Multiple DNA-binding sites in Tetrahymena telomerase

Telomerase is a ribonucleoprotein enzyme that maintains chromosome ends through de novo addition of telomeric DNA. The ability of telomerase to interact with its DNA substrate at sites outside its catalytic centre (‘anchor sites’) is important for its unique ability to undergo repeat addition processivity. We have developed a direct and quantitative equilibrium primer-binding assay to measure DNA-binding affinities of regions of the catalytic protein subunit of recombinant Tetrahymena telomerase (TERT). There are specific telomeric DNA-binding sites in at least four regions of TERT (the TEN, RBD, RT and C-terminal domains). Together, these sites contribute to specific and high-affinity DNA binding, with a Kd of ∼8 nM. Both the Km and Kd increased in a stepwise manner as the primer length was reduced; thus recombinant Tetrahymena telomerase, like the endogenous enzyme, contains multiple anchor sites. The N-terminal TEN domain, which has previously been implicated in DNA binding, shows only low affinity binding. However, there appears to be cooperativity between the TEN and RNA-binding domains. Our data suggest that different DNA-binding sites are used by the enzyme during different stages of the addition cycle

National Evaluation of the Culture Collective programme Part one: ‘Unprecedented and revitalising’ - Emerging Impacts and Ways of Working: Reflections from the first year of the Culture Collective, Reporting from Queen Margaret University March 2023

David Stevenson - ORCID: 0000-0002-8977-1818 https://orcid.org/0000-0002-8977-1818Anthony Schrag - ORCID: 0000-0001-8660-7572 https://orcid.org/0000-0001-8660-7572The Culture Collective is a network of 26 participatory arts projects, shaped by local communities alongside artists and creative organisations, and funded by Scottish Government emergency COVID-19 funds through Creative Scotland. This report captures a snapshot of the programme a year into their work.https://www.creativescotland.com/resources-publications/research/archive/2023/national-evaluation-of-the-culture-collective-programmepubpu

Development of an insulin-prescribing chart for paediatric diabetes

Aim: Our aim was to design a new insulin prescribing tool in compliance with the Irish Medicines Safety Network recommendations. Methods: In 2015, we undertook a review of the existing paediatric subcutaneous insulin-prescribing sheet introduced to Cork University Hospital in 2013. This involved a retrospective analysis of 15 consecutive in-patient insulin prescribing charts and a questionnaire distributed to health professionals. Following this a new insulin prescribing chart was designed and implemented in 2016 and a re-audit was performed in 2017. Results: The 2017 re-audit demonstrated that the new insulin chart was viewed as easier (95% of previous users n=18) and safer (n=16) to use. There was less confusion (2017: 28%, n=11/39 vs 2015: 50%, n=17/34 2015) and the ALERT system helped staff standardise hypo/hyperglycaemia management (71%, n=28). Conclusion: The new paediatric insulin prescribing chart has improved safety and ease of prescribing insulin. The colour coded quasi graph and ALERT system has made it easier to appreciate capillary blood glucose trends and manage them safely

Direct involvement of the TEN domain at the active site of human telomerase

Telomerase is a ribonucleoprotein that adds DNA to the ends of chromosomes. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is important for activity and processivity. Here we describe a mutation in the TEN domain of human TERT that results in a greatly increased primer Kd, supporting a role for the TEN domain in DNA affinity. Measurement of enzyme kinetic parameters has revealed that this mutant enzyme is also defective in dNTP polymerization, particularly while copying position 51 of the RNA template. The catalytic defect is independent of the presence of binding interactions at the 5′-region of the DNA primer, and is not a defect in translocation rate. These data suggest that the TEN domain is involved in conformational changes required to position the 3′-end of the primer in the active site during nucleotide addition, a function which is distinct from the role of the TEN domain in providing DNA binding affinity

Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin

Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3′ terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was ‘supershifted’ with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions

ZIP8 Zinc Transporter: Indispensable Role for Both Multiple-Organ Organogenesis and Hematopoiesis In Utero

Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn2+/(HCO3–)2 symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects–proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis

A Non-Canonical Function of Zebrafish Telomerase Reverse Transcriptase Is Required for Developmental Hematopoiesis

Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from “authentic” telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words

Mechanisms of human telomerase reverse transcriptase (hTERT) regulation: clinical impacts in cancer

Background Limitless self-renewal is one of the hallmarks of cancer and is attained by telomere maintenance, essentially through telomerase (hTERT) activation. Transcriptional regulation of hTERT is believed to play a major role in telomerase activation in human cancers. Main body The dominant interest in telomerase results from its role in cancer. The role of telomeres and telomere maintenance mechanisms is well established as a major driving force in generating chromosomal and genomic instability. Cancer cells have acquired the ability to overcome their fate of senescence via telomere length maintenance mechanisms, mainly by telomerase activation. hTERT expression is up-regulated in tumors via multiple genetic and epigenetic mechanisms including hTERT amplifications, hTERT structural variants, hTERT promoter mutations and epigenetic modifications through hTERT promoter methylation. Genetic (hTERT promoter mutations) and epigenetic (hTERT promoter methylation and miRNAs) events were shown to have clinical implications in cancers that depend on hTERT activation. Knowing that telomeres are crucial for cellular self-renewal, the mechanisms responsible for telomere maintenance have a crucial role in cancer diseases and might be important oncological biomarkers. Thus, rather than quantifying TERT expression and its correlation with telomerase activation, the discovery and the assessment of the mechanisms responsible for TERT upregulation offers important information that may be used for diagnosis, prognosis, and treatment monitoring in oncology. Furthermore, a better understanding of these mechanisms may promote their translation into effective targeted cancer therapies. Conclusion Herein, we reviewed the underlying mechanisms of hTERT regulation, their role in oncogenesis, and the potential clinical applications in telomerase-dependent cancers.info:eu-repo/semantics/publishedVersio