A single microRNA miR-195 rescues the arrested B cell development induced by EBF1 deficiency
- Department of Innovative Medical Science, Tokai University School of Medicine, Japan
- Department of Regulation of Infectious Cancer, Research Institute for Microbial Diseases, The University of Osaka, Japan
- Division of Immunology and Allergy, Research Institute for Biomedical Sciences, Tokyo University of Science, Japan
- Department of Immunology, Graduate School of Medicine, Chiba University, Japan
- Department of Experimental Immunology, Graduate School of Medicine, Chiba University, Japan
- Laboratory for Transcriptional Regulation, RIKEN Center for Integrative Medical Sciences, Japan
- Department of Immunology, Tokai University School of Medicine, Japan
- Department of Molecular Life Science, Tokai University School of Medicine, Japan
- Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Japan
- Department of Gastroenterology and Hepatology, Okayama University Hospital, Japan
- Project for Cancer Epigenomics, Cancer Institute, Japanese Foundation for Cancer Research, Japan
- Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Japan
- Laboratory of Lymphocyte Differentiation, WPI Immunology Frontier Research Center, Osaka University, Japan
- Laboratory for Infectious Disease Education and Research, Osaka University, Japan
- Laboratory for Lymphocyte Differentiation, RIKEN Center for Integrative Medical Sciences, Japan
- Laboratory of Immunology, Institute for Life and Medical Sciences, Kyoto University, Japan
Figures
Figure 1
Mir195 promotes hematopoietic progenitor cells (HPCs) to differentiate into the pro-B cell stage without EBF1.
(A) Flow cytometry analysis of control and Mir195-expressing Lin– cells. HPCs from fetal livers of wild-type mice were cultured for 7 days on OP9 with SCF, Flt3-ligand, and IL-7, after infection with control or Mir195 retrovirus. Representative result of control (upper panel) and Mir195 (lower panel) viral infections is shown (n=3). (B) Outline of the in vitro culture system of Ebf1-/- HPCs. (C) Flow cytometry analysis of control and Mir195-expressing Ebf1-/- HPCs. Shown data is representative of n=3. (D) Microarray analysis of Mir195-expressing Ebf1-/- HPCs. Log2 fold-changes in the expression levels of genes related to B (left panel), T (middle-upper panel), NK (middle-lower panel), and myeloid (right panel) cell lineages were classified and are shown as colored columns. The analysis was carried out in duplicates.
Figure 2 with 1 supplement
Mir195 leads Ebf1-deficient hematopoietic progenitor cells (HPCs) to mature into B cells with bone marrow niche assistance.
(A) In vivo analysis of B cell development of Ebf1-/- HPCs. (B) Flow cytometry analysis of control and Mir195-expressing Ebf1-/- HPCs in the bone marrow collected at 7 days after transplantation. (C) Using droplet digital PCR, VJ region fragments were amplified from the genomic DNA of B220+ cells in the bone marrow of mice transplanted with control and Mir195-expressing Ebf1-/- HPCs. (D) Flow cytometry analysis of control and Mir195-expressing Ebf1-/- HPCs in the bone marrow (BM) and spleen (SP), at 10 days after transplantation. (E) Flow cytometry analysis of class-switch recombination. Splenocytes of mice transplanted with control and Mir195-expressing Ebf1-/- HPCs were cultured for 72 hr with IgG1 class-switch stimuli, LPS, and IL-4. Each flow cytometric data is representative of n=3.
Figure 2—figure supplement 1
Raw data of Figure 2C droplet digital PCR (ddPCR).
Amplitude (A) and concentration (B) of ddPCR on VJ recombination.
Figure 3
Several B cell populations are disturbed in the Mir195-deficient mice.
Flow cytometry data of B cell lineage populations in Mir195-/- and littermate WT mice. Representative plots (left side) and mean ± SD of relative population rates in each littermate WT mice (right side) are shown. (A) Analysis of early B cell populations in the bone marrow. Pre-pro-B (B220+ IgM- CD43+ CD19-); pro-B (B220+ IgM– CD43+ CD19+); pre-B (B220+ IgM- CD43- CD19+); n=5. (B) Analysis of hematopoietic progenitor populations in the bone marrow; n=5. (C) Analysis of B cell populations in the spleen. FO B (CD19+ IgM+ CD21/35low-middle); MZ B (CD19+ IgM+ CD21/35high); n=8. (D) Analysis of B cell populations in the peritoneal cavity: B-1 (B220+ CD11b+); B-2 (B220+ CD11b-); n=7. Statistical significance was tested using one-sample t-test. *p<0.05; **p<0.01. WT, wild-type.
Figure 4 with 1 supplement
FOXO1 phosphorylation pathways are key targets of Mir195 for promotion of B cell development.
(A) Relative luciferase inhibitory rates of Mir195 onto predicted target 3′UTR were analyzed using Dual-Luciferase reporter assay. (B) Relative expression rate of Mir195 and predicted target genes were compared between control (EMPTY) and Mir195-expressing Ebf1-/- hematopoietic progenitor cells (HPCs). (C) Western blot of FOXO1 and phosphorylated FOXO1 (pFOXO1) in control and Mir195-expressing Ebf1-/- HPCs. Quantification of FOXO1 and phospho-FOXO1 band intensities from three independent experiments is shown in the bar graph. Data are presented as mean ± SD. Shown data is representative of n=3. (D) Flow cytometry analysis of control and Foxo1-expressing Ebf1-/- HPCs. Shown data is representative of n=3. Statistical significance was tested using a one-sample t-test. *p<0.05, n=3.
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Figure 4—source data 1
PDF file containing original western blots for Figure 4C, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/101510/elife-101510-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4C.
- https://cdn.elifesciences.org/articles/101510/elife-101510-fig4-data2-v1.zip
Figure 4—figure supplement 1
Western blot analysis of other known B-lineage regulators in Ebf1-/- hematopoietic progenitor cells (HPCs) with or without Mir195 transduction.
(A–B) PAX5 and β-actin western blot images (A). Three pairs of control (left) and Mir195 (right) transduced Ebf1-/- HPCs were analyzed. Quantification of β-actin and PAX5 band intensities from three independent samples is shown in the bar graph (B). Data are presented as mean ± SD, n=3. No significant difference in PAX5 protein level was observed. (C–D) ERG and β-actin western blot images (C). ERG was analyzed the same as PAX5, and quantification of β-actin and ERG band intensities is also shown in the bar graph (D). Data are presented as mean ± SD, n=3.
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Figure 4—figure supplement 1—source data 1
PDF file containing original western blots for Figure 4—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/101510/elife-101510-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/101510/elife-101510-fig4-figsupp1-data2-v1.zip
Figure 5
ATAC-seq analysis of Ebf1-/- CD19-positive B cells differentiated by Mir195.
(A) Outline of analysis of open chromatin regions in Mir195-expressing Ebf1-/- cells. (B) Venn diagram of numbers of genes in which DNA regions of open chromatin peaks were detected by means of peak call analysis. The analyses were examined between CD19-negative (FrA) and -positive (FrB, FrC, and FrD) stages of B cell development (GSE100738; upper red circle); wild-type (WT) and Ebf1-/- pro-B cells (GSE92434; left-lower blue circle); B220+ CD19- cells of control and B220+ CD19+-positive miR-195-expressing Ebf1-/- cells (right-lower green circle). Overlapping regions in the Venn diagram are interpreted as follows: the intersection of WT and Rescue represents canonical EBF1-regulated regions; the overlap between Rescue and Mir195 indicates partial mimicry by Mir195; and regions unique to Mir195 may reflect EBF1-independent chromatin changes. (C and D) Venn diagram of numbers of enriched known motifs detected using HOMER find motif analysis (C) and lists of high p-value motifs, up to rank 10 (D).
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/101510/elife-101510-mdarchecklist1-v1.pdf
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Supplementary file 1
Lists of high-p-value pathways detected by means of targetome analysis of human Mir195 with TargetScan and starBase in the KEGG pathway.
- https://cdn.elifesciences.org/articles/101510/elife-101510-supp1-v1.zip
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Supplementary file 2
Lists of high-p-value pathways detected by means of targetome analysis of murine Mir195 with TargetScan and starBase in the KEGG pathway.
- https://cdn.elifesciences.org/articles/101510/elife-101510-supp2-v1.zip
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Supplementary file 3
List of genes with detected peaks in the DNA region common in the three analyses.
- https://cdn.elifesciences.org/articles/101510/elife-101510-supp3-v1.zip
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Supplementary file 4
Targetome analysis of human Mir195 with TargetScan.
- https://cdn.elifesciences.org/articles/101510/elife-101510-supp4-v1.zip
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Supplementary file 5
Targetome analysis of murine Mir195 with TargetScan.
- https://cdn.elifesciences.org/articles/101510/elife-101510-supp5-v1.zip
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A single microRNA miR-195 rescues the arrested B cell development induced by EBF1 deficiency
eLife 13:RP101510.
https://doi.org/10.7554/eLife.101510.3
