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A. Brown et al.
that not the 7 bp insertion but the additional 675 bp region drive elevated levels of Pttg1 expression in D2 or A cells (Figure 3E). We also identified several exon-specific SNP causing amino acid substitutions in Pttg1. Using 3D in silico models that predict the protein structure of PTTG1, no obvious difference in the structure was observed between the B6 and D2 variants besides a slight increase in 310 helices, a common secondary structure, which renders an additional contribution of the coding SNP of Pttg1 to the phenotype less likely (Online Supplementary Figure S4A).
To test whether Pttg1 is indeed the QTL gene within the described locus, and thus whether the increased HU-sensi- tivity of HSPC is caused by elevated Pttg1 levels, we over-
expressed a Pttg1-Egfp fusion gene by lentiviral transduc- tion in B6 HSPC. The level of expression of the transgene was within the range of the difference in gene expression between B6 and D2 HSPC and thus in a physiological range (Figure 4A, left panel). Transduced BM cells were transplanted into B6 recipients for their in vivo expansion. We sorted GFP+ BM cells five weeks post transplantation to analyze the susceptibility of HSPC to HU with the CAFC assay. BM cells of the transplanted mice were presented with similar rates of transduction (GFP+ cells), excluding a potential bias of certain subpopulations upon transduction (Online Supplementary Figure S4B and C). Elevated expres- sion of Pttg1 in B6 HSPC resulted in a significant increase
A
B
C
Figure 4. Pttg1 promotes hydroxyurea (HU) sensitivity of hematopoietic stem and progenitor cells (HSPC) and influences epigenetic aging. (A) HSPC from B6 mice were cytokine-stimulated and transduced with lentiviruses mediating stable endogenous Pttg1-Egfp (PTTG1 OE) or Egfp (control) overexpression. After transplantation into B6 recipients, total BM GFP+ cells were isolated, treated with HU or its solvent and processed for the cobblestone area-forming cell (CAFC) assay. (Left) Real-time-poly- merase chain reaction (RT-PCR) analysis of transduced (GFP+) HSPC. (Right top) Representative pictures of transduced day 7 cobblestones. (Right bottom) Quantification of the frequency of HU-sensitive CAFC. n=3. (B and C) Epigenetic age predictions were determined based on DNA methylation at three CpG sites (Prima1, Hsf4, Kcns1). For B6 mice they followed a linear regression curve, whereas for D2 it followed a logarithmic trend, as previously described.10 The deviance is the difference of the calculated age and the “real” chronological age of the four mouse strains. Mice per group: 26-40. (B) Regression curves and (C) dot plots of the corresponding methy- lation analyses. *P<0.05; ***P<0.001. PH: phase contrast; ns: not significant.
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haematologica | 2020; 105(2)