Figure 6.
PLAG1-S activates imprinted loci to support human HSPCs. (A) Heatmap of top 10 differentially expressed transcripts in the transcriptome of PLAG1-SOE Lin−CD34+ HSPCs. (B) Intracellular flow cytometry of components of the PI3K signaling pathway, including phospho-S473 AKT, phospho-S2448 mTOR, and phospho-Thr37/46 4EBP1, in PLAG1-SOE Lin−CD34+ cells on day 4 of culture. Numbers above PLAG1-SOE bars show the paired Student t-test P value relative to control (n = 3, ph-4EBP1 n = 5). (C) Total nucleated cell (top) and CD34+ cell (bottom) fold change in Lin−CD34+BFP+ cultures overexpressing either PLAG1-S or Luciferase control and treated with 50 nM rapamycin (RAPA), 1 μM AKT inhibitor (AKTi), or vehicle (DMSO) (n = 4). Student t-test P values in red are relative to Cntrl-DMSO and in black are relative to PLAG1-SOE-DMSO. (D) CD34 positivity in PLAG1-SOE or control HSPCs following 4 and 8 days of ex vivo culture with RAPA, AKTi, or vehicle (n = 4). Student t-test P values in red are relative to Cntrl-DMSO and in black are relative to PLAG1-SOE-DMSO. (E) OP-Puro incorporation by PLAG1-SOE HSPCs cultured in the presence of RAPA, AKTi, or vehicle on day 4 of culture (n = 4). (F) Schematic of the imprinted human DLK1/MEG3 locus, which encodes miRNA mega-clusters miR127/136 (7 miRNAs) and miR-379/410 (39 miRNAs). (G) RNA-seq read tracks for miRNA transcripts from this locus detected in PLAG1-SOE HSPCs. (H) Overlap of the PLAG1-S overexpression gene set enrichment map (P < .025) to signatures of miR-127-5p and miR-127-3p validated targets (Mann-Whitney U test, P < .05). (I-J) Schematic of lentivectors used for dual PLAG1-S overexpression and miR127-5p inhibition via a sponge consisting of multiple bulged 26-mer target sequences (miR127TB) or miR127 overexpression. (I) CD34+ cell fold change ex vivo when PLAG1-S and the miR127-5p inhibitor are coexpressed in Lin−CD34+ cells (n = 3). (J) CD34+ cell fold change ex vivo when miR127 is overexpressed in Lin−CD34+ cells (n = 3); and OP-Puro incorporation Lin−CD34+ cells overexpressing miR127 (n = 3). Data are presented as average ± SEM unless otherwise indicated. Each point represents an individual CB unit. ***P < .005, **P < .01, *P < .05. n.s., not significant. See also supplemental Figure 6.

PLAG1-S activates imprinted loci to support human HSPCs. (A) Heatmap of top 10 differentially expressed transcripts in the transcriptome of PLAG1-SOE Lin−CD34+ HSPCs. (B) Intracellular flow cytometry of components of the PI3K signaling pathway, including phospho-S473 AKT, phospho-S2448 mTOR, and phospho-Thr37/46 4EBP1, in PLAG1-SOE Lin−CD34+ cells on day 4 of culture. Numbers above PLAG1-SOE bars show the paired Student t-test P value relative to control (n = 3, ph-4EBP1 n = 5). (C) Total nucleated cell (top) and CD34+ cell (bottom) fold change in Lin−CD34+BFP+ cultures overexpressing either PLAG1-S or Luciferase control and treated with 50 nM rapamycin (RAPA), 1 μM AKT inhibitor (AKTi), or vehicle (DMSO) (n = 4). Student t-test P values in red are relative to Cntrl-DMSO and in black are relative to PLAG1-SOE-DMSO. (D) CD34 positivity in PLAG1-SOE or control HSPCs following 4 and 8 days of ex vivo culture with RAPA, AKTi, or vehicle (n = 4). Student t-test P values in red are relative to Cntrl-DMSO and in black are relative to PLAG1-SOE-DMSO. (E) OP-Puro incorporation by PLAG1-SOE HSPCs cultured in the presence of RAPA, AKTi, or vehicle on day 4 of culture (n = 4). (F) Schematic of the imprinted human DLK1/MEG3 locus, which encodes miRNA mega-clusters miR127/136 (7 miRNAs) and miR-379/410 (39 miRNAs). (G) RNA-seq read tracks for miRNA transcripts from this locus detected in PLAG1-SOE HSPCs. (H) Overlap of the PLAG1-S overexpression gene set enrichment map (P < .025) to signatures of miR-127-5p and miR-127-3p validated targets (Mann-Whitney U test, P < .05). (I-J) Schematic of lentivectors used for dual PLAG1-S overexpression and miR127-5p inhibition via a sponge consisting of multiple bulged 26-mer target sequences (miR127TB) or miR127 overexpression. (I) CD34+ cell fold change ex vivo when PLAG1-S and the miR127-5p inhibitor are coexpressed in Lin−CD34+ cells (n = 3). (J) CD34+ cell fold change ex vivo when miR127 is overexpressed in Lin−CD34+ cells (n = 3); and OP-Puro incorporation Lin−CD34+ cells overexpressing miR127 (n = 3). Data are presented as average ± SEM unless otherwise indicated. Each point represents an individual CB unit. ***P < .005, **P < .01, *P < .05. n.s., not significant. See also supplemental Figure 6.

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