Page 67 - Haematologica - Vol. 105 n. 6 - June 2020

DKC1 drives upregulation of telomerase in normal human erythroblasts
whether they were generated ex vivo or in vivo. Consistent with these findings, publicly available microarray data from fractionated bone marrow showed high levels of DKC1 expression in proerythroblasts.
In addition to regulating TERT, MYC has been shown to bind and activate the DKC1 promoter in tumor cell lines.26,27 The present study adds to this knowledge by demonstrating that MYC binds the DKC1 promoter in pri- mary human hematopoietic cells. Notably however, MYC binding at the DKC1 promoter appeared to decline, while DKC1 was upregulated during erythroid commitment and expansion. These results suggest that MYC plays a less prominent role in driving DKC1 expression in erythro- blasts compared with undifferentiated HSPC. Instead, our study highlights a potential role for GATA1 in the regula- tion of DKC1 in the erythroid lineage. This was evidenced by enrichment of GATA1 at the DKC1 promoter in GLYA+ erythroblasts, and an apparent transcriptional requirement for GATA binding sites in the proximal region of the DKC1 promoter.
Consistent with the known role of dyskerin in stabiliz- ing TERC,38 we consistently observed an increase in TERC in parallel with upregulation of endogenous DKC1. The magnitude of this effect was moderate, yet consis- tent with results from ectopic overexpression of DKC1, and converse to the observed effect of shRNA-mediated downregulation of DKC1. Overexpression of TERC was previously shown to be sufficient to elevate telomerase and hyper-extend telomeres.39-41 Nevertheless it seems unlikely that the modest elevation in endogenous TERC observed in primary erythroblasts was the singular cause of the dramatic increase in telomerase activity observed in the erythroblasts. In addition to stabilizing TERC, dyskerin may hyperactivate telomerase through its intrin- sic pseudouridine synthase activity. This could involve targeting and enzymatically modifying TERC moieties with structural or functional roles, or indirectly through functional modification of rRNA or splicesomal RNA.42-46 There is also scope for dyskerin to modulate telomerase activity through direct interactions with H/ACA box RNA that have been ascribed roles in post-transcriptional regulation of gene expression.47-53 Clarification of the functional significance of pseudouridylation of TERC and other non-coding RNA that interact with dyskerin will be valuable in understanding the full extent of dyskerin’s influence on telomerase activity.
Gene suppression experiments reported here demon- strate that high DKC1 expression is required for prolifer- ation of erythroblasts but is dispensable for erythroid dif- ferentiation. The apparent requirement for high expres- sion of DKC1 to sustain erythroblast proliferation may
reflect dependence on telomerase as well as the telom- erase-independent function of dyskerin in ribosome bio- genesis.2 During erythropoiesis, erythroblasts undergo a period of intense ribosome biogenesis that is necessary for synthesis of large quantities of hemoglobin.54 An abundance of dyskerin may be necessary to support this process. Consistent with this possibility, zebra fish and murine models have shown that hypomorphic DKC1 mutations impaired rRNA processing.50-53 Ribosomal stress has also been demonstrated in MYC-transformed cancer cells subjected to shRNA-mediated suppression of dyskerin.27 The implications of dyskerin’s function in ribosome biogenesis are yet to be fully elucidated in rela- tion to the pathogenesis of dyskeratosis congenita when DKC1 is mutated. However, it is worth noting that the hematologic deficiencies observed in dyskeratosis con- genita are also primary characteristics of the bone mar- row failure disorders referred to as ribosomopathies, which feature impaired ribosome biogenesis as an under- lying cause.55 Ribosome dysfunction in these disorders is usually attributed to mutations in genes with known roles in ribosome biogenesis. However, the discovery of GATA1 mutations in the ribosomopathy Diamond Blackfan anemia raises the possibility that dyskerin insuf- ficiency may contribute to the pathogenesis of this genet- ic subtype.56
Collectively the results from these investigations reveal a novel mechanism of telomerase regulation in primary human erythroblasts which contrasts with the estab- lished paradigm centered on MYC-mediated regulation of TERT expression in HSPC, lymphocytes and myelomono- cytic cells. Notwithstanding the requirement for a rate- limiting amount of TERT for telomerase activity,57 this study shows that DKC1 expression levels are a critical determinant of telomerase enzyme levels in proliferating erythroblasts. Evidence provided herein that GATA1 con- tributes to the regulation of DKC1 has implications in hematopoietic disorders that feature DKC1 mutations, GATA1 deregulation and/or telomerase insufficiency.
Acknowledgments
We thank midwives, mothers and neonates at Royal North Shore Hospital and Royal Hospital for Women for cord blood donations to this project. We also thank Prof Emery Bresnick from University of Wisconsin School of Medicine and Public Health for the GATA1 antibody, and Prof Inderjeet Dokal, Queen Mary University of London, for the pCL10.1-DKC1 lentiviral vector. This project was funded by the National Health and Medical Research Council (1007911, 510378, RG150480, RG170246), Cancer Council NSW (RG08-03), Cancer Institute NSW and the Anthony Rothe Memorial Fund.
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