Early B cell development requires a balance of antagonistic cell programs that drive cellular proliferation as well as maturation. In large pre-B cells, pre-B cell receptor (pre-BCR) signaling activates the ETS family transcription factor, PU.1, and the tyrosine kinase, SYK, which together promote cell proliferation. Subsequently, cells undergo cell cycle arrest and transition to the small pre-B cell stage where immunoglobulin light chain (Igl) gene rearrangement is initiated. Igl gene assembly proceeds through DNA double-stranded breaks (DSBs) generated by the RAG endonuclease. These RAG DSBs induce cellular programs that inhibit pre-BCR signaling through activation of the transcription factor NFkB2. NFkB2 upregulates expression of the SPIC/BCLAF1 transcriptional repressor complex, which displaces PU.1 from the Syk promoter, resulting in reduction of SYK and enforcement of cell cycle arrest. Pediatric pre-B cell leukemia can be driven by errant pre-BCR signaling and increased SYK. We propose that B cell development relies on signals from RAG DSBs to counter pre-BCR signaling and attenuate SYK expression, thereby enforcing a pre-B cell checkpoint that promotes Igl rearrangement and suppresses leukemic transformation. To examine coordination between pre-BCR and the RAG DSB signals in vivo, we developed a novel reporter mouse with a knock-in allele at the endogenous Syk locus encoding an N-terminal fusion of mCherry to SYK (SykmCh). Heterozygous SykmCh/+ mice express equivalent SYK and mCherry-SYK proteins and have normal SYK activity as evidenced by phosphorylation of BLNK, a downstream target of the SYK kinase. To determine if SYK levels are modulated by RAG DSB signals, we generatedRag1-/-:SykmCh/+:IgH:Bcl2 and Art-/-:SykmCh/+:IgH:Bcl2 mice. Rag1-/- mice cannot make DSBs whereas Art-/- mice cannot repair DSBs, due to lack of the DNA damage response protein, Artemis. IgH transgene promotes development to pre-B cell stage and Bcl2 supports survival to permit assessment of DNA damage responses. Flow cytometry revealed lower mCherry-SYK levels in Art-/- small pre-B cells compared to Rag1-/- small pre-B cells, which supports that SYK is suppressed in vivo by RAG DSBs. We next assessed SYK levels during normal B cell development. mCherryhigh and mCherrylow small pre-B cells from SykmCh/+mice were sorted and demonstrated an inverse correlation between SYK and DSB signals. Specifically, relative to mCherryhigh small pre-B cells, mCherrylow small pre-B cells have lower SYK and increased NFkB2 activation, indicating concurrent repressed pre-BCR signals and active RAG DSB signals. Notably, mCherryhigh small pre-B cells have expression of DSB-induced genes Spic, Pim2, and Cd40. Together, these results demonstrate that RAG DSB signals are initiated in SYK-high small pre-B cells and then subsequently promote transition to SYK-low small pre-B cells. To investigate consequences of altered SYK regulation, we generated p53f/f:Bclaf1f/f:Mb1-cre mice, which have selective loss of p53 and Bclaf1 in early B cells. We previously showed that Bclaf1flox/flox:Mb1-cre mice have increased numbers of large pre-B cells with higher Syk expression. However, there were no abnormalities in immature B cell numbers, suggesting additional mechanisms, such as activation of p53 by RAG DSBs, may complement BCLAF1 to enforce B cell development. Indeed, we find that all p53flox/flox:Bclaf1flox/flox:Mb1-cre mice die between 9-12 months of age with malignant expansion of B cells in both spleen and bone marrow. In contrast, no p53flox/flox:Mb1-cre, Bclaf1flox/flox:Mb1-cre, or Bclaf1flox/flox:p53flox/flox mice died or developed abnormal B cells in this time period. The atypical B cells in the p53:Bclaf1 double-deficient mice have altered B220 expression, normal expression of IgM and absence of CD43, consistent with an aberrant immature B cell phenotype. These results demonstrate that RAG DSB signals function to preserve pre-B cell checkpoint and maintain normal B cell maturation. Collectively, these studies establish a novel system for quantitating DNA damage signaling in vivo and for assessing dynamic changes in SYK during B cell maturation. Further, our findings demonstrate a critical function of RAG DSB signals in regulation of early B cell development.
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Sep 14, 2023
Tianle Tang + 6
Open Access
