American Society of Hematology

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.

This Feature Is Available To Subscribers Only