Figure 1.
SAMD1 alters erythroid progenitor gene transcription. (A) Heat map of SAM-containing protein mRNA transcript levels in FAC-sorted mouse bone marrow–derived hematopoietic cells. Transcript levels per gene normalized to maximum expression level in all hematopoietic cell types: LT-HSC, HSC, MPP, CLP, CMP, GMP, MF, Gn, mono, NK cells, MEP, and EryA and EryB. RNA-seq data from Lara-Astiaso et al.20 (B) Quantitation of SAM-containing protein mRNA transcript levels in FAC-sorted mouse bone marrow–derived MEPs. RNA-seq data from Lara-Astiaso et al.20 (C) Quantitation of Samd1 and Lsd1 mRNA transcript levels in FAC-sorted mouse bone marrow–derived hematopoietic cells. RNA-seq data from Lara-Astiaso et al.20 (D) Quantitation of SAMD1 and LSD1 protein copy number per cell. Relative protein levels measured by mass spectrometry from Gautier et al for human erythroid progenitors.27 Prog1: Band3−CD71medGPA−, Prog2: Band3−CD71highGPA−, ProE: Band3−CD71highGPAlow, Baso1: Band3lowCD71highGPAmed, Baso2: Band3medCD71highGPAhighCD49dhigh, Poly: Band3medCD71highGPAhighCD49dmed, and Ortho: Band3highCD71medGPAhigh. (E) Experimental layout of 17-day erythroid differentiation of CD34+ HSPCs. (F) T7 endonuclease assay of day 12 CD34+ progenitors Cas9 infected with scramble (sgControl) or SAMD1 knockout (sgSAMD1) sgRNAs. (G) Western blot of SAMD1 and β-actin in day-12 CD34+ progenitors. (H) Volcano plot of RNA-seq from sgSAMD1 vs sgControl erythroid progenitors. Cutoff: P < .01 and P < .5 log2 fold change (n = 4). Enrichr analysis of 154 genes downregulated after SAMD1 knockout (“SAMD1 activated”), using data from the Reactome and MSigDB catalogs (left). Enrichr analysis of 162 genes upregulated after SAMD1 knockout (“SAMD1 repressed”), using data from the Reactome and MSigDB catalogs (right). Baso1/2, basophilic erythroblast; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; EryA/B, erythroblast A/B; GMP, granulocyte-monocyte progenitor; Gn, granulocyte; HSC, hematopoietic stem cell; LT-HSC, long-term HSC; MF, macrophage; Mono, monocyte; MPP, multipotent progenitor; NK, natural killer; Ortho, orthochromatic erythroblast; Poly, polychromatic erythroblast; ProE, proerythroblast; Prog1/2, progenitor; Puro, puromycin; RNA-seq, RNA sequencing; T7E1, endonuclease 1.

SAMD1 alters erythroid progenitor gene transcription. (A) Heat map of SAM-containing protein mRNA transcript levels in FAC-sorted mouse bone marrow–derived hematopoietic cells. Transcript levels per gene normalized to maximum expression level in all hematopoietic cell types: LT-HSC, HSC, MPP, CLP, CMP, GMP, MF, Gn, mono, NK cells, MEP, and EryA and EryB. RNA-seq data from Lara-Astiaso et al.20 (B) Quantitation of SAM-containing protein mRNA transcript levels in FAC-sorted mouse bone marrow–derived MEPs. RNA-seq data from Lara-Astiaso et al.20 (C) Quantitation of Samd1 and Lsd1 mRNA transcript levels in FAC-sorted mouse bone marrow–derived hematopoietic cells. RNA-seq data from Lara-Astiaso et al.20 (D) Quantitation of SAMD1 and LSD1 protein copy number per cell. Relative protein levels measured by mass spectrometry from Gautier et al for human erythroid progenitors.27 Prog1: Band3−CD71medGPA−, Prog2: Band3−CD71highGPA−, ProE: Band3−CD71highGPAlow, Baso1: Band3lowCD71highGPAmed, Baso2: Band3medCD71highGPAhighCD49dhigh, Poly: Band3medCD71highGPAhighCD49dmed, and Ortho: Band3highCD71medGPAhigh. (E) Experimental layout of 17-day erythroid differentiation of CD34+ HSPCs. (F) T7 endonuclease assay of day 12 CD34+ progenitors Cas9 infected with scramble (sgControl) or SAMD1 knockout (sgSAMD1) sgRNAs. (G) Western blot of SAMD1 and β-actin in day-12 CD34+ progenitors. (H) Volcano plot of RNA-seq from sgSAMD1 vs sgControl erythroid progenitors. Cutoff: P < .01 and P < .5 log2 fold change (n = 4). Enrichr analysis of 154 genes downregulated after SAMD1 knockout (“SAMD1 activated”), using data from the Reactome and MSigDB catalogs (left). Enrichr analysis of 162 genes upregulated after SAMD1 knockout (“SAMD1 repressed”), using data from the Reactome and MSigDB catalogs (right). Baso1/2, basophilic erythroblast; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; EryA/B, erythroblast A/B; GMP, granulocyte-monocyte progenitor; Gn, granulocyte; HSC, hematopoietic stem cell; LT-HSC, long-term HSC; MF, macrophage; Mono, monocyte; MPP, multipotent progenitor; NK, natural killer; Ortho, orthochromatic erythroblast; Poly, polychromatic erythroblast; ProE, proerythroblast; Prog1/2, progenitor; Puro, puromycin; RNA-seq, RNA sequencing; T7E1, endonuclease 1.

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