A Spic/BCLAF1 Complex Suppresses Pre-B Cell Receptor Signaling in Response to RAG-Mediated DNA Breaks

Abstract B cell development is carefully orchestrated to generate a diverse antibody repertoire while preventing malignant transformation. At early developmental stages, the pre-B cell receptor (pre-BCR) is an essential regulator of B cell maturation, coordinating both clonal expansion and immunoglobulin light chain gene (Igl) assembly. Igl gene rearrangement proceeds through V(D)J recombination, which requires the generation of DNA double-stranded breaks (DSBs) and the subsequent joining of two distant DNA segments. The DSBs necessary for Igl gene rearrangements are generated by the RAG1 and RAG2 proteins, which form the RAG endonuclease. Once RAG DSBs are generated, cells must be prevented from re-entering cell cycle where DSBs can be aberrantly repaired as deleterious translocations. Thus, temporal segregation of pre-BCR driven proliferation and Igl gene assembly is essential for normal maturation and prevention of errors that could lead to leukemic transformation. The pre-BCR signals through the SYK tyrosine kinase to both drive proliferation and initiate Igl gene rearrangement. Unopposed activation of the pre-BCR, particularly increased SYK activity, blocks B cell development and supports leukemic transformation. Furthermore, pre-B leukemic blasts depend on SYK for proliferation and survival. Tight regulation of pre-BCR signaling is essential for enforcing normal maturation and suppressing leukemic transformation. We previously showed that pre-BCR signaling is inhibited by signals from RAG DSBs. Specifically, RAG DSBs trigger ATM-dependent induction of SPIC, an ETS family transcriptional repressor with significant homology to PU.1 and SPIB. SPIC inhibits expression of the SYK tyrosine kinase resulting in suppression of pre-BCR signaling, inhibition of pre-B cell proliferation, and limitation of additional RAG DSB generation. In order to determine the mechanism of SPIC-mediated gene regulation and to determine the role of SPIC in early B cell development, we compared SPIC and PU.1 binding throughout the genome in early B cells. Chromatin-immunoprecipitation followed by sequencing (ChIP-seq) identified shared binding sites for SPIC and PU.1 throughout the genome in pre-B cells suggesting that both transcription factors regulate a similar cohort of genes. Interestingly, binding of SPIC to the Syk promoter and the Igl(kappa) enhancer results in reduction of PU.1 binding at these sites and suppression of transcription of the respective genes. These results demonstrate that SPIC antagonizes PU.1 function to repress pre-BCR signaling in early B cells. SPIC shares a DNA binding domain motif with the other ETS-family transcription factors, PU.1 and SPIB, but has a unique transactivation domain, which could recruit transcriptional repressors to gene regulatory regions. To determine the unique binding partners of SPIC and PU.1, we generated pre-B cells expressing tetracycline-inducible FLAG-SPIC and FLAG-PU.1. The tagged proteins were purified from nuclei using tandem affinity purification. Mass spectrometry of the precipitated samples identified BCLAF1 (Bcl2 associated transcription factor 1) as a unique SPIC interactor. In contrast, IRF4 binds to PU.1 but not to SPIC. In pre-B cells, BCLAF1 binds to regulatory regions of a similar set of genes as SPIC. Consistent with this, BCLAF1 binds to the Syk promoter only in cells with RAG DSBs, which correlates with binding of SPIC and loss of PU.1 at this site. RNAi-mediated knockdown of BCLAF1 results in increased Syk mRNA expression. However, loss of BCLAF1 does not affect SPIC occupancy at the Syk promoter. Thus, SPIC binds DNA but only represses transcription if BCLAF1 is also recruited. Collectively, our findings reveal that, in response to RAG DSBs, SPIC and BCLAF1 cooperate to antagonize PU.1 function and regulate expression of a broad genetic program that modulates pre-BCR signaling to enforce normal B cell maturation. Disclosures No relevant conflicts of interest to declare.