American Society of Hematology

Figure 4

$Figure 4. The mErg +85 enhancer targets fetal and adult hematopoietic stem/progenitors and shows selective activity in lineage committed cells. (A) Cross species sequence conservation at the ERG locus. The top panel shows a VISTA plot of human/mouse sequence conservation > 50% across the ERG locus. Noncoding sequences with > 75% conservation are shaded in pink, exons in magenta, and the untranslated regions in cyan; arrow indicates location of the +85 element. Bottom schematic shows the Venus reporter construct driven by the SV40 promoter and mouse Erg +85 element. (B) Erg +85 activity in E11.5 fetal liver (FL) cells. Fetal livers were dissociated and single cell suspensions stained with fluorescent-labeled antibodies and FACS analyzed to assess Venus reporter expression in fetal liver cells. Enhancer activity is highest in c-Kit+Ter119− FL stem/progenitors and decreases in c-Kit−Ter119+ erythroid cells. (C) Erg expression in bone marrow (BM) cells. Bone marrow cells were harvested from approximately 3-month-old C57BL/6J mice, lineage depleted, stained with fluorescent-labeled antibodies, and FACS sorted into various stem/progenitor fractions for RNA isolation. Erg expression was quantified by SYBR Green qPCR and is expressed relative to GAPDH. Erg expression is higher in bone marrow Lin−Sca1+c-Kit+ (LSK) stem progenitor cells than in whole bone marrow. (D) Erg +85 activity in lineage negative adult bone marrow cells. Bone marrow from +85 enhancer transgenic mice was harvested and sorted into lineage-negative and -positive fractions using magnetic beads. The lineage-negative fraction was FACS analyzed following incubation with fluorescent-labeled antibodies to establish Venus reporter expression in gated cell fractions. Erg +85 is most active in c-Kit+ stem/progenitors. (E) Erg +85 activity in lineage positive adult bone marrow cells. Unsorted Lin+ cells show mid-low reporter activity (F) Erg +85 activity in specific subpopulations of lineage committed bone marrow cells. (i) Erg +85 is active in a subset of Mac1+/Gr1+ macrophage/granulocyte precursors. (ii) Erg +85 has low activity in a small fraction of Ter119 erythrocyte precursors. (iii) Erg +85 is inactive in the B-cell lineage. (iv) Erg +85 has low activity in a subset of CD3+ T cells. (G) Erg +85 activity during early T-cell development. The thymus was dissected from 6-week-old +85 enhancer transgenic mice and single-cell suspensions stained for FACS analysis. Venus reporter activity is absent during the DN1 stage, then rapidly increases in DN2 cells before reducing in DN3 and DN4 cells.$

The mErg +85 enhancer targets fetal and adult hematopoietic stem/progenitors and shows selective activity in lineage committed cells. (A) Cross species sequence conservation at the ERG locus. The top panel shows a VISTA plot of human/mouse sequence conservation > 50% across the ERG locus. Noncoding sequences with > 75% conservation are shaded in pink, exons in magenta, and the untranslated regions in cyan; arrow indicates location of the +85 element. Bottom schematic shows the Venus reporter construct driven by the SV40 promoter and mouse Erg +85 element. (B) Erg +85 activity in E11.5 fetal liver (FL) cells. Fetal livers were dissociated and single cell suspensions stained with fluorescent-labeled antibodies and FACS analyzed to assess Venus reporter expression in fetal liver cells. Enhancer activity is highest in c-Kit⁺Ter119⁻ FL stem/progenitors and decreases in c-Kit⁻Ter119⁺ erythroid cells. (C) Erg expression in bone marrow (BM) cells. Bone marrow cells were harvested from approximately 3-month-old C57BL/6J mice, lineage depleted, stained with fluorescent-labeled antibodies, and FACS sorted into various stem/progenitor fractions for RNA isolation. Erg expression was quantified by SYBR Green qPCR and is expressed relative to GAPDH. Erg expression is higher in bone marrow Lin⁻Sca1⁺c-Kit⁺ (LSK) stem progenitor cells than in whole bone marrow. (D) Erg +85 activity in lineage negative adult bone marrow cells. Bone marrow from +85 enhancer transgenic mice was harvested and sorted into lineage-negative and -positive fractions using magnetic beads. The lineage-negative fraction was FACS analyzed following incubation with fluorescent-labeled antibodies to establish Venus reporter expression in gated cell fractions. Erg +85 is most active in c-Kit⁺ stem/progenitors. (E) Erg +85 activity in lineage positive adult bone marrow cells. Unsorted Lin⁺ cells show mid-low reporter activity (F) Erg +85 activity in specific subpopulations of lineage committed bone marrow cells. (i) Erg +85 is active in a subset of Mac1⁺/Gr1⁺ macrophage/granulocyte precursors. (ii) Erg +85 has low activity in a small fraction of Ter119 erythrocyte precursors. (iii) Erg +85 is inactive in the B-cell lineage. (iv) Erg +85 has low activity in a subset of CD3⁺ T cells. (G) Erg +85 activity during early T-cell development. The thymus was dissected from 6-week-old +85 enhancer transgenic mice and single-cell suspensions stained for FACS analysis. Venus reporter activity is absent during the DN1 stage, then rapidly increases in DN2 cells before reducing in DN3 and DN4 cells.

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