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. Author manuscript; available in PMC: 2014 Mar 14.
Published in final edited form as: Nat Med. 2013 Jul 14;19(8):1014–1022. doi: 10.1038/nm.3247

Figure 3. Bach2 mediates V(D)J recombination and μ-heavy chain checkpoint control during early B cell development.

Figure 3

(a) Using a classical pre-B cell differentiation model based on tyrosine kinase inhibition (Imatinib; IM) of BCR-ABL1-transformed pre-B cells12,13, gene expression changes upon inducible differentiation of Bach2-/- and Bach2+/+ pre-BI cells were measured by microarray analysis. (b) Increased expression of the early progenitor antigen Ly6f (Sca-1) and reduced expression of the pre-B cell antigen Il2rα (CD25) in Bach2-deficient Pre-BI cells was validated by flow cytometry. (c) Reduced mRNA levels of Rag1 and Rag2 in Bach2-deficient Pre-BI ALL (BCR-ABL1) cells in the presence and absence of IM were validated by qRTPCR. (d) The effect of inducible overexpression of Bach2 on Rag1 and Rag2 mRNA levels was measured by qRT-PCR. (e) Single-locus ChIP analysis using IgG and BACH2-specific antibodies depicting the binding of BACH2 to Rag1 and Rag2 promoters, which is further enhanced by IM-treatment.

(f) To test functional consequences of defective expression of Rag1/Rag2, Bach2+/+ and Bach2-/- pre-BI cells were transduced with a puromycin-selectable V(D)J recombination substrate carrying an inverted GFP flanked by recombination signal sequences (RSS). Recombination activity of the RSS substrate in Bach2+/+ and Bach2-/- pre-BI cells was measured by flow cytometry (inversion of the GFP cassette in the correct orientation; percentages of GFP+ cells are given) in the presence and absence of IM treatment. (g) Comparison of the composition of B cell progenitor populations in the bone marrow of Bach2+/+ and Bach2-/- mice by flow cytometry. Fragment length analysis revealed a size peak distribution of VH-DJH junctions that were indeed selected for multiples of 3 bp among Bach2+/+ compared to random distribution in Bach2-/- pre-B cells.

(h-i) IL7-dependent Bach2+/+ and Bach2-/- pre-B cells were transduced with tamoxifen (4-OHT)-inducible Bach2-ERT2 and ERT2 empty vector controls. Cells were treated with 4-OHT for 24 hours and sequence analysis of VH-DJH junctions was performed. (i) Amino acid sequences of VH-DJH junctions with non-functional VH-DJH rearrangements shaded in gray.