Margaret A. Strong

Studying essential genes required for dynamic processes in live mice is challenging as genetic perturbations are irreversible and limited by slow protein depletion kinetics. The auxin-inducible degron (AID) system is a powerful tool for analyzing inducible protein loss in vitro, but it is toxic to mice. Here, we use an optimized second-generation AID system to achieve the conditional and reversible loss of the essential centrosomal protein CEP192 in live mice. We show that the auxin derivative 5-phenyl-indole-3-acetic acid is well tolerated over 2 weeks and drives near-complete CEP192 degradation in less than 1 hour in vivo. CEP192 loss did not affect centriole duplication but decreased γ-tubulin recruitment to centrosomes impairing mitotic spindle assembly. Sustained CEP192 loss in vivo led to cell division failure and cell death in proliferative tissues. Thus, the second-generation AID system is well suited for rapid and/or sustained protein depletion in live mice to study essential functions in vivo.

Hydrolethalus syndrome (HLS) is a lethal, autosomal recessive ciliopathy caused by the mutation of the conserved centriole protein HYLS1. How HYLS1 controls centriole function is poorly understood. Here, we show that mice harboring the HYLS1 disease mutation die shortly after birth and exhibit developmental defects that recapitulate several manifestations of HLS. These phenotypes arise from a loss of centriole integrity that causes tissue-specific defects in cilia assembly and function. We show that HYLS1 is recruited to the centriole by CEP120 and stabilizes the localization of centriole inner scaffold proteins that ensure the integrity of the centriolar microtubule wall. The HLS disease mutation reduced the centriole localization of HYLS1 and caused degeneration of the centriole distal end. We propose that tissue-specific defects in centriole integrity caused by the HYLS1 mutation prevent ciliogenesis and contribute to HLS phenotypes.

Hydrolethalus Syndrome (HLS) is a lethal, autosomal recessive ciliopathy caused by the mutation of the conserved centriole protein HYLS1. However, how HYLS1 facilitates the centriole-based templating of cilia is poorly understood. Here, we show that mice harboring the HYLS1 disease mutation die shortly after birth and exhibit developmental defects that recapitulate several manifestations of the human disease. These phenotypes arise from tissue-specific defects in cilia assembly and function caused by a loss of centriole integrity. We show that HYLS1 is recruited to the centriole by CEP120 and functions to recruit centriole inner scaffold proteins that stabilize the centriolar microtubule wall. The HLS mutation disrupts the interaction of HYLS1 with CEP120 leading to HYLS1 displacement and degeneration of the centriole distal end. We propose that tissue-specific defects in centriole integrity caused by the HYLS1 mutation prevent ciliogenesis and drive HLS phenotypes.

TIN2 is an important regulator of telomere length, and mutations in TINF2, the gene encoding TIN2, cause short-telomere syndromes. While the genetics underscore the importance of TIN2, the mechanism through which TIN2 regulates telomere length remains unclear. Here, we tested the effects of human TIN2 on telomerase activity. We identified a new isoform in human cells, TIN2M, that is expressed at levels similar to those of previously studied TIN2 isoforms. All three TIN2 isoforms localized to and maintained telomere integrity in vivo, and localization was not disrupted by telomere syndrome mutations. Using direct telomerase activity assays, we discovered that TIN2 stimulated telomerase processivity in vitro. All of the TIN2 isoforms stimulated telomerase to similar extents. Mutations in the TPP1 TEL patch abrogated this stimulation, suggesting that TIN2 functions with TPP1/POT1 to stimulate telomerase processivity. We conclude from our data and previously published work that TIN2/TPP1/POT1 is a functional shelterin subcomplex.

Telomere length (TL) predicts the onset of cellular senescence in vitro but the diagnostic utility of TL measurement in clinical settings is not fully known. We tested the value of TL measurement by flow cytometry and FISH (flowFISH) in patients with mutations in telomerase and telomere maintenance genes. TL had a discrete and reproducible normal range with definable upper and lower boundaries. While TL above the 50th age-adjusted percentile had a 100% negative predictive value for clinically relevant mutations, the lower threshold in mutation carriers was age-dependent, and adult mutation carriers often overlapped with the lowest decile of controls. The extent of telomere shortening correlated with the age at diagnosis as well as the short telomere syndrome phenotype. Extremely short TL caused bone marrow failure and immunodeficiency in children and young adults, while milder defects manifested as pulmonary fibrosis-emphysema in adults. We prospectively examined whether TL altered treatment decisions for newly diagnosed idiopathic bone marrow failure patients and found abnormally short TL enriched for patients with mutations in some inherited bone marrow failure genes, such as RUNX1, in addition to telomerase and telomere maintenance genes. The result was actionable, altering the choice of treatment regimen and/or hematopoietic stem cell donor in one-fourth of the cases (9 of 38, 24%). We conclude that TL measurement by flowFISH, when used for targeted clinical indications and in limited settings, can influence treatment decisions in ways that improve outcome. Significance This study defines clinical indications for using telomere length (TL) measurement as a diagnostic tool in a hospital setting. It shows that, in contrast to other methods, TL measurement by flow cytometry and FISH (flowFISH) can be standardized, and has reproducible and definable upper and lower normal boundaries. In telomerase mutation carriers and carriers of other mutant telomere maintenance genes, TL had prognostic value, correlating with the age of onset of short telomere syndrome phenotypes, as well as the predominant complication. In a prospective study, TL results were actionable in one-fourth of cases with idiopathic bone marrow failure affecting the stem cell donor choice and/or treatment regimen. The data show that, for targeted clinical indications, and in a hospital setting, TL measurement by flowFISH informs patient care decisions.

Cancer cells maintain telomere length equilibrium to avoid senescence and apoptosis induced by short telomeres, which trigger the DNA damage response. Limiting the potential for telomere maintenance in cancer cells has been long been proposed as a therapeutic target. Using an unbiased shRNA screen targeting known kinases, we identified bromodomain-containing protein 4 (BRD4) as a telomere length regulator. Four independent BRD4 inhibitors blocked telomere elongation, in a dose-dependent manner, in mouse cells overexpressing telomerase. Long-term treatment with BRD4 inhibitors caused telomere shortening in both mouse and human cells, suggesting BRD4 plays a role in telomere maintenance in vivo. Telomerase enzymatic activity was not directly affected by BRD4 inhibition. BRD4 is in clinical trials for a number of cancers, but its effects on telomere maintenance have not been previously investigated.

Telomeres, specialised structures that protect chromosome ends, play a critical role in preserving chromosome integrity. Telomere dynamics in the Tasmanian devil (Sarcophilus harrisii) are of particular interest in light of the emergence of devil facial tumour disease (DFTD), a transmissible malignancy that causes rapid mortality and threatens the species with extinction. We used fluorescent in situ hybridisation to investigate telomere length in DFTD cells, in healthy Tasmanian devils and in four closely related marsupial species. Here we report that animals in the Order Dasyuromorphia have chromosomes characterised by striking telomere length dimorphism between homologues. Findings in sex chromosomes suggest that telomere length dimorphism may be regulated by events in the parental germlines. Long telomeres on the Y chromosome imply that telomere lengthening occurs during spermatogenesis, whereas telomere diminution occurs during oogenesis. Although found in several somatic cell tissue types, telomere length dimorphism was not found in DFTD cancer cells, which are characterised by uniformly short telomeres. This is, to our knowledge, the first report of naturally occurring telomere length dimorphism in any species and suggests a novel strategy of telomere length control. Comparative studies in five distantly related marsupials and a monotreme indicate that telomere dimorphism evolved at least 50 million years ago.

Telomerase is essential for telomere length maintenance. Mutations in either of the two core components of telomerase, telomerase RNA (TR) or the catalytic protein component telomerase reverse transcriptase (TERT), cause the genetic disorders dyskeratosis congenita, pulmonary fibrosis, and other degenerative diseases. Overexpression of the TERT protein has been reported to have telomere length-independent roles, including regulation of the Wnt signaling pathway. To examine the phenotypes of TERT haploinsufficiency and determine whether loss of function of TERT has effects other than those associated with telomere shortening, we characterized both mTERT^+/− and mTERT^−/− mice on the CAST/EiJ genetic background. Phenotypic analysis showed a loss of tissue renewal capacity with progressive breeding of heterozygous mice that was indistinguishable from that of mTR-deficient mice. mTERT^−/− mice, from heterozygous mTERT^+/− mouse crosses, were born at the expected Mendelian ratio (26.5%; n = 1,080 pups), indicating no embryonic lethality of this genotype. We looked for, and failed to find, hallmarks of Wnt deficiency in various adult and embryonic tissues, including those of the lungs, kidneys, brain, and skeleton. Finally, mTERT^−/− cells showed wild-type levels of Wnt signaling in vitro. Thus, while TERT overexpression in some settings may activate the Wnt pathway, loss of function in a physiological setting has no apparent effects on Wnt signaling. Our results indicate that both TERT and TR are haploinsufficient and that their deficiency leads to telomere shortening, which limits tissue renewal. Our studies imply that hypomorphic loss-of-function alleles of hTERT and hTR should cause a similar disease spectrum in humans.

Telomerase-mediated telomere addition counteracts telomere shortening due to incomplete DNA replication. Short telomeres are the preferred substrate for telomere addition by telomerase; however, the mechanism by which telomerase recognizes short telomeres is unclear. In yeast, the Ataxia telangiectasia mutated (Atm) homolog, Tel1, is necessary for normal telomere length regulation likely by altering telomere structure, allowing telomerase recruitment to short telomeres. To examine the role of Atm in establishing preference for elongation of short telomeres in mice, we examined telomerase-mediated elongation of short dysfunctional telomeres in the presence or absence of Atm. Here we show that Atm is dispensable for elongation of short telomeres by telomerase, suggesting that telomerase recruitment in mammalian cells and in yeast may be regulated differently.