Runx1-Cbfβ facilitates early B lymphocyte development by regulating expression of Ebf1 - PubMed (original) (raw)

Runx1-Cbfβ facilitates early B lymphocyte development by regulating expression of Ebf1

Wooseok Seo et al. J Exp Med. 2012.

Abstract

Although Runx and Cbfβ transcription factor complexes are involved in the development of multiple hematopoietic lineages, their precise roles in early mouse B lymphocyte differentiation remain elusive. In this study, we examined mouse strains in which Runx1, Runx3, or Cbfβ were deleted in early B lineage progenitors by an mb1-cre transgene. Loss of Runx1, but not Runx3, caused a developmental block during early B lymphopoiesis, resulting in the lack of IgM(+) B cells and reduced V(H) to DJ(H) recombination. Expression of core transcription factors regulating early B cell development, such as E2A, Ebf1, and Pax5, was reduced in B cell precursors lacking Runx1. We detected binding of Runx1-Cbfβ complexes to the Ebf1 proximal promoter, and these Runx-binding motifs were essential to drive reporter gene expression. Runx1-deficient pro-B cells harbored excessive amounts of the repressive histone mark H3K27 trimethylation in the Ebf1 proximal promoter. Interestingly, retroviral transduction of Ebf1, but not Pax5, into Runx1-deficient progenitors restored not only development of B220(+) cells that underwent V(H) to DJ(H) rearrangement but also expression of B lineage signature genes. Collectively, these results demonstrate that Runx1-Cbfβ complexes are essential to facilitate B lineage specification, in part via epigenetic activation of the Ebf1 gene.

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Figures

Figure 1.

Figure 1.

Defective early B lymphocyte development caused by conditional inactivation of Runx1 or Cbfβ genes. (A–C) Dot plots showing IgM and CD19 expression profiles in splenocytes (SPL; A) and IgM and B220 or CD43 and B220 expression profiles in total bone marrow (B) from mice of the indicated genotypes. In C, BP-1 and HSA expression profiles in CD43+ B220+ bone marrow cells are shown. (D) Absolute numbers of spleen B220+, bone marrow B220+, CD43− B220hi immature B, CD43− B220int pre-B, and CD43+ B220int pro-B cells from each mouse strain are shown. Each symbol represents one mouse. (E) PCR analysis of _VH_-DJH (VH558 or VH7183 families) and _DH_-JH rearrangement in sorted pro-B cell populations from Runx1+/F;mb1-cre and Runx1F/F;mb1-cre mice. The ThPOK silencer region was used as the loading control (Cont.). (F) Dot plots showing CD43 and B220 expression profiles in bone marrow from CbfβF/F;mb1-cre and CbfβF/−;mb1-cre mice (top). The bottom graph shows the percentages of CD43− B220hi immature B and CD43− B220int pre-B cells in bone marrow of CbfβF/F;mb1-cre and CbfβF/−;mb1-cre mice. (G) Deletion efficiencies of floxed alleles in the indicated cells are shown. Controls are Runx1F/− or CbfβF/− genotypes. (A–F) Representative data of four (A–E) or three (F) independent experiments are shown.

Figure 2.

Figure 2.

Direct activation of the Ebf1 proximal β promoter by Runx1. (A) Real-time PCR analysis showing messenger RNA (mRNA) expression levels of several B cell signature genes in subsets of pro-B cells (HSA+ BP-1−, HSA+ BP-1+, and HSAhi BP-1+ in the order of development) from Runx1F/F;mb-1-cre mice. Data were normalized to HPRT and are shown as fold changes to wild-type control. (B) A ChIP-on-chip assay was performed with anti-Cbfβ antibody to evaluate Runx–Cbfβ binding to the promoter regions of E2a, Pax5, and Ebf1 genes in B220+ bone marrow cells. Positions of putative RRSs within the Ebf1 proximal β promoter are indicated. One representative of two independent experiments is shown. (C) Analytical ChIP assay using B220+ bone marrow cells. The mb1 promoter was used as a positive control for Cbfβ binding. One representative result from three independent experiments is shown. (D) Schematic overview of the Ebf1 proximal β promoter is shown with three predicted RRSs. +1 indicates the transcription start site. The top panel shows the structure of each reporter construct, and the bottom panel indicates relative luciferase activity from each construct in a transfection assay in the Ba/F3 cell line. Values are shown in relative light units (RLU). (E) Relative H3K4Me3 (K4) and H3K27Me3 (K27) histone modification levels at the Ebf1 proximal β promoter, the mb1 promoter, and ThPOK silencer regions in Runx1-deficient pro-B cells relative to wild-type pro-B cells. Data are represented as relative fold changes as in A. (A, D, and E) Error bars represent mean ± SD of three independent experiments.

Figure 3.

Figure 3.

Rescue of B cell development from Runx1-defcient progenitors by EBF1 transduction. Purified hematopoietic progenitors, which were enriched by negative selection of lineage markers from the indicated mouse strains, were co-cultured with TSt-4 stromal cells for 3 d after transduction with retrovirus encoding GFP alone, Ebf1-IRES-GFP, or Pax5-IRES-GFP. GFP+ cells were sorted and further co-cultured with TSt-4 stromal cells to facilitate B cell differentiation. (A and B) At 7 or 14 d after cell sorting, cells were analyzed for surface expression of CD19 and MacI by flow cytometry (A) and the absolute numbers of B220+ cells (B). Dot blots in A are data at 14 d. (C–E) At day 17, VH558 to JH3, VH7183 to JH3, and DH to JH3 joining (the Thpok silencer region was used as the loading control [Cont.]; C), expressions of genes known to be important for B cell development (D), and endogenous Ebf1 expression relative to Runx1F/F cells transduced with GFP vector (E) were measured in purified B220+ cells. One representative result from two independent transduction experiments with duplicate is shown.

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