Although the total number of B220+ B cell lineage cells was only slightly reduced in BCL6?/? compared with wild-type bone marrow, the frequency of IgM+ and light chain+ immature B cells was significantly diminished (Fig. a transcriptional repressor (Chang et al., 1996; Seyfert et al., 1996; Shaffer et al., 2000) in normal and malignant germinal center (GC) B cells, and belongs to the BTB/POZ (Bric–brac, tramtrack, broad complex/Pox virus zinc finger) zinc finger family of proteins. In diffuse large B cell lymphomas (DLBCLs), is frequently translocated into the Ig heavy or light chain loci (e.g., t(3;14)(q27;q32); Ye et al., 1993). During normal B cell development, BCL6 expression was only found in GC B cells (Cattoretti et al., 1995; Allman et al., 1996), in which BCL6 is critical for survival and proliferation. In the absence of BCL6, GC formation is abrogated (Dent et al., 1997; Ye et al., 1997). This is mainly attributed to the central negative regulatory effect of BCL6 on DNA damage response genes in GC B cells (Ranuncolo et al., 2007). Through somatic hypermutation and DNA double-strand break (DSB) events resulting from class-switch recombination in GCs combined with replication errors owing to a high proliferation rate, GC B cells are exposed to a high level of DNA damage stress (Schlissel et al., 2006; Liu et al., 2008). Therefore, the ability of BCL6 to suppress DNA damage response and checkpoint genes (Shaffer et al., 2000; Shvarts et al., 2002; Phan and Dalla-Favera, 2004; Phan et al., 2005, Ranuncolo et al., 2008) as well as the DNA damage sensor ATR (Ranuncolo et al., 2007) is essential for GC B cell proliferation and survival. Extensive DNA damage not only occurs in GCs but also CTX 0294885 during early B cell development in the bone marrow (Schlissel CTX 0294885 et al., 2006). However, previous studies focused on the function of BCL6 within GCs, and a role of BCL6 in early B cell development was not examined in detail. Non-GC B cells, such as preCB cells, sustain DNA damage owing to DNA DSBs during V(D)J recombination and replication errors linked to their high proliferation rate. In preCB cells, DNA DSBs during V(D)J recombination first target one DH and JH and then multiple VH segments. This is followed by V-J gene rearrangement and potentially multiple additional rearrangements targeting the -deleting element (ranked first in the analysis (Fig. 1 B). Of note, the protooncogene was among the genes on the opposite extreme of this analysis. Silencing of and de novo expression CTX 0294885 of upon inhibition of IL-7 or BCR-ABL1 signaling was confirmed at the protein level by Western blot analysis and correlated with STAT5 dephosphorylation at Y694 (Fig. 1 C). BCL6 is expressed at very high levels in GC B cells and serves a critical role in GC B cell survival (Dent et al., 1997; CTX 0294885 Ye et al., 1997; Phan and Dalla-Favera, 2004). Likewise, BCL6 functions as a protooncogene in DLBCL cells, where it is often expressed at very high levels owing to the translocation (t(3;14)(q27;q32); Ye CTX 0294885 et al., 1993). For these reasons, we studied BCL6 protein levels in preCB cells upon IL-7 withdrawal as compared with GC B cells and DLBCLs by Western blotting (Fig. 1 D). Of note, withdrawal of IL-7 resulted in dramatic up-regulation of BCL6 protein expression, which reached levels comparable to both DLBCLs and GC B cells. Open Rabbit Polyclonal to EPB41 (phospho-Tyr660/418) in a separate window Figure 1. Regulation of BCL6 during inducible preCB cell differentiation. (A) IL-7Cdependent and BCR-ABL1Ctransformed preCB cells were induced to differentiate by withdrawal of 10 ng/ml IL-7 and ABL1 kinase inhibition (2 mol/liter STI571), respectively. Cell size (FSC) and light chain surface expression were monitored by flow cytometry (= 5). Numbers indicate percentages. (B) To identify genes that are differentially regulated during induced preCB cell differentiation, we studied preCB cells stimulated to differentiate in a microarray analysis. Genes were sorted based on the ratio of gene expression values observed upon withdrawal of IL-7 from IL-7Cdependent preCB cells. (C) Likewise, protein lysates from preCB cells in the presence or absence of induced differentiation (treatment with 10 mol/liter STI571 or withdrawal of IL-7 for 24 h) were analyzed by Western blotting using antibodies against STAT5, phosphorylated STAT5 at Y694, BCL6 (clone N3), MYC, and an ACTB antibody as loading control (= 6). The asterisk denotes a nonspecific band that is consistently observed with the N3 BCL6 antibody. Of note, BCR-ABL1 kinase signaling results in stronger STAT5 tyrosine phosphorylation at.