cells. In the current study, we found that multiple 42-bp repeats of VNTR2-1 activated luciferase gene in reporter plasmids. Mutation of the predicted cis-regulatory elements within the 42-bp repeats, including a E-box motif, resulted in a partial or complete loss of its enhancer activity. Moreover, MYC family proteins, c-MYC, MAX, and MNT, regulated hTERT gene transcription through both VNTR2-1 and E-boxes at the proximal hTERT promoter. Chromatin segmentation analysis of published ChIP-sequencing data from K562 cells indicated that VNTR2-1 was a bivalent enhancer. In telomerase-expressing human melanoma cell line MelJuSo, deletion of VNTR2-1 caused the hTERT promoter chromatin status to change from an active state to a repressed state, accompanied by increases of H3K27me3 and H3K9me3 marks. Therefore, we provided additional evidence for VNTR2-1 as a functional regulatory element that regulated hTERT expression by MYC family transcription factors. These results have improved our knowledge on the functions of repetitive genomic DNAs and the regulatory mechanisms of human telomerase gene.

Melanoma models for the next generation of therapies.

There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.

Transcriptional silencing of a novel hTERT reporter locus during in vitro differentiation of mouse embryonic stem cells.

The human telomerase reverse transcriptase hTERT is highly expressed in undifferentiated embryonic cells and silenced in the majority of somatic cells. To investigate the mechanisms of hTERT silencing, we have developed a novel reporter using a bacterial artificial chromosome (BAC) that contained the entire hTERT gene and its neighboring loci, hCRR9 and hXtrp2. Firefly and Renilla luciferases were used to monitor transcription from the hTERT and hCRR9 promoters, respectively. In mouse embryonic stem cells stably integrated with the BAC reporter, both hTERT and hCRR9 promoters were highly expressed. Upon differentiation into embryoid bodies and further into mineral-producing osteogenic cells, the hTERT promoter activity decreased progressively, whereas the hCRR9 promoter remained highly active, both resembling their endogenous counterparts. In fully differentiated cells, the hTERT promoter was completely silenced and adopted a chromatin structure that was similar to its native counterpart in human cells. Inhibition of histone deacetylases led to the opening of the hTERT promoter and partially relieved repression, suggesting that histone deacetylation was necessary but not sufficient for hTERT silencing. Thus, our result demonstrated that developmental silencing of the human TERT locus could be recapitulated in a chromosomal position-independent manner during the differentiation of mouse embryonic stem cells.