RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals

Abstract

DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre-B cell receptor (pre-BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre-BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre-B cells. Here, we show that RAG DSBs inhibit pre-BCR signals through the ATM- and NF-κB2-dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre-BCR signaling. This regulatory circuit prevents the pre-BCR from inducing additional Igl chain gene rearrangements and driving pre-B cells with RAG DSBs into cycle. We propose that pre-B cells toggle between pre-BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes.

Figure 1.

RAG DSBs regulate the genetic program in small pre–B cells. (A) Southern blot of SacI and EcoRI digested genomic DNA from Rag1^−/−:μIgH:Bcl2, Art^−/−:μIgH:Bcl2 and Art^−/−:Atm^−/−:μIgH:Bcl2 small pre–B cells in IL-7 (0 d) and after withdrawal of IL-7 (IL-7 wd) for the indicated days. Bands reflecting the germline (GL) Igk locus and Jk coding ends (Jk1, Jk2, Jk4, and Jk5 CE) are indicated as are molecular weight markers in kilobases. (B) Western blot of phosphorylated p53 (p-p53) in pre–B cells in IL-7 (0 d) and after withdrawal of IL-7 (IL-7 wd) for the indicated days. GAPDH is shown as a protein loading control. Data in A and B are representative of at least three independent experiments. (C) Heat map of gene expression changes in Rag1^−/−:μIgH:Bcl2 and Art^−/−:μIgH:Bcl2 pre–B cells in the presence of IL-7 (+ IL-7) and 2 d after IL-7 withdrawal (− IL-7). Columns represent three independent cell cultures of each genotype. Representative genes are delineated to the right. (D and E) RT-PCR validation of representative RAG DSB-independent (D) or RAG DSB-dependent (E) gene expression changes from C. mRNA expression was assessed in Rag1^−/−:μIgH:Bcl2 (black) and Art^−/−:μIgH:Bcl2 (red) pre–B cells in the presence of IL-7 (+ IL-7) and 2 d after IL-7 withdrawal (− IL-7). Data are mean and standard error for three replicates. (F) Venn diagram comparing gene expression changes that occur during the transition of large to small pre–B cells in the bone marrow of WT mice (ImmGen; blue) and gene expression changes we observe in small pre–B cells after withdrawal of IL-7 (red). The ImmGen data represents all genes that were significantly changed (p-value < 0.05) between large and small pre–B cells. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005; ****, P ≤ 0.0001 by Student’s t test.

Figure 2.

Signals from RAG DSBs activate NF-κB2. (A) Nfkb2 and Relb mRNA expression in Rag1^−/−:μIgH:Bcl2 (black), Art^−/−:μIgH:Bcl2 (red), and Art^−/−:Atm^−/−;μIgH:Bcl2 (blue) small pre–B cells 2 d after IL-7 withdrawal. Data are mean and standard error for three replicates. (B) Nfkb2 and Relb mRNA expression in sorted small pre–B cells from Rag1^−/−:μIgH:Bcl2, WT, and Atm^−/− mice. Data are mean and standard error from three independent mice of each genotype. (C) Western blot of NF-κB2 (p100 and p52) and RELB in pre–B cells in IL-7 (0 d) and after withdrawal of IL-7 (IL-7 wd) for the indicated days. GAPDH is shown as a protein loading control. (D) Western blot of NF-κB2 (p100 and p52) and RELB incytoplasmic andnuclear fractions from pre–B cells in IL-7 (0 d) and after withdrawal of IL-7 for the indicated days. LaminB and GAPDH are shown as protein loading controls for nuclear and cytoplasmic fractions, respectively. (E) Western blot of NF-κB2 (p100 and p52) in pre–B cells in IL-7 (0 d) and after withdrawal of IL-7 (IL-7 wd) for the indicated days. GAPDH is shown as a protein loading control. Data in all panels are representative of at least three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005 by Student’s t test.

Figure 3.

Signals from RAG DSBs induce SPIC expression. (A) Heat map of RAG DSB-dependent gene expression changes that require NF-κB2. Art^−/−:μIgH:Bcl2 pre–B cells were compared with Rag1^−/−:μIgH:Bcl2 pre–B cells to identify RAG DSB-dependent gene expression changes followed by comparison of Art^−/−:μIgH:Bcl2 and Art^−/−:Nfkb2^−/−:μIgH:Bcl2 pre–B cells to identify NF-κB2–dependent changes. Columns represent three independent cell cultures of each genotype. Representative genes are delineated to the right. (B) Spic mRNA expression in Rag1^−/−:μIgH:Bcl2 (black), Art^−/−:μIgH:Bcl2 (red), Art^−/−:Atm^−/−:μIgH:Bcl2 (blue), and Art^−/−:Nfkb2^−/−:μIgH:Bcl2 (green) small pre–B cells 2 d after IL-7 withdrawal. Data are mean and standard error for three replicates. (C) Flow cytometric analysis of CD40 (y-axis) and EGFP (x-axis) expression in bone marrow small pre–B cells (B220^loCD43⁻IgM⁻) from Rag1^−/−:Spic^igfp/igfp:μIgH:Bcl2 and Art^−/−:Spic^igfp/igfp:μIgH:Bcl2 mice. Inset shows percent of cells in each quadrant. Data are representative of three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005 by Student’s t test.

Figure 4.

RAG DSBs suppress pre–BCR signaling. (A) Western blot of phosphorylated BLNK (p-BLNK) and total BLNK in pre–B cells in IL-7 (0 d) and following IL-7 withdrawal (IL-7 wd) for the indicated days. GAPDH is shown as a protein loading control. (B and C) Western blot of SYK in pre–B cells cultured in IL-7 (+) and 3 d after IL-7 withdrawal (–). GAPDH is shown as a protein loading control. (D and E) Blnk, Syk and Btk mRNA expression in Rag1^−/−:μIgH:Bcl2 (black), Art^−/−:μIgH:Bcl2 (red), and Art^−/−:Atm^−/−;μIgH:Bcl2 (blue) small pre–B cells 3 d after IL-7 withdrawal. Data are mean and standard error for three replicates. (F) Western blot of BTK in pre–B cells cultured in IL-7 (+) and 3 d after IL-7 withdrawal (–). GAPDH is shown as a protein loading control. Data in A, B, C, and F are representative of at least three independent experiments. *, P ≤ 0.05; ***, P ≤ 0.005; ****, P ≤ 0.0001 by Student’s t test.

Figure 5.

SPIC inhibits expression of Syk and Blnk. (A) Rag1^−/−:μIgH:Bcl2 small pre–B cells were transduced with an empty retrovirus or a retrovirus expressing SPIC. Blnk and Syk mRNA expression was assessed 2 d after IL-7 withdrawal. Data are mean and standard error for three replicates. (B) Blnk and Syk mRNA expression in EGFP-negative (–) and EGFP-expressing (+) small pre–B cells sorted from Spic^igfp/igfp mice. Data are the mean and standard error from three independent mice. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005 by Student’s t test.

Figure 6.

SPIC displaces PU.1 from Syk and Blnk. (A) Western blot of PU.1 in pre–B cells cultured in IL-7 (+) and 3 d after IL-7 withdrawal (–). GAPDH is shown as a protein loading control. (B) ChIP-qPCR of PU.1 binding at the Blnk promoter in Rag1^−/−:μIgH:Bcl2 and Art^−/−:μIgH:Bcl2 small pre–B cells 3 d after IL-7 withdrawal. (C–E) Rag1^−/−:μIgH:Bcl2 small pre–B cells were transduced with an empty retrovirus, a retrovirus expressing SPIC, or a retrovirus expressing a DNA binding–deficient SPIC (SPIC^R175G). (C) Western blot of FLAG was performed 2 d after IL-7 withdrawal. GAPDH is shown as a protein loading control. ChIP-qPCR of SPIC binding (D) and PU.1 binding (E) at the Blnk promoter and at a putative regulatory element located 10-kb upstream of the transcription start site for Syk was performed 2 d after IL-7 withdrawal. Data in all panels are representative of two independent experiments.

Figure 7.

SPIC inhibits Igl transcription and rearrangement. (A) Irf4 and germline Igk mRNA expression in Rag1^−/−:μIgH:Bcl2 and Art^−/−:μIgH:Bcl2 small pre–B cells assessed 3 d after IL-7 withdrawal. Data are mean and standard error for three replicate experiments. (B) Western blot of IRF4 in pre–B cells in IL-7 (0 d) and after IL-7 withdrawal (IL-7 wd) for the indicated days. GAPDH is shown as a protein loading control. Data are representative of three independent experiments. (C) Rag1^−/−:μIgH:Bcl2 small pre–B cells were transduced with an empty retrovirus or a retrovirus expressing SPIC. Irf4 and germline Igk mRNA expression were assessed 2 d after IL-7 withdrawal. Data are mean and standard error for three replicate experiments. (D) ChIP-qPCR of PU.1 binding at the 3′ Igk enhancer in Rag1^−/−:μIgH:Bcl2 and Art^−/−:μIgH:Bcl2 small pre–B cells 3 d after IL-7 withdrawal. (E and F) Rag1^−/−:μIgH:Bcl2 small pre–B cells were transduced with an empty retrovirus or a retrovirus expressing SPIC. ChIP-qPCR of SPIC binding (E) and PU.1 binding (F) at the 3′ Igk enhancer was performed 2 d after IL-7 withdrawal. (G) Southern blot of SacI and EcoRI digested genomic DNA from Art^−/−:Atm^−/−:μIgH:Bcl2 small pre–B cells transduced with an empty retrovirus or a retrovirus expressing SPIC 3 d after IL-7 withdrawal. Bands reflecting the germline (GL) Igk locus and Jk coding ends (Jk1, Jk2, Jk4, and Jk5 CE) are indicated as are molecular weight markers in kilobases. Data in D–G are representative of two independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005 by Student’s t test.

Figure 8.

RAG DSBs inhibit pre–BCR-driven proliferation. (A and B) Rag1^−/−:μIgH:Bcl2 small pre–B cells were transduced with an empty retrovirus or a retrovirus expressing NF-κB2 (A) or SPIC (B). Flow cytometric analysis of BrdU incorporation (y axis) and DNA content (7AAD; x axis) was performed after IL-7 withdrawal. Percentage of cells that entered S-phase during BrdU labeling (box) is indicated. Data are representative of at least three independent experiments. (C) Cell cycle analysis was performed as in A on Art^−/−:μIgH:Bcl2 small pre–B cells with RAG DSBs (CD40 pos) and those without RAG DSBs (CD40 neg) after IL-7 withdrawal. Percentage of cells that entered S-phase during BrdU labeling (box) is indicated. Data are representative of at least three independent experiments. (D) Cell cycle analysis of CD40 neg and CD40 pos Art^−/−:μIgH:Bcl2 and Art^−/−:p53^−/−:μIgH small pre–B cells as in C. Graph represents the percentage of cells that entered S-phase. Data are mean and standard error for two independent experiments. (E and F) Art^−/−:p53^−/−:μIgH (E) and Art^−/−:μIgH:Bcl2 (F) small pre–B cells were transduced with an empty retrovirus or a retrovirus expressing SYK. Cell cycle analysis of CD40 neg and CD40 pos pre–B cells was performed as in C. Graphs represent the percentage of cells that entered S-phase. Data in both panels are representative of three independent experiments. **, P ≤ 0.01; ****, P ≤ 0.0001 by Student’s t test.