Page 60 - Haematologica - Vol. 105 n. 6 - June 2020

L.A. Richards et al.
phomas.4-7 It also plays an important role in the function of normal hematopoietic stem and progenitor cells (HSPC).8- 11 Telomerase activity is low in quiescent hematopoietic stem cells, and is upregulated by self-renewal cytokines that promote cell cycling.11,12 It is then downregulated to undetectable levels as HSPC differentiate into granulo- cytes, monocytes and macrophages.9,11,12 Telomerase is similarly upregulated in lymphoid cells upon exposure to mitogens but is barely detectable in mature resting lym- phocytes and peripheral blood mononuclear cells.9,13 Studies of telomerase in erythroid lineage cells are limited; however, our previous investigation demonstrated that in contrast to the downregulation of telomerase observed during myelomonocytic differentiation, telomerase was robustly upregulated as umbilical cord blood (CB)-derived HSPC underwent commitment and expansion along the erythroid lineage.14 Supporting the functional significance of this finding, a strong correlation was demonstrated between the level of telomerase in human HSPC and the proliferative potential of erythroblasts. In contrast, there was no correlation between telomerase in HSPC and expansion along granulocytic or monocytic lineages. A recent study demonstrating that telomerase knockout mice have more pronounced defects in erythroid progeni- tors than in granulocyte-macrophage progenitors further supports an important role for telomerase in the erythroid lineage.15 The mechanism responsible for the upregulation of telomerase during erythropoiesis is currently unknown.
Insufficient telomerase due to mutations in telomerase- associated genes is causally involved in inherited bone marrow failure syndromes including dyskeratosis con- genita.16 Dyskeratosis congenita manifests with mucocu- taneous symptoms and multiple organ dysfunction; how- ever, anemia is prevalent and bone marrow failure is the most common cause of death among patients with telom- erase mutations.17 Patients with telomerase insufficiency syndromes exhibit pancytopenias and have fewer circulat- ing hematopoietic progenitors than do healthy individu- als.18 Understanding the mechanisms that regulate telom- erase activity in human hematopoietic cells is a crucial step toward the development of effective treatment of hematologic conditions associated with insufficient telomerase. Past studies along these lines have attributed the modulation of telomerase activity in hematopoietic cells to transcriptional regulation of TERT, the rate-limit- ing component of the telomerase holoenzyme.19-21 These studies focused on telomerase regulation in progenitors, lymphoid and myelomonocytic cells. There is no prior study of telomerase regulation in normal erythroid lineage cells. The prevalence of anemia in telomerase insufficien- cy syndromes and the need for new treatments for these disorders, provided the impetus for investigation in this area.
Here we show that the increase in telomerase activity that occurs as human HSPC commit to the erythroid line- age is a result of upregulation of the DKC1 gene in the presence of limiting amounts of TERT mRNA. It is shown for the first time that the DKC1 gene is a direct transcrip- tional target of the erythroid-specific transcription factor GATA1 and that high expression of DKC1 is required for efficient production of glycophorin A-positive (GLYA+) erythroblasts. These results provide a novel mechanistic explanation for high levels of telomerase in GLYA+ ery- throblasts and the heightened vulnerability of the ery- throid compartment to telomerase insufficiency.
Methods
Cord blood cell isolation and culture of CD34+ and glycophorin A+ cells
CB was obtained from the Royal North Shore Hospital and the Australian Cord Blood Bank. Ethical approval for the use of CB was obtained from the Human Research Ethics Committees of the relevant hospitals and the University of New South Wales (approval numbers: HREC 05188, NSCCH 0602-004M, SESIAHS 08/190). Bone marrow mononuclear cells were obtained from Lonza (Mt Waverly Australia). CB processing and isolation of CD34+ HSPC and GLYA+ cells are described in the Online Supplementary Methods. CD34+ HSPC were expanded for 1 week in Isocove modified Dulbecco media (Life Technologies, Carlsbad, CA, USA) with 20% fetal bovine serum (Trace Scientific, Melbourne, Australia), 100 ng/mL stem cell factor (SCF, Amgen, Thousand Oaks, CA, USA), 100 ng/mL thrombopoietin (Peprotech, Rocky Hill, NJ, USA), 100 ng/mL Flt-3 ligand (FLT-3L, Amgen) (STF), 50 μg/mL gentamycin and 200 mM glutamine. The cells were then cultured in cytokine combinations that force expansion and differentiation along specific lineages as described in our previous study (Online Supplementary Table S1).14 Differentiation was assessed by fluorescence activated cell sorting (FACS) analysis after staining cells with the conjugated antibodies detailed in Online Supplementary Table S2. Green fluorescent pro- tein-positive (GFP+), GLYA+ and CD13+ subpopulations were puri- fied by FACS using a FACS Diva (Becton Dickinson).
DKC1 gene suppression and overexpression
The viral vectors and methods used for suppression and over- expression of DKC1 are described in the Online Supplementary
Methods.
Telomerase enzyme assays and telomere length measurements
Telomerase enzyme activity was quantified using the real-time polymerase chain reaction (PCR)-based telomeric amplification protocol (qTRAP) as described elsewhere.22 Mean telomeric restriction fragment length was measured using the TeloTAGGG Telomere Length Assay kit (Roche, Mannheim, Germany) as pre- viously described and detailed in the Online Supplementary Methods.23
Gene and protein expression analyses
Quantitative real-time PCR (qRT-PCR) and western blot analy- sis were performed according to standard protocols described in the Online Supplementary Methods.
Chromatin immunoprecipitation and reporter assays
Chromatin immunoprecipitation (ChIP) assays were performed as previously described.24 Briefly, 2 x 107 cells were treated with 1% formaldehyde, then cross-linked chromatin was sonicated to obtain 300-500 bp fragments. Chromatin was immunoprecipitat- ed with antibodies detailed in Online Supplementary Table S2 and subjected to qRT-PCR using Express SYBR Green (Life Technologies) and the primers described in Online Supplementary Table S3. Values were normalized to products from immunopre- cipitation with control IgG antibody.
A DKC1 promoter reporter construct (pGL2-DKC1L) was made by cloning a DKC1 sequence spanning +211 to -1113 bp from the DKC1 transcription start site into Xho1 and HindIII sites of the pGL2 vector encoding luciferase. Two proximal GATA sites were mutated by site-directed mutagenesis using the QuikChange Site- directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) with the primers listed in Online Supplementary Table S3. The mutated
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haematologica | 2020; 105(6)