Epigenetic silencing of miR-125b is required for normal B-cell development

Abstract

Deregulation of several microRNAs (miRs) can influence critical developmental checkpoints during hematopoiesis as well as cell functions, eventually leading to the development of autoimmune disease or cancer. We found that miR-125b is expressed in bone marrow multipotent progenitors and myeloid cells but shut down in the B-cell lineage, and the gene encoding miR-125b lacked transcriptional activation markers in B cells. To understand the biological importance of the physiological silencing of miR-125b expression in B cells, we drove its expression in the B-cell lineage and found that dysregulated miR-125b expression impaired egress of immature B cells from the bone marrow to peripheral blood. Such impairment appeared to be mediated primarily by inhibited expression of the sphingosine-1-phosphate receptor 1 (S1PR1). Enforced expression of S1PR1 or clustered regularly interspaced short palindromic repeats/Cas9-mediated genome editing of the miR-125b targeting site in the S1PR1 3' untranslated region rescued the miR-125b-mediated defect in B-cell egress. In addition to impaired B-cell egress, miR-125b dysregulation initially reduced pre-B-cell output but later induced pre-B-cell lymphoma/leukemia in mice. Genetic deletion of IRF4 was found in miR-125b-induced B-cell cancer, but its role in oncogenic miR-125b-induced B-cell transformation is still unknown. Here, we further demonstrated an interaction of the effects of miR-125b and IRF4 in cancer induction by showing that miR125b-induced B-cell leukemia was greatly accelerated in IRF4 homozygous mutant mice. Thus, we conclude that physiological silencing of miR-125b is required for normal B-cell development and also acts as a mechanism of cancer suppression.

Graphical abstract

Figure 1.

Epigenetic regulation of miR-125b expression in B lymphocytes. (A) Relative miR-125b expression in the bone marrow, spleen, and various immune cells. The expression of miR-125b-5p and miR-125b-1-3p was measured using TaqMan reverse transcription qPCR (n = 3). (B) Relative miR-125b expression in HSPCs (L⁻S⁺K⁺) and various B-lymphocyte subsets. (C) Modification patterns of H3K14Ac, H3K36Ac, H3K4me3, H3K79me2, H4K91ac, and H3K27me3, as well as PolII and p300 recruitment in the loci encoding miR-125b and miR-15a/16-1 in resting mature B cells. IL, interleukin; LPS, lipopolysaccharide.

Figure 2.

Reduced number of B cells in peripheral tissues of Eμ/miR-125b-Tg mice. (A) Frequency of B220⁺ B cells in peripheral tissues from 8-week-old Eμ/miR-125b-Tg and control mice. Lymphocytes from the bone marrow, peripheral blood, and spleen and PLNs from control and Eμ/miR-125b-Tg mice were analyzed by flow cytometry. (B) Total number of leukocytes (CD45⁺) and B cells (B220⁺), T cells (CD4⁺ or CD8⁺), or myeloid cells (CD11b⁺) counted in each organ or per microliter of peripheral blood. Bars represent mean values of pooled data. Data are pooled from 2 to 3 experiments and represented as mean ± SEM (n = 10-12 mice per group). LN, lymph node. *P < .05; **P < .01; ***P < .001; NS, not significant.

Figure 3.

Reduced number of immature B cells in the blood of Eμ/miR-125b-Tg mice. (A) Representative flow cytometric analysis of Hardy fractions for B cell progenitor subpopulations in the bone marrow of 8-week-old Eμ/miR-125b-Tg and control mice. Hardy fractions in bone marrow (A-F) were gated as follows: fraction A, pre/pro-B cell (B220⁺CD43⁺BP-1⁻CD24⁻); fraction BC, pro-B cells (B220⁺CD43⁺BP-1⁻CD24⁺ and B220⁺CD43⁺BP-1⁺CD24⁺); fraction D, pre-B cells (B220⁺CD43⁻IgM⁻IgD⁻); fraction E, immature B cells (B220⁺CD43⁻IgM⁺IgD⁻); and fraction F, mature B cells (B220⁺CD43⁻IgM⁺IgD⁺); the C′ fraction (B220⁺CD43⁺BP-1⁺CD24^hi) was not resolved. (B) The frequency and number of each B-cell subpopulation in Eμ/miR-125b-Tg and control mice. (C) Representative flow cytometric analysis of immature and mature B-cell populations in peripheral blood of 8-week-old Eμ/miR-125b-Tg and control mice. Immature B cells were identified as B220⁺IgD^lowIgM^high, B220⁺CD93⁺, and B220⁺CD62L^low, and mature B cells were identified as B220⁺IgD^highIgM^low, B220⁺CD93⁻, and B220⁺CD62L^high. (D) The number and frequency of immature B cells (B220⁺CD93⁺) in the blood of Eμ/miR-125b-Tg and control mice. Bars represent mean values of pooled data. Data are pooled from 2 to 3 experiments and represent mean ± SEM (n = 8 mice per group). *P < .05; ***P < .001; NS, not significant.

Figure 4.

Impaired release of immature B cells in Eμ/miR-125b-Tg mice. (A) Normal proliferation of bone marrow B cells in Eμ/miR-125b-Tg mice. 8-week-old Eμ/miR-125b-Tg and control mice were injected with BrdU for 48 hr. Bone marrow cells from these mice were stained with anti-B220 antibodies in combination with BrdU detection methodology. Percentages in the bar graph indicate BrdU-positive cells in the gated B200⁺ cell subpopulations. (B) Distribution and number of the indicated B-lineage populations in the bone marrow parenchyma (CD45.2⁻) and sinusoids (CD45.2⁺) of 8-week-old Eμ/miR-125b-Tg and control mice. (C) Representative flow cytometric analysis of bone marrow B cells for staining of apoptotic B cells using anti-annexin-V and 7-AAD. (D) Number of total B cells (B220⁺), immature B cells (B220⁺CD93⁺), T cells (CD3⁺), and myeloid cells (CD11b⁺) in 300 μL blood from Eμ/miR-125b-Tg and control mice that were injected with the AMD3100 or phosphate-buffered saline alone (n = 5 mice per group). *P < .05; **P < .01; ***P < .001; NS, not significant.

Figure 5.

miR-125b inhibits B-cell release from the bone marrow through direct targeting of S1PR1. (A) Schematic representation of the S1PR1 3′ UTR showing the conserved miR-125b seed region. (B) S1PR1 transcript expression levels in B cells purified from the bone marrow of Eμ/miR-125b-Tg and control mice. (C) Relative luciferase expression in HEK293T cells transfected with luciferase reporter construct bearing the S1PR1 3′ UTR immediately downstream (S1PR1UTR) and either a miR-125b overexpression vector (MG-125b) or a control vector (MG). (D) S1pr1 protein expression levels in B cells purified from the bone marrow of Eμ/miR-125b-Tg and control mice. Data represent 2 independent experiments. (E) Enumeration of B cells (B220⁺) and immature B cells (B220⁺CD93⁺) in the peripheral blood of reconstituted mice at 7 weeks after reconstitution (n = 10 mice per group). (F) Schematic representation of small gRNA target (red) and protospacer adjacent motif (green) sequence designed to edit genomic sequence in the mouse S1PR1 3′UTR locus. (G) Detection of genome editing in the S1PR1 3′ UTR locus using a surveyor assay. (H) Enumeration of B cells (B220⁺) in peripheral blood of reconstituted mice at 7 weeks after reconstitution with CRISPR/Cas9-edited HSPCs (n = 10-12 mice per group). Data represent 2 independent experiments and represent mean ± SEM. *P < .05; ***P < .001; NS, not significant.

Figure 6.

miR-125b–induced B-cell leukemia is accelerated in IRF4-deficient mice, and enforced expression of IRF4 inhibits miR-125b–induced development of spontaneous B-cell cancers in mice. (A) Survival curve of Eμ/miR-125b-Tg mice, CD19cre IRF4^flox/flox Tg mice, double-Tg mice, and control mice. The genotypes and number of mice in each group are indicated on the plot. (B) Some Tg mice developed lymphoma. Lymphomas are shown at the superficial cervical or inguinal lymph node sites by red arrows. (C) Representative flow cytometric analysis of leukemic B cells in the blood, bone marrow, and spleen of moribund Eμ/miR-125b, CD19cre IRF4^flox/flox and double-Tg mice. (D) Survival curve of the secondary recipient mice transplanted with bone marrow cells from moribund Eμ/miR-125b-Tg mice, CD19cre IRF4^flox/flox Tg mice, double-Tg mice, and control mice (n = 6 mice per group). (E) Spleen weight of the secondary recipients described in panel D. (F) Frequency of bone marrow B cells (B220⁺) in mice receiving Eμ/miR-125b-Tg donor cells that were transduced with either an IRF4-expressing vector or a control vector. NS, not significant.