|
Fig. 4 REs as Erv elicit a virus-like response in HSPCs promoting B2m expression. (A) Flow cytometry showing that ~20% of runx1+23mCherry+ HSPCs express Isg15 (gating strategy is shown in fig. S4A). Data points represent a pool of ~200 embryos acquired in three independent experiments. Data are shown as means ± SEM. Data were analyzed with Mann-Whitney test. P = 0.003. (B) Positive correlation between B2m and Isg15 levels. Data were analyzed by Pearson correlation. Data points represent a pool of ~200 embryos acquired in three independent experiments. (C) Left: representative image of a Isg15+-HSPC interaction. HSPCs are shown in red, macrophages in blue, and isg15 in green. Right: HSPCs-Isg15+ cells showing lower dooming ratios. (D) Sorted isg15+ HSPCs showing higher b2m and RE expression. Data points represent a pool of ~200 embryos. Experiments were performed twice. The y axis depicts the relative gene expression. (E) Uniform manifold and projection (UMAP) of sorted runx1+23-mcherry HSPCs in standard morpholino (gray, control sdM) and irf8-depleted embryos (yellow, irf8 sdKD). [Original data are from Wattrus et al. (6)]. (F) Cluster 2 (C2), enriched for control HSPCs, showing higher expression of RE. A total of 808 cells were analyzed. (G) Treating 2-dpf embryos with CM272 (5 uM) led to higher number of isg15+HSPCs in the 3-dpf CHT. Data were quantified by live-cell imaging and analyzed by Mann-Whitney test. *P = 0.02. Data points represent an embryo. Experiments were performed twice. (H) EdU staining of runx1+23:mCherry embryos treated with CM272 identifies a significant increase in proliferating HSPCs at 3 dpf. Data were analyzed by Student?s t test. Data normality was enquired by the Shapiro-Wilk test. **P = 0.0061. Data are shown as means ± SEM. Control, n = 5; CM272, n = 7. (I) CM272-treated embryos showed lower dooming ratios. Data points represent an embryo. Percentage was calculated by quantification of Edu+ Runx1+ cells divided by the total number of runx1+ cells. The plot depicts two independent experiments. Control, n = 6; CM272, n = 10. (J) Gini coefficient in control (DMSO), CM272, and CM272 b2m-knockdown zebrafish. Data were analyzed by Kruskall-Wallis test followed by Dunn?s multiple-comparisons test. **P = 0.0043. Data points represent an adult fish. DMSO, n = 6; CM272, n = 5; and b2m sgRNA, n = 5. The plot depicts two independent experiments. (K) b2m-TWISTR Zebrabow-M;draculin:CreERT2 treated CM272 exhibits reduced numbers of HSC clones in the adult marrow. Data were analyzed by Kruskall-Wallis test followed by Dunn?s multiple-comparisons test. *P = 0.023. Data points represent an adult fish. DMSO, n = 6; CM272, n = 5; and b2m sgRNA, n = 5. The plot shows two independent experiments. (L) ISG15 expression positively correlates with transposable element (TE) expression in human HSCs. Previously published single-cell RNA-seq datasets of human bone marrow CD34+ stem and progenitors [original data from Nam et al. (90), n = 4 samples] were aligned to a TE reference using CellRanger v3.2. TE expression was normalized by total RNA counts. Pearson correlation was performed between TE expression and ISG15 gene expression of individual HSCs, represented in each data point. (M) Human bone marrow HSCs from the same dataset as in (L) were grouped based on their ISG15 expression, where in the upper quartile was considered the threshold for high and the lower quartile as low. Normalized expression of B2M is shown for individual cells. ISG15 high cells showed higher B2M expression. Only cells with at least one transcript of ISG15 were included in the analysis. High, n = 203 cells; low, n = 608 cells from n = 4 samples. P = 0.02 using a linear mixed model, with sample as a random effects variable. Box plot represents the median; in the bottom and top quartiles, whiskers correspond to 1.5× the interquartile range. (N) Human Erv overexpression was up-regulated the B2M levels on human CD34+ cells. Data are shown as means ± SEM. Data points depict a CD34 donor. Data were collected from two independent donors. (O) EdU staining of MFG+ mice treated with DL-PPMP identifies an increase in proliferating HSPCs 24 hours after the treatment. Percentage was determined by the total number of living cells. Data points depict a mouse. (P) DL-PPMP treatment stimulates B2M expression in murine HSPCs. Flow cytometry was performed using the cells from the tibia and femur (for gating, see fig. S4Q). Data were analyzed by Student?s t test. *P = 0.02. Data are shown as means ± SEM. n = 3 mice per condition. (Q) Schematic overview of the proposed molecular pathway resulting in higher surface B2m.