![Figure 1. Setd1a expression in postnatal hematopoietic cells is crucial for survival. (A) Normalized RNA-sequence counts for Mll1, Mll2, Mll3, Mll4, Setd1a, and Setd1b in KSL Slam LT-HSCs (lineage− = Lin−, Kit+ Sca-1+ [KSL] CD48/41− CD150+ CD34− CD135−). Shown is the average of 3 replicates of 1 experiment. (B) qPCR of Setd1a transcripts in LT-HSCs (KSL CD34− CD135−), ST-HSCs (KSL CD34+ CD135−), MPPs (KSL CD34+ CD135+), common myeloid progenitors (CMPs; Lin− Kit+ Sca-1− [MP] CD34+ CD16/32−), megakaryocyte erythroid progenitors (MEPs; MP CD34− CD16/32−), and granulocyte macrophage progenitors (GMPs; MP CD34+ CD16/32+) from wild-type mice. Setd1a transcript expression is shown in relation to transcripts of Rpl19. (C) Survival curve of R.26-CreERT2+;Setd1aF/F (red line; n = 7) and control mice (blue line; n = 15) after tamoxifen induction. Data are pooled from 2 independent experiments. (D) Survival curve of Vav-Cre+;Setd1aF/F (red line; n = 17) and control (blue line; n = 15) mice. (E) Experimental outline using R.26-CreERT2+ deleter mice. (F) White and red blood cell counts, hematocrit, hemoglobin, and platelet values from R.26-CreERT2+;Setd1aF/F (red lines; n = 10) or R.26-CreERT2+;Setd1a+/+ (blue lines; n = 10) chimeric mice before (0 day) and after TAM treatment or untreated wild-type (gray lines; n = 3) controls. (G) Plot shows colony growth 8 days (BFU-E) and 12 days (G, M, GM, and GEMM) after culture of 104 BM cells from R.26-CreERT2+;Setd1aF/F or control mice (n = 6) that were TAM induced 8 days beforehand. Data are pooled from 2 independent experiments. (H) Western blot of H3K4me1, H3K4me2, and H3K4me3 in Lin− cells from R.26-CreERT2+;Setd1aF/F or control mice after 4 days in vivo and 2 days in vitro TAM induction (n = 3 each). Data are representative of 3 independent experiments. (I) Representative fluorescence-activated cell scan (FACS) plots resolve thymocytes from CD4-Cre+;Setd1aF/F and control mice for the expression of CD4 and CD8. Plots are gated on 4′,6-diamidino-2-phenylindole (DAPI) negative cells. (J) Quantification of CD4 and CD8 single positive thymocytes. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Hb, hemoglobin; HCT, hematocrit; ns, not significant; Plt, platelet; RBC, red blood cell; SP, single positive; WBC, white blood cell.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/131/12/10.1182_blood-2017-09-806844/4/blood806844f1.jpeg?Expires=1724884848&Signature=r1O3niIbNljsvuJUJkaoQ2HycdmK5pcevsO9Sz1~VFN-erw2jX9iL24YSatbUJ8S48x8nyT7xbIJiCvfzR9umaEBI9FdwpcMRQ0e27l-x0rWXaLKK5VkciMaOszq5yy~yF35MsI93XqA1OkwbwtlWqUaDhmKNP6sbNKzYcGtqHx0NzhmSBIAayhBG2ke3rHXzdIJVw2Sx1ploCP5IRJWz2GFn9cWjSW~mFyqbGSSUK-vzPHkzhiJSQRd1oz8IhEi~Np-qJyGDg4oIxfLaBYVnIQsw6QKhbjI~qx~8bMkCkVBvELuujt9LFGSuWC6EXQXBwopKBweg2NqrCxMhH7xNA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Setd1a expression in postnatal hematopoietic cells is crucial for survival. (A) Normalized RNA-sequence counts for Mll1, Mll2, Mll3, Mll4, Setd1a, and Setd1b in KSL Slam LT-HSCs (lineage− = Lin−, Kit+ Sca-1+ [KSL] CD48/41− CD150+ CD34− CD135−). Shown is the average of 3 replicates of 1 experiment. (B) qPCR of Setd1a transcripts in LT-HSCs (KSL CD34− CD135−), ST-HSCs (KSL CD34+ CD135−), MPPs (KSL CD34+ CD135+), common myeloid progenitors (CMPs; Lin− Kit+ Sca-1− [MP] CD34+ CD16/32−), megakaryocyte erythroid progenitors (MEPs; MP CD34− CD16/32−), and granulocyte macrophage progenitors (GMPs; MP CD34+ CD16/32+) from wild-type mice. Setd1a transcript expression is shown in relation to transcripts of Rpl19. (C) Survival curve of R.26-CreERT2+;Setd1aF/F (red line; n = 7) and control mice (blue line; n = 15) after tamoxifen induction. Data are pooled from 2 independent experiments. (D) Survival curve of Vav-Cre+;Setd1aF/F (red line; n = 17) and control (blue line; n = 15) mice. (E) Experimental outline using R.26-CreERT2+ deleter mice. (F) White and red blood cell counts, hematocrit, hemoglobin, and platelet values from R.26-CreERT2+;Setd1aF/F (red lines; n = 10) or R.26-CreERT2+;Setd1a+/+ (blue lines; n = 10) chimeric mice before (0 day) and after TAM treatment or untreated wild-type (gray lines; n = 3) controls. (G) Plot shows colony growth 8 days (BFU-E) and 12 days (G, M, GM, and GEMM) after culture of 104 BM cells from R.26-CreERT2+;Setd1aF/F or control mice (n = 6) that were TAM induced 8 days beforehand. Data are pooled from 2 independent experiments. (H) Western blot of H3K4me1, H3K4me2, and H3K4me3 in Lin− cells from R.26-CreERT2+;Setd1aF/F or control mice after 4 days in vivo and 2 days in vitro TAM induction (n = 3 each). Data are representative of 3 independent experiments. (I) Representative fluorescence-activated cell scan (FACS) plots resolve thymocytes from CD4-Cre+;Setd1aF/F and control mice for the expression of CD4 and CD8. Plots are gated on 4′,6-diamidino-2-phenylindole (DAPI) negative cells. (J) Quantification of CD4 and CD8 single positive thymocytes. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Hb, hemoglobin; HCT, hematocrit; ns, not significant; Plt, platelet; RBC, red blood cell; SP, single positive; WBC, white blood cell.
