Journal: Cell reports

Article Title: Identification of the global miR-130a targetome reveals a role for TBL1XR1 in hematopoietic stem cell self-renewal and t(8;21) AML.

doi: 10.1016/j.celrep.2022.110481

Figure Lengend Snippet: Figure 2. Enforced expression of miR-130a expands HSCs by forcing them into cell cycle (A) Representative flow plots of HSPC populations from control and miR-130a OE xenografts at 12 weeks. (B) Proportion of CD34+CD38 compartment and frequency of LT-HSC, ST-HSC, and MLP (multi-lymphoid progenitor) cell populations (n = 4, each replicate contains pooled RF and BM from 2 to 4 individual mice). (C) Proportion of CD34+CD38+ compartment and frequency of CMP (common myeloid progenitor), MEP (megakaryocyte-erythroid progenitor), and GMP (granulocyte-macrophage progenitor) cell populations (n = 4, each replicate contains pooled RF and BM from 2 to 4 individual mice). (D) Secondary transplantation of CD45+mO+ from 12 week NSG mice at limiting dilution doses. Frequency of HSCs was evaluated from the engraftment in secondary mice (>0.05% CD45+mO+ cells in BM, n = 19–31 mice from two independent experiments). (E) Normalized enrichment score (NES) of the gene sets significantly different between miR-130a OE and control-transduced HSCPs (n = 3). (F) GSEA plots of HSC cell-cycle-primed gene sets and MYC targets in the transcriptome profile following miR-130a OE in CD34+ HSPCs. (G) Cell-cycle analysis of LT-HSCs, ST-HSCs, and CD34+CD38+ cells transduced with miR-130a or control lentiviruses (n = 3). (B and C) Unpaired t test, all error bars indicate ±SEM, *p < 0.05.

Article Snippet: Quantitative RT-PCR for expression levels of miR-130a in CBF AML Peripheral blood samples from t(8;21) and inv(16) patients (Table S5) were thawed as described and CD34+ blasts were enriched with Direct CD34+ Progenitor Isolation Kit (Miltenyi) or sorted by FACS.

Techniques: Expressing, Control, Transplantation Assay, Cell Cycle Assay, Transduction

Journal: Cell reports

Article Title: Identification of the global miR-130a targetome reveals a role for TBL1XR1 in hematopoietic stem cell self-renewal and t(8;21) AML.

doi: 10.1016/j.celrep.2022.110481

Figure Lengend Snippet: Figure 3. Mass spectrometry and chimeric AGO2 eCLIP identify miR-130a targetome in human HSPCs (A) Heatmap of downregulated pathways and proteins following miR-130a OE in CD34+ CB cells; Wilcoxon one-sided test, n = 3, p < 0.05. (B) List of miR-130a downregulated proteins that are predicted TarBase and mirDIP targets, limma t test. (C) Western blot showing repression of miR-130a targets. (D) Enrichment map showing gene sets containing miR-130a targets listed in (B); node size is proportional to NES; Wilcoxon one-sided test, p < 0.05. (E) GSEA plots of miR-130a targets from chimeric AGO2 eCLIP in proteome profile following miR-130a OE in CD34+ HSPCs. (F) Genome browser tracks from chimeric AGO2 eCLIP-seq in CD34+ HSPCs. (G) Enrichment map of miR-130a targets from chimeric AGO2 eCLIP in downregulated protein sets following miR-130a OE; false discovery rate (FDR) < 0.05, node size is proportional to NES, Wilcoxon one-sided test; p < 0.05.

Article Snippet: Quantitative RT-PCR for expression levels of miR-130a in CBF AML Peripheral blood samples from t(8;21) and inv(16) patients (Table S5) were thawed as described and CD34+ blasts were enriched with Direct CD34+ Progenitor Isolation Kit (Miltenyi) or sorted by FACS.

Techniques: Mass Spectrometry, Western Blot

Journal: Cell reports

Article Title: Identification of the global miR-130a targetome reveals a role for TBL1XR1 in hematopoietic stem cell self-renewal and t(8;21) AML.

doi: 10.1016/j.celrep.2022.110481

Figure Lengend Snippet: Figure 4. Repression of TBL1XR1 impairs B lymphoid differentiation and expands LT-HSC (A) Western blot showing TBL1XR1 protein levels in xenografts. (B) Human CD45+ chimerism in the right femur (RF) and bone marrow (BM) at 24 weeks post-transplantation (n = 2 biological experiments, 6–8 mice/experimental group). (C) Fold change in BFP+CD45+ cells in RF and BM at 24 weeks post-transplantation compared with input levels. (D) Lineage distribution of BFP+ xenografts. (E) Flow cytometry plots of HSPC populations, flow plots are representative of 3 samples overlaid together. (F) Distribution of BFP+CD34+CD38 cell populations from 24 week xenografts (n = 5, each replicate contains pooled RF from 2 to 4 individual mice), unpaired t test, *p < 0.05. (G) Secondary transplantation of CD45+BFP+ from 24 week NSG mice at limiting dilution doses. Frequency of HSC was evaluated from the engraftment in NSG- GF secondary mice (>0.05% CD45+BFP+cell in BM, n = 11–16 mice/experimental group). (H) Enrichment map of upregulated (red nodes) and downregulated (blue nodes) pathways following TBL1XR1 KD intersected with miR-130a targets from eCLIP, hypergeometric t test, p < 0.05. (I) GSEA plots of proliferative and quiescence genes in transcriptome profile following TBL1XR1 KD. (J) Western blot showing protein levels of NCoR1 and TBL1XR1 following TBL1XR1 KD in CD34+ CB cells. (K) GSEA plots of genes upregulated and downregulated by RA in the transcriptome profile following TBL1XR1 KD. (B–D) Unpaired Mann-Whitney U test, all error bars indicate ±SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Quantitative RT-PCR for expression levels of miR-130a in CBF AML Peripheral blood samples from t(8;21) and inv(16) patients (Table S5) were thawed as described and CD34+ blasts were enriched with Direct CD34+ Progenitor Isolation Kit (Miltenyi) or sorted by FACS.

Techniques: Western Blot, Transplantation Assay, Flow Cytometry, MANN-WHITNEY

Journal: Cell reports

Article Title: Identification of the global miR-130a targetome reveals a role for TBL1XR1 in hematopoietic stem cell self-renewal and t(8;21) AML.

doi: 10.1016/j.celrep.2022.110481

Figure Lengend Snippet: Figure 5. Loss of function of miR-130a and TBL1XR1 OE induces differentiation of t(8;21) AML (A) Clustering of TCGA AMLs into four groups based on scRNA-seq data (n = 173). (B) Expression of miR-130a in AML subtypes from the TCGA dataset (n = 170). (C) Kaplan-Meier curve showing correlation between miR-130a expression and CBF AML patient survival (n = 48). (D) qRT-PCR showing expression levels of miR-130a in CD34+ blasts from t(8;21) AML compared with healthy controls (n = 20) and CD34 cells from the same patient samples (n = 24).

Article Snippet: Quantitative RT-PCR for expression levels of miR-130a in CBF AML Peripheral blood samples from t(8;21) and inv(16) patients (Table S5) were thawed as described and CD34+ blasts were enriched with Direct CD34+ Progenitor Isolation Kit (Miltenyi) or sorted by FACS.

Techniques: Expressing, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-1 was located on the upstream of miR-23a∼27a∼24-2 cluster and activated its expression during erythropoiesis. ( A ) Q-PCR analysis of the primary transcript of the miR-23a∼27a∼24-2 cluster in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( B ) Q-PCR analysis of the mature transcripts of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( C ) Northern blot analysis of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation. U6 snRNA was used as a loading control. ( D ) Q-PCR analysis of mature miR-27a and miR-24 expression in CD34+ HPCs undergoing day 4, 7, 11, 15 and 18 of E culture. ( E ) Representation of the human −603 bp miR-23a∼27a∼24 cluster promoter fragments. ( F ) ChIP-PCR and ChIP-qPCR analysis of the GATA-1 hit on the −557 site at the miR-23a∼27a∼24 cluster promoter in K562s. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01. ( G ) Immunoblot analysis of GATA-1 expression in K562s treated with siRNAs specific to GATA-1 (si_GATA-1) for 48 h or K562s transfected with a construct overexpressing GATA-1 (over_GATA-1) for 48 h, respectively. ( H ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (G). ( I ) Immunoblot analysis of GATA-1 expression in K562s undergoing erythroid differentiation. ( J ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( K ) Functional activity of GATA-1 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Northern Blot, Control, ChIP-qPCR, Standard Deviation, Western Blot, Transfection, Construct, Functional Assay, Activity Assay, Mutagenesis, Luciferase

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: MiR-27a and miR-24 promoted erythroid differentiation in CD34+ HPCs. ( A ) Monitoring of the GFP + population (left panel) and the CD235a stained GFP + fraction (medium panel) of Lenti-miRNA-transduced CD34+ HPCs on day 15 of E culture. The morphology (May-Grunwald Giemsa staining) of CD34+ HPCs derivatives on day 15 is shown in the right panel. A 400X magnification of a representative field is shown. ( B ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. Percentages of basophilic (Bas), polychromatophilic (Pol), orthochromatic (Ort) erythroblasts and erythrocytes (Ery) were determined by May-Grunwald/Giemsa staining of cytospin preparations. ( C ) Q-PCR analysis of gamma-globin mRNA expression in CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. ( D ) A comparison of the erythroid colony-forming capacity (CFU-E, BFU-E) of CD34+ HPCs transduced with Lenti-miRNAs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) FACS monitoring of CD34+ HPCs transduced with Zip-miRNA or Zip-GFP as described in (A). ( F ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Zip-27a, Zip-24 or Zip-GFP as described in (B). ( G ) Detection of gamma-globin mRNA level in CD34+ HPCs transduced with Zip-miRNA. ( H ) Colony-forming assay of CD34+ HPCs transduced with Zip-miRNA as described in (D).

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Staining, Transduction, Expressing, Comparison, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-2 was post-transcriptionally regulated by miR-27a and miR-24 during erythropoiesis. ( A ) A computer prediction of conserved and mutated binding sites within the 3′ UTR of GATA-2 mRNA for miR-27a and miR-24. ( B ) Relative luciferase activity of the indicated GATA-2 reporter constructs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( C ) Immunoblot analysis of GATA-2 in K562s transfected with scramble or miRNA mimics (miR-27a, miR-24) or inhibitors (Anti-27a, Anti-24). ( D ) Immunoblot analysis of GATA-2 in CD34+ HPCs transduced with Lenti-GFP control and lentivirus expressing miR-27a or miR-24 (Lenti-27a or Lenti-24). ( E, F ) ‘Rescue’ assays for miRNAs and GATA-2 during erythroid differentiation. Immunoblot analysis of GATA-2 in K562s treated with scramble or Anti-27a/24 (E) for 24 h. These cells were subsequently treated for another 24 h with control siRNAs or siRNAs specific to GATA-2 and were then treated with hemin treatment for 0, 48 and 72 h. (F) FACS analysis of K562s stained for CD71 and CD235a expression after 48 h of hemin induction as described earlier in the text.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Standard Deviation, Western Blot, Transfection, Transduction, Control, Expressing, Staining

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: The GATA switch regulated miR-27a and miR-24 expression. ( A ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1, GATA-2 and Pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( B ) Immunoblot analysis of GATA-1 and GATA-2 expression in K562s undergoing erythroid differentiation for 0, 24, 48 and 72 h. ( C ) Functional activity of GATA-1 and GATA-2 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( D ) Immunoblot analysis of GATA-2 in K562s treated with siRNAs specific to GATA-2 (si_GATA-2) for 48 h or K562s transfected with a construct overexpressing GATA-2 (over_GATA-2) for 48 h, respectively. ( E ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (D). ( F ) ChIP-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with control siRNAs or siRNAs specific to GATA-1/GATA-2 or K562s transfected with empty pcDNA3.1 vectors or pcDNA3.1_GATA-1/GATA-2. ( G ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in CD34+ HPCs of E culture. ( H ) Q-PCR analysis of Pri-27a-24 abundance in CD34+ HPCs of E culture (Top panel). Error bars represent the standard deviation obtained from three independent experiments. An immunoblot analysis of GATA-1 and GATA-2 expression in CD34+ HPCs of E culture (Bottom panel). ( I ) Q-PCR (left panel) and immunoblot (right panel) analysis of GATA-1 and GATA-2 expression in CD34+ HPCs transduced with lentivirus-expressing siRNAs against GATA-1 or control on day 11 of E culture. Error bars represent standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( J ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in CD34+ HPCs treated as described in (I). Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Standard Deviation, Western Blot, Functional Assay, Activity Assay, Mutagenesis, Luciferase, Transfection, Construct, Control, Transduction

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: A regulatory circuit involving GATA-1, GATA-2 and miR-27a/24 in erythropoiesis. ( A ) A schematic representation of the regulatory circuit comprised GATA-1, GATA-2, miR-27a and miR-24 in erythroid differentiation. ( B , C ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with scramble or miRNA mimics and inhibitors (B, r miR-27a and C, miR-24). ( D ) Q-PCR analysis of Pri-27a∼24 abundance in K562s treated as described in (B) and (C). Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Lenti-miR-27a/24 or Lenti-GFP control for 11 days. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( F ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Zip-miRNA or Zip-GFP for 11 days. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Transfection, Standard Deviation, Transduction, Control