The Ets factor Spi-B is a direct critical target of the coactivator OBF-1

Abstract

OBF-1 (Bob.1, OCA-B) is a lymphoid-specific transcriptional coactivator that associates with the transcription factors Oct-1 or Oct-2 on the conserved octamer element present in the promoters of several ubiquitous and lymphoid-specific genes. OBF-1-deficient mice have B cell-intrinsic defects, lack germinal centers, and have severely impaired immune responses to T cell-dependent antigens. Crucial genes that are regulated by OBF-1 and that might explain the observed phenotype of OBF-1 deficiency have remained elusive to date. Here we have generated transgenic mice expressing OBF-1 specifically in T cells and examined these together with mice lacking OBF-1 to discover transcriptional targets of this coactivator. Using microarray analysis, we have identified the Ets transcription factor Spi-B as a direct target gene critically regulated by OBF-1 that can help explain the phenotype of OBF-1-deficient mice. Spi-B has been implicated in signaling pathways downstream of the B cell receptor and is essential for germinal center formation and maintenance. The present findings establish a hierarchy between these two factors and provide a molecular link between OBF-1 and B cell receptor signaling.

Fig. 1.

Expression of Spi-B, from the octamer site containing P₂, is proportional to OBF-1 levels. (A) The Spi-B mRNA level is proportional to the amount of OBF-1 expression in thymocytes. Thymocytes were isolated from OBF-1-deficient WT or lck-OBF-1 transgenic mice, and total RNA was analyzed by Northern blotting with a Spi-B-specific probe (Left). The membrane was rehybridized with a probe for β-actin as a control for loading. Quantification by PhosphorImaging and normalization to the expression level of β-actin are shown (Right). (B) Schematic representation of the mouse Spi-B gene in the promoter region. The structure of the mRNAs originating from P₁ (upstream) or P₂ (downstream) is indicated. Exon sequences are depicted as boxes. The sequence surrounding the octamer site in P₂ is shown at the bottom of B. (C) Spi-B P₂ is under the control of OBF-1. An RNase protection assay with RNA from thymocytes of the indicated genotypes is shown (Left). A probe for S16 was included as a control in the hybridization reaction, and its signal was used for the normalized expression presented (Right).

Fig. 2.

Critical role of the octamer motif for activation of the Spi-B P₂. (A) Luciferase reporter constructs under the control of the Spi-B P₁ or P₂ were transfected into 293T cells together with an empty expression vector (filled bars) or an OBF-1 expression vector (open bars). The histograms represent the mean ± SD of three independent experiments. (B) The reporter constructs were transfected into the K46 B cell line expressing endogenous OBF-1. The histograms represent the mean ± SD of three independent experiments. (C) EMSA with a WT (P₂) or mutated (P_2-S, P_2-M) octamer site DNA probe derived from the Spi-B promoter and in vitro translated proteins. Either full-length Oct-1 (lanes 4–9) or its isolated POU domain (lanes 10–15) was used for complex formation.

Fig. 3.

In vivo OBF-1 is bound on the Spi-B P₂. (A) Cross-linked chromatin was isolated from WT or lck-OBF-1 thymocytes and immunoprecipitated with an anti-HA antibody. The precipitated material was used as a template for a PCR with primers derived from Spi-B P₂ or from the BLR1 promoter. (Top and Middle) The precipitated material was used as a template for a PCR with primers derived from Spi-B P2 or from the BLR1 promoter, as indicated. (Bottom) The panel labeled “input” shows PCR amplification with the chromatin before immunoprecipitation. (B) Cross-linked chromatin was isolated from WT or OBF-1^−/− Abelson virus-transformed pro-B cells and immunoprecipitated with antibodies against OBF-1. A PCR with primers derived from Spi-B P₂ is shown for the precipitate (Upper) and the input (Lower).

Fig. 4.

OBF-1 is required for Spi-B expression in B cells. (A) OBF-1 is necessary for STI571-induced expression of Spi-B in Abelson pro-B cells. WT or OBF-1^−/− Abl pro-B cells were cultured in the presence (open bars) or absence (solid bars) of STI571 (10 nM) for 10 h, and total RNA was extracted. After cDNA synthesis, expression of the Spi-B gene was measured by real-time PCR, with primers specific for RNA originating from P₁ or P₂. The histograms show the mean ± SD of three independent experiments. (B) Mature B cells from OBF-1^−/− mice have reduced levels of Spi-B mRNA originating from promoter P₂. Real-time PCR was performed on sorted CD43⁺-depleted mature splenocytes of OBF-1^+/− and OBF-1^−/− mice. (C) Rescue of Spi-B transcription by expression of OBF-1 in a mature OBF-1^−/− B cell line. (Upper) The Northern blot shows the OBF-1-deficient BM4 parental cell line and two clones thereof expressing an inducible OBF-1/estrogen receptor fusion protein (lanes 1 and 2); all samples were treated with hormone before RNA isolation. (Lower) The blot was reprobed with β-actin as a loading control.

Fig. 5.

Absence of Spi-B expression in OBF-1^−/− follicular B cells. (A) Ten days after immunization with 2,4-dinitrophenyl-conjugated keyhole limpet hemocyanin (DNP-KLH) splenic cryosections were prepared. Formation of GCs was monitored by staining of B cells with an anti-B220 antibody (green) together with PNA (red). Spi-B expression was detected by immunohistochemistry with a specific riboprobe (antisense, blue), and nuclei were counterstained with Nuclear Fast red (red). A control with a sense Spi-B probe is shown. (B) (Upper) Spi-B expression in GC and follicular B cells of immunized WT mice. Splenic GC (GL7⁺ PNA⁺ B220⁺) and non-GC (GL7⁻ PNA⁻ B220⁺) B cells of WT mice were FACS-sorted 10 days after immunization, and Spi-B mRNA expression was measured by semiquantitative RT-PCR with primers specific for RNA originating from P₁ or P₂. GAPDH expression was used for normalization. Three experiments were performed with identical results. (Lower) RT-PCR analysis of AID expression, a GC-specific enzyme, demonstrated the purity of the sorted GC B cell population (lane 1) compared with the non-GC B cells (lane 3). Nontemplate controls (without reverse transcription) are shown in lanes 2 and 4, respectively.