Compound haploinsufficiencies of Ebf1 and Runx1 genes impede B cell lineage progression

Abstract

Early B cell factor (EBF)1 is essential for B lineage specification. Previously, we demonstrated the synergistic activation of Cd79a (mb-1) genes by EBF1 and its functional partner, RUNX1. Here, we identified consequences of Ebf1 haploinsufficiency together with haploinsufficiency of Runx1 genes in mice. Although numbers of "committed" pro-B cells were maintained in Ebf1(+/-)Runx1(+/-) (ER(het)) mice, activation of B cell-specific gene transcription was depressed in these cells. Expression of genes encoding Aiolos, kappa0 sterile transcripts, CD2 and CD25 were reduced and delayed in ER(het) pro-B cells, whereas surface expression of BP-1 was increased on late pro-B cells in ER(het) mice. Late pre-B and immature and mature B cells were decreased in the bone marrow of Ebf1(+/-) (E(het)) mice and were nearly absent in ER(het) mice. Although we did not observe significant effects of haploinsuficiencies on IgH or Igkappa rearrangements, a relative lack of Iglambda rearrangements was detected in E(het) and ER(het) pre-B cells. Together, these observations suggest that B cell lineage progression is impaired at multiple stages in the bone marrow of E(het) and ER(het) mice. Furthermore, enforced expression of EBF1 and RUNX1 in terminally differentiated plasmacytoma cells activated multiple early B cell-specific genes synergistically. Collectively, these studies illuminate the effects of reduced Ebf1 dosage and the compounding effects of reduced Runx1 dosage. Our data confirm and extend the importance of EBF1 in regulating target genes and Ig gene rearrangements necessary for B cell lineage specification, developmental progression, and homeostasis.

Fig. 1.

Development of bone marrow B cells in WT and haploinsufficient mice. All mice were 4–6 weeks old (n ≥ 5 per group). (A) Numbers of B220⁺ splenocytes as determined by flow cytometry. (B) Numbers of B220⁺CD19⁺ bone marrow cells as determined by flow cytometry. (C–F) Representative flow cytometry assessing (C) CD117 (c-kit) and CD19 expression on IgM^– bone marrow cells, (D) BP-1 and CD25 expression on CD19⁺IgM^– bone marrow cells, (E) B220 and IgM expression on CD43^– bone marrow cells, and (F) IgM and IgD expression on CD19⁺ bone marrow cells.

Fig. 2.

Expression of stage-specific markers in CD117⁺CD19⁺mIgM^– and CD117^–CD19⁺mIgM^– bone marrow cells of WT and haploinsufficient mice. (A) Quantitative PCR (qRT-PCR) of transcripts in CD117⁺CD19⁺mIgM^– “committed” pro-B cells (n = 3–5 mice per group). All transcript levels were normalized to levels of β-actin transcripts. (B) qRT-PCR of transcripts in CD117^–CD19⁺mIgM^– late pro-B/total pre-B cells (n = 3–5 mice per group). All transcript levels were normalized to levels of Hprt transcripts. (C) Analysis of CD2 and CD19 expression on B220⁺IgM^– cells by flow cytometry.

Fig. 3.

Ig λ light chain gene rearrangements are reduced by Ebf1 haploinsufficiency. (A) Frequencies of Igκ and Igλ gene rearrangements in pre-B cells of WT and haploinsufficient mice. (B) Reduced frequencies of Igλ gene rearrangements including Jλ1, Jλ2, and Jλ3 in pre-B cells of E^het and ER^het mice (n = 3 mice per group).

Fig. 4.

Enforced expression of EBF1 and RUNX1 activates expression of early B cell-specific genes in plasmacytoma cells. μM.2 plasmacytoma cells were transduced with retroviruses for expression of EBF1 and/or RUNX1 as shown. Transduced cells were purified 48 h postinfection and analyzed using qRT-PCR. Data were normalized to control Hprt transcripts and represent three to five independent experiments.