Project description: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).

Project description:Background: Calcific aortic valve disease (CAVD) is the pathological remodeling of valve leaflets. The initial steps in the osteogenic reprogramming of the leaflet are not fully understood. Studies have shown that TERT overexpression primes mesenchymal stem cells to differentiate into osteoblasts, and we investigated whether TERT contributes to the osteogenic reprogramming of valve interstitial cells (VICs). Methods: Human control and CAVD aortic valve leaflets and patient-specific hVICs were used in in vivo and in vitro calcification assays. Loss of function experiments in hVICs and cells isolated from Tert-/- and Terc-/- mice were used for mechanistic studies. In silico modeling, proximity ligation and co-immunoprecipitation assays defined novel TERT interacting partners. Chromatin immunoprecipitation and CUT&TAG sequencing defined protein-DNA interactions. Results: TERT protein was highly expressed in calcified valve leaflets without changes in telomere length, DNA damage, or senescence markers, and these features were retained in isolated primary hVICs. TERT expression increased with osteogenic stimuli, while knock-down or genetic deletion of TERT prevented calcification. Mechanistically, TERT was required to initiate osteogenic reprogramming independent of its canonical telomere-extending activity and the lncRNA TERC. TERT’s osteogenic functions via binding with Signal Transducer and Activator of Transcription 5 (STAT5). Depletion and inhibition of STAT5 prevented calcification. STAT5 was found to bind the promoter region of Runt-Related Transcription Factor 2 (RUNX2), the master regulator of osteogenic reprogramming. Finally, we demonstrate that TERT and STAT5 are upregulated and colocalized in CAVD tissue. Conclusions: TERT's non-canonical activity is required to initiate VIC calcification. TERT partners with STAT5 to bind the RUNX2 gene promoter. These data identify a novel mechanism and potential therapeutic target to decrease vascular calcification.

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Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro.

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Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro.

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