Page 63 - Haematologica - Vol. 105 n. 6 - June 2020

DKC1 drives upregulation of telomerase in normal human erythroblasts
and monocyte colony-stimulating factor (SFM) for mono- cytic differentiation, to SCF, FLT-3L and granulocyte colony-stimulating factor (SFG) for granulocytic expan- sion and to SCF, thrombopoietin and interleukin-6 (ST6) to promote megakaryocyte differentiation (Online Supplementary Figure S2). qRT-PCR analysis showed that in contrast to the upregulation of DKC1 observed in cells cul- tured with SE (P<0.01), there was no significant induction of DKC1 expression in cells grown under conditions favoring monocytic, granulocytic or megakaryocytic dif- ferentiation (Figure 2B). Western blot analysis further demonstrated high expression of dyskerin protein only in cell populations produced under erythroid conditions (Figure 2C).
Upregulation of DKC1 bolsters telomerase activity and promotes telomere lengthening
To confirm that DKC1 expression was upregulated in erythroblasts produced in vivo as well as in ex vivo-generat- ed erythroid cultures, DKC1 expression was assessed in GLYA+ cells isolated from uncultured CB and bone mar- row mononuclear cells. Results from qRT-PCR analysis consistently showed higher DKC1 mRNA in the GLYA+ fraction than in the GLYA- fraction of cells isolated from four samples (Figure 2D). Consistent with these results, gene expression data, collected through meta-analysis of multiple independent studies (available through the BioGPS Primary Cell Atlas dataset), showed DKC1 expression to be upregulated in proerythroblasts relative to hematopoietic stem cells and myeloid lineage cells (Figure 2E).25 The upregulation of DKC1 mRNA in bone marrow proerythroblasts appeared to be transient, as DKC1 levels in intermediate erythroblasts were similar to those in hematopoietic stem cells. Collectively these data provide strong evidence of DKC1 upregulation during ery- throid commitment of human HSPC.
To test whether induction of DKC1 was sufficient for the upregulation of telomerase activity observed in erythrob- lasts, HSPC were transduced with lentiviral vectors encod- ing DKC1 cDNA plus GFP (MSCV-DKC1) or GFP alone (MSCV-GFP). Robust upregulation of DKC1 mRNA in MSCV-DKC1-transduced cells was demonstrated by qRT- PCR analysis of GFP+ cells isolated by FACS (P<0.01, Student t test) (Figure 3A). Telomerase enzyme activity was also substantially increased in MSCV-DKC1 cells relative to control vector-transduced cells (P<0.01, Student t test) (Figure 3B). There were no significant differences in the expression of TERT and TERC between MSCV-DKC1 and control vector-transduced cells (Figure 3A), although TERC expression tended to be higher in the MSCV-DKC1 cells.
To enable analysis of the effect of DKC1 upregulation over a time course sufficient for assaying telomere length changes, we also overexpressed DKC1 in the immortal erythroleukemia cell line, HEL 92.1.7 (Figure 3C). Consistent with the CB experiments, overexpression of DKC1 in HEL 92.1.7 cells caused a substantial elevation of telomerase activity without an apparent change in TERT mRNA expression (Figure 3C, D). Southern blot-based analysis of telomere length showed that ectopic expres- sion of DKC1 resulted in telomere lengthening at a rate of approximately 500 bp over a 2-month period, and 900 bp over 7 months (Figure 3E). To verify that DKC1 upregula- tion is sufficient for telomere extension, we also overex- pressed DKC1 in HL-60 cells. Consistent results were obtained, showing that DKC1 overexpression resulted in
robust upregulation of telomerase and elongated telom- eres (Online Supplementary Figure S4). These data show that upregulation of DKC1 results in an accumulation of functional telomerase complexes capable of telomere elongation.
High expression of DKC1 is necessary for erythroblast proliferation
We next tested whether elevated expression of DKC1 was necessary for erythroid cell proliferation and differen- tiation. For these investigations CB-derived HSPC were transduced with retroviral vectors encoding one of two different shRNA targeting DKC1 mRNA (D2 and D3) or a non-silencing shRNA (NS) plus GFP. HSPC were pre-stim- ulated and transduced in medium supplemented with STF and then FACS-sorted for GFP+ cells, which were then cul- tured in SE. qRT-PCR analysis confirmed effective sup- pression of DKC1 and corresponding downregulation of telomerase activity in erythroid cells transduced with D2 and D3 vectors relative to control vector-transduced cells (Figure 4A, B). The suppression of telomerase activity was not attributable to reduced expression of TERT, which was expressed at equivalent levels in D2-, D3- and NS- transduced cells (Figure 4C). TERC levels varied among the independent experiments, although they tended to be lower in D2- and D3-transduced cells relative to control cells (Figure 4D), consistent with the known role of dyskerin as a stabilizing scaffold for TERC.
Weekly counts of transduced cells revealed that shRNA- mediated suppression of DKC1 expression inhibited pro- liferation in SE-driven cultures (Figure 4E). Since there was no apparent difference in the proportion of GLYA+ cells in D2 and D3 cultures compared to NS cultures, the reduc- tion in GLYA+ cell number did not appear to be due to impaired erythroid differentiation (Figure 4F). When plat- ed in methylcellulose, D2- and D3-transduced cells formed erythroid colonies with normal burst-forming unit-erythroid (BFU-E) size and morphology; however, significantly fewer colonies were generated by D2 and D3 cultures than by NS (Figure 4G). In contrast to the effect of DKC1 knockdown on BFU-E colony numbers, there was no discernible effect on colony-forming units of granulo- cyte-monocyte or granulocyte-erythrocyte-macrophage- megakaryocyte. Together, the results demonstrate a criti- cal role for DKC1 expression in erythroblast proliferation that is independent of differentiation.
GATA1 contributes to transcriptional regulation of DKC1 in erythroblasts
The DKC1 promoter was previously shown to be a tar- get of MYC family oncoproteins in MYC-driven can- cers.26,27 Since MYC is also expressed in erythroid progeni- tors,28 we next investigated whether MYC or other ery- throid transcription factors, namely GATA1 and TAL1, play a role in the regulation of DKC1. Western blot analy- sis of uncultured CB CD34+ HSPC and ex vivo-expanded CB cells revealed MYC expression in unstimulated CB HSPC and in cells harvested from STF, STM and SE cul- tures (Figure 5A). In contrast, expression of GATA1 and TAL1 was confined to cells cultured in SE. Consistent with the qRT-PCR results (Figure 2B), dyskerin protein was detectable in undifferentiated cells cultured in STF and erythroid cells at weeks 2 and 3, but was not detected in cells from monocyte-enriched cultures (Figure 5A).
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