miR-195 promotes HPCs to differentiate into the pro-B cell stage without EBF1.

(A) Endogenous expression levels of miR-195 were measured by means of quantitative RT-PCR. RNAs were collected from the following hematopoietic lineage cells in 8-week-old C57BL/6 mice. KSL (c-kit+ Sca1+ Lin−); lymphoid-primed multipotent progenitors (LMPP: C-kit+ Sca1+ Lin− Flt3+ IL7Rα−); common lymphoid progenitors (CLP: CD43− IL7Rα+); pro-B (B220+ CD43+ IgM−); pre-B (B220+ CD43− IgM−); mature B (B220high CD43− IgM+); double-negative T (DN: CD3+ CD4− CD8−); common myeloid progenitor (CMP: c-kit+ Sca1− CD11b+); myeloid cell (Mac1: c-kit− Sca1− CD11b+). Data are shown as mean ± S.D. (n=3). (B) Flow cytometry analysis of control and miR-195-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 miR-195 retrovirus. Representative result of control (upper panel) and miR-195 (lower panel) viral infections is shown (n=3). (C) Outline of the in vitro culture system of Ebf1−/− HPCs. (D) Flow cytometry analysis of control and miR-195-expressing Ebf1−/− HPCs. Shown data is representative of n=3. (E) Microarray analysis of miR-195-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.

miR-195 leads Ebf1-deficient 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 miR-195-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 miR-195-expressing Ebf1−/− HPCs. (D) Flow cytometry analysis of control and miR-195-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 miR-195-expressing Ebf1−/− HPCs were cultured for 72 hrs with IgG1 class-switch stimuli, LPS, and IL-4. Each flow cytometric data is representative of n=3.

Several B cell populations are disturbed in the miR-195-deficient mouse.

Flow cytometry data of B cell lineage populations in miR-195−/− and littermate WT mice. Representative plots (left side) and mean ± S.D. of relative population rates in each littermate WT mouse (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 two-tailed Student’s t test. *p<0.05; **p<0.01. WT, wild-type.

FOXO1 phosphorylation pathways are key targets of miR-195 for promotion of B cell development.

(A) Relative expression rate of miR-195 and predicted target genes were compared between control (EMPTY) and miR-195-expressing Ebf1−/− HPCs. (B) Relative luciferase inhibitory rates of miR-195 onto predicted target 3′-UTR were analyzed using Dual-Luciferase® reporter assay. (C) Western blot of FOXO1 and phosphorylated FOXO1 (pFOXO1) in control and miR-195-expressing Ebf1−/− HPCs. 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. Statical significance was tested using two-tailed Student’s t test. *p<0.05, n=3.

ATAC-seq analysis of Ebf1−/− CD19-positive B cells differentiated by miR-195.

(A) Outline of analysis of open chromatin regions in miR-195-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 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). (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).