Figure 1
Figure 1. JmjC gene expression in HSC/progenitor cell populations and selection for RNAi screen. (A) Transcript levels of HDM in HSC-enriched cell populations. Results show ΔCT values determined by qRT-PCR assays (with respect to endogenous Hprt expression levels, Ct∼22) and represent average ± standard error of the mean (SEM) of 5 independently sorted HSC populations (bone marrow, n = 3; E14.5 d.p.c. fetal liver, n = 2). Frequencies of long-term–repopulating HSCs in these populations are shown in supplemental Table 1. (B) Comparison of HDM transcript levels detected in HSCs and total bone marrow cell populations. Relative transcript quantities (RQ) are shown in log2 scale and represent the ΔCT (HSC)/ΔCT(bone marrow) ratio determined by qRT-PCR assays (average ± SEM, n = 3). An HSC expression score was implemented based on expression levels (ΔCT) and differential expression (RQ) of individual HDM to rank gene candidates according to relative expression in HSC vs mature cells: gene not expressed in HSC, −2; less (< ) expressed in HSC vs mature cells, −1; more (> ) expressed in HSC, +1; equally expressed, 0. (C) List of the 23 HDM candidates tested in primary screen. HDMs subfamilies sharing similarities outside the catalytic domain are denoted by different shading. Left column, the revised current terminology; center column, synonyms; right column, proposed substrate specificity.10 Four HDM genes were excluded from the screen: Jarid1d and Uty map to chromosome Y and are thus likely not required for regulation of HSC activity; Pla3g4b belongs to the cytosolic phospholipase A2 family; Jhdm1e knockdown could not be achieved by any hairpin in 2 independent experiments. (D) Schema of shRNA retroviral vector backbone (top) and experimental outline (bottom) of the primary screen at the bottom. At 16 weeks after transplantation, an increase in the proportion of GFP+ peripheral blood leukocytes above their input levels reflects knockdown of a negative regulator of HSC activity (green box), and the inverse outcome denotes a positive HSC regulator (red box).

JmjC gene expression in HSC/progenitor cell populations and selection for RNAi screen. (A) Transcript levels of HDM in HSC-enriched cell populations. Results show ΔCT values determined by qRT-PCR assays (with respect to endogenous Hprt expression levels, Ct∼22) and represent average ± standard error of the mean (SEM) of 5 independently sorted HSC populations (bone marrow, n = 3; E14.5 d.p.c. fetal liver, n = 2). Frequencies of long-term–repopulating HSCs in these populations are shown in supplemental Table 1. (B) Comparison of HDM transcript levels detected in HSCs and total bone marrow cell populations. Relative transcript quantities (RQ) are shown in log2 scale and represent the ΔCT (HSC)/ΔCT(bone marrow) ratio determined by qRT-PCR assays (average ± SEM, n = 3). An HSC expression score was implemented based on expression levels (ΔCT) and differential expression (RQ) of individual HDM to rank gene candidates according to relative expression in HSC vs mature cells: gene not expressed in HSC, −2; less (< ) expressed in HSC vs mature cells, −1; more (> ) expressed in HSC, +1; equally expressed, 0. (C) List of the 23 HDM candidates tested in primary screen. HDMs subfamilies sharing similarities outside the catalytic domain are denoted by different shading. Left column, the revised current terminology; center column, synonyms; right column, proposed substrate specificity.10  Four HDM genes were excluded from the screen: Jarid1d and Uty map to chromosome Y and are thus likely not required for regulation of HSC activity; Pla3g4b belongs to the cytosolic phospholipase A2 family; Jhdm1e knockdown could not be achieved by any hairpin in 2 independent experiments. (D) Schema of shRNA retroviral vector backbone (top) and experimental outline (bottom) of the primary screen at the bottom. At 16 weeks after transplantation, an increase in the proportion of GFP+ peripheral blood leukocytes above their input levels reflects knockdown of a negative regulator of HSC activity (green box), and the inverse outcome denotes a positive HSC regulator (red box).

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