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HoxC4 binds to the Aicda promoter to induce AID expression, class switch DNA recombination and somatic hypermutation
Associated Data
Abstract
AID is critical for immunoglobulin class switch DNA recombination (CSR) and somatic hypermutation (SHM). Here we showed that AID expression was induced by the HoxC4 homeodomain transcription factor, which bound to a highly conserved HoxC4-Oct site in the Aicda promoter. This site functioned in synergy with a conserved Sp-NF-κB-binding site. HoxC4 was preferentially expressed in germinal center B cells and was upregulated by CD154:CD40 engagement, lipopolysaccharide and interleukin-4. HoxC4 deficiency resulted in impaired CSR and SHM, due to decreased AID expression and not other putative HoxC4-dependent activity. Enforced expression of AID in Hoxc4−/− B cells fully restored CSR. Thus, HoxC4 directly activates the Aicda promoter, thereby inducing AID expression, CSR and SHM.
CSR and SHM are critical for the maturation of antibody responses to foreign and self-antigens. CSR recombines switch (S) region DNA located upstream of constant heavy chain (CH) region exons, thereby changing immunoglobulin (Ig) CH regions and endowing antibodies with new biological effector functions. SHM introduces mainly point mutations in Ig variable regions, thereby providing the structural substrate for selection of higher affinity antibody mutants by antigen. In spite of the recent advances made in the identification of some factors involved in CSR and SHM, the intimate mechanisms of these processes remain elusive. Both CSR and SHM require activation-induced cytidine deaminase (AID), which is expressed by activated B cells, mainly in germinal centers (GCs) of peripheral lymphoid organs1,2. AID initiates CSR and SHM by deaminating dC residues to yield dU:dG mispairs in DNA3–8. These dU:dG mispairs trigger DNA repair processes entailing introduction of mutations in V(D)J regions or DNA breaks, including double-stranded DNA breaks, which lead to non-classic non-homologous end-joining and CSR3,5,9–14.
The mechanisms governing the transcriptional regulation of the gene encoding AID (AICDA in the human and Aicda in the mouse) remain to be elucidated. A conserved region in the first intron of Aicda containing two E-boxes, the consensus sequence for E2A (http://www.signaling-gateway.org/molecule/query?afcsid=A000804) binding, has been suggested to contribute to Aicda transcription regulation through recruitment of the E2A helix-loop-helix (HLH) transcription factor E47 and the inhibitor of DNA-binding HLH protein Id3, respectively15. Pax5 has been suggested to cooperate with E2A proteins in controlling Aicda transcription16. However, this could not be confirmed by another study, which rather suggested a role for the Sp1 family of ubiquitous zinc-finger transcription factors. These regulate various promoters by binding to dGdC, dGdA or dGdT boxes, in activating the Aicda promoter17.
Hox proteins are highly conserved HLH homeodomain-containing transcription factors that regulate cellular differentiation and organogenesis18,19. Hox genes, which are chromosomally clustered, are expressed in a temporally and spatially regulated fashion20,21. Among human HOX, HOXC genes, particularly HOXC4, are predominantly expressed in lymphoid cells22. HOXC4 gene expression increases through sequential stages of B cell development22–25, from non-committed hematopoietic progenitors in the bone marrow to mature B cells in the periphery, particularly when activated and proliferating. Malignant B cells including mantle cell lymphoma, Burkitt's lymphoma and B cell chronic lymphocytic leukemia, express aberrant AID26, 27 and abundant HoxC422,28. HoxC4 induces the human 3' Eα enhancer elements, particularly DNAse I hypersensitive sites hs1,2, in a B cell development stage-specific fashion25. HoxC4 binds to a HoxC4-Oct motif 5'-ATTTGCAT-3' site in hs1,224,25, which is conserved in the human, mouse, rat and rabbit, and synergizes with the Oct1/Oct2 (http://www.signaling-gateway.org/molecule/query?afcsid=A001704) homeodomain proteins and the OcaB (http://www.signaling-gateway.org/molecule/query?afcsid=A001696) co-activator to induce this enhancer in B cells24,25. HOXC4 expression is induced by stimuli that induce GC B cell differentiation and AICDA expression24,25, such as CD154 (http://www.signaling-gateway.org/molecule/query?afcsid=A000536) and interleukin 4 (IL-4) (http://www.signaling-gateway.org/molecule/query?afcsid=A001262), suggesting a role of HoxC4 in CSR and SHM.
In this study, we tested the hypothesis that HoxC4 regulates AID expression in human and mouse B cells. We showed that HoxC4 bound to a HoxC4-octamer motif in the AICDA and Aicda promoters that is conserved in humans, chimps, mice, rats, dogs and cows. Binding of HoxC4 to this cis-element activated the AID gene promoter and induced AID expression, thereby inducing CSR and SHM. In this function, HoxC4 synergized with an equally conserved upstream Sp-NF-κB site in the AID gene promoter.
RESULTS
HoxC4 and AID are induced in GC B cells
We have shown that HoxC4 is upregulated in human IgD−CD38+ GC B cells24,25, which express AID and undergo CSR and SHM. Stimulation of human IgD+CD38− naive B cells with an agonistic CD40 monoclonal antibody (mAb) and huIL-4 upregulated HoxC4 and induced AID expression24,25. We further analyzed the expression of Hoxc4 and Aicda in bone marrow, thymus, spleen, Peyer’s patches, liver and heart of wild-type C57BL/6 mice. Real-time quantitative qRT-PCR revealed that like Aicda, Hoxc4 was preferentially expressed in the spleen and Peyer’s patches, which contain a large proportion of hypermutating and switching B cells, but not in non-lymphoid organs, such as the liver or the heart (Fig. 1a). To further address the correlation between HoxC4 and AID expression, we isolated PNAhiB220+ GC and PNAloB220+ (non-GC) B cells from spleen of 8- to10-week-old C57BL/6 mice, 14 d after immunized with 4-hydroxy-3-nitrophenyl acetyl coupled to chicken γ-globulin (NP16-CGG), and analyzed the amount of the two proteins, as well as PCNA, which is a multi-functional protein critical for DNA replication and repair and is highly expressed in actively dividing cells. HoxC4 was specifically expressed in PNAhiB220+ GC B cells, where AID and PCNA were also highly expressed (Fig. 1b). Stimulation of mouse spleen B cells with bacterial lipopolysaccharide (LPS) and IL-4 or CD154 and IL-4, which induce GC B cell differentiation and Aicda expression upregulated Hoxc4 expression by 10 to 15 fold (Fig. 1c) and induced CSR to IgG1 (not shown), indicating that HoxC4 plays a role in inducing AID expression.
Hoxc4 expression correlates with Aicda expression. (a) Hoxc4 and Aicda transcripts in bone marrow, thymus, spleen, Peyer’s patches, liver or heart of C57BL/6 mice, as measured by real-time qRT-PCR performed in triplicate samples using SYBR-green. In each sample, mRNA expression was normalized to Gapdh expression. Data are means ± s.e. (bars) of fold mRNA in the indicated tissue compared to bone marrow of 3 independent experiments. (b) HoxC4, AID, PCNA and β-actin protein expression in PNAhiB220+ (GC) B cells and PNAloB220+ (non-GC) B cells from spleen of NP16-CGG immunized mice, as detected by immunoblotting. Data are from 2 independent experiments. (c) Increased expression of Hoxc4 and Aicda mRNA in LPS and IL-4- or CD154 and IL-4-stimulated spleen B cells, as determined by real-time qRT-PCR. Spleen B cells from 3 C57BL/6 mice were cultured with nil, LPS plus IL-4 or CD154 plus IL-4, and harvested after 3 d to extract RNA and perform real-time qRT-PCR. mRNA expression was normalized to CD79b transcripts. Data are from 3 independent experiments. In each experiment, values are means ± s.e. (bars) of fold mRNA levels in B cells stimulated as indicated, as compared to unstimulated B cells (nil).
HoxC4 deficiency impairs antibody response to NP-CGG
We used Hoxc4−/− mice to address the role of HoxC4 in CSR and SHM. Two lines of HoxC4-deficient mice were independently generated and homozygous mutants of both lines displayed esophageal defects and abnormal cervical and thoracic vertebral development and suffered high post-natal mortality rates29,30. In these mice, the expression of Hoxc5 and Hoxc6, which lie in the same gene cluster as Hoxc4, was reduced, likely due to the neighboring impact of the neomycin-selection (neor) cassette inserted into the Hoxc4 locus29,30. To obviate this, a third Hoxc4−/− strain, in which the neor cassette was deleted by Cre recombinase through two flanking loxP sites in the germline (Supplementary Fig. 1 online), thereby leaving Hoxc5 and Hoxc6 expression unaltered (Boulet and Capecchi unpublished). Such mice were since lost, but frozen Hoxc4+/− sperm on the C57BL/6 background was preserved. Using Hoxc4+/− sperm, we re-derived Hoxc4+/− mice by in vitro fertilization and bred them to obtain Hoxc4−/− mice. These Hoxc4−/− mice are born at Mendelian ratio, do not suffer the high post-natal mortality rate of the earlier HoxC4-deficient mouse lines29,30 and develop to adulthood.
In non-immunized Hoxc4−/− mice, serum IgM titers were normal. However, the average serum IgG1 concentration was less than 0.6 mg/ml, as compared to 1.2 mg/ml in their Hoxc4+/+ littermates (not shown), suggesting an impairment of CSR. We immunized four pairs of 8–10-week-old littermate Hoxc4−/− and Hoxc4+/+ mice with NP16-CGG and analyzed blood from these mice for IgM and IgG1 titers, NP30-binding IgM and IgG1, and high-affinity NP3-binding IgM and IgG1 (Fig. 2a). Total IgM and NP30-binding IgM were not significantly different in Hoxc4−/− from Hoxc4+/+ mice. Hoxc4−/− mice, however, showed some decrease in NP3-binding IgM and significantly lower total IgG1, NP30-binding IgG1 as well as high affinity NP3-binding IgG1 titers. The defective antibody response to NP-CGG did not reflect an altered plasma cell or memory B cell development, as the proportions of B220loCD138+ cells and CD38hi B cells among NP-binding IgG1 B cells in NP16-CGG-immunized Hoxc4−/− mice were comparable to those of their Hoxc4+/+ littermates (Fig. 2b). Rather, it reflected the decreased overall IgG1 levels and, together with the slightly lower NP3-binding IgM activity, a decreased binding affinity for NP3.
Impaired antibody response in Hoxc4−/− mice. (a) Four pairs of littermate Hoxc4+/+ and Hoxc4−/− mice were immunized with NP16-CGG and boost-injected 21 d later. Circulating IgM and IgG1, NP30-binding IgM and IgG1, and (high-affinity) NP3-binding IgM and IgG1 were measured 7 d after the boost-injection, and, their levels were expressed as 50% effective concentration (EC50) units. These were defined as the number of dilutions needed to reach 50% of saturation binding. (b and c) Normal plasma cell and memory B cell development in Hoxc4−/− mice 14 d after immunization with NP16-CGG. (b) Surface CD138 and B220 expression in spleen cells from immunized littermate Hoxc4+/+ and Hoxc4−/− mice. Numbers in boxes are B220lo CD138+ (plasma) cells, as percentage of total B220+ cells. (c) Spleen cells from immunized littermate Hoxc4+/+ and Hoxc4−/− mice were analyzed by flow cytometry after staining with FITC-PNA, APC-anti-mouse IgG1 mAb, PE-NP and PECy7-anti-mouse CD38 mAb. Insets in left panels show NP-binding surface IgG1+ B cells. Right panels show CD38 expression by the gated NP-binding IgG1+ B cells. Data are representative of 3 independent experiments (b and c).
HoxC4 deficiency does not alter GC formation
The defective antibody response to NP-CGG in Hoxc4−/− mice was not due to obvious defects in lymphoid differentiation. In these mice, the size of the spleen, and the number and the size of Peyer’s patches were comparable to those in Hoxc4+/+ mice (not shown). Moreover, the number of B and T cells, the proportion of CD4+ T cells, and death of B and T cells in the spleen and Peyer’s patches, as analyzed by staining with 7-amino-actinomycin (7-AAD), were also comparable to those of Hoxc4+/+ mice (Fig. 3a–d). After stimulation with LPS and IL-4, Hoxc4−/− B lymphocytes were comparable in cell cycle, as analyzed by propidium iodide (PI) staining, and cell division rate, as measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) vital dye incorporation, to Hoxc4+/+ cells (Fig. 3e,f). In Hoxc4−/− mice, the number and architecture of the GCs in the spleen, the proportions of proliferating B cells, as shown by in vivo bromodeoxyuridine (BrdU) incorporation, and the proportion of PNAhi GC B cells in both spleen and Peyer’s patches were comparable to those of Hoxc4+/+ mice (Fig. 3g,h,i), suggesting that the defective antibody response to NP16-CGG in Hoxc4−/− mice reflected an intrinsic impairment of the CSR and SHM machineries.
HoxC4 deficiency does not affect B and T cell numbers, CD4+ T cell numbers, B and T cell death in spleen and Peyer’s patches, B cell cycle or division, nor does it alter GC formation and in vivo B cell proliferation. (a–d) Flow cytometric profiles of cells from spleen and Peyer’s patches stained with (a) PE-anti-B220 mAb and FITC-anti-CD3 mAb, (b) FITC-anti-CD3 mAb and PerCP-anti-CD4 mAb, (c) 7-AAD and FITC-anti-CD3 mAb, and (d) 7-AAD and PE-anti-B220 mAb. (e) Cell cycle analysis of Hoxc4+/+ and Hoxc4−/− B cells. Spleen Hoxc4+/+ and Hoxc4−/− B cells were stimulated with LPS and IL-4 and harvested after 1 and 2 d for PI staining and flow cytometry analysis to measure DNA content and enumerate cells in G0/G1, S and G2/M phases. (f) Cell division in Hoxc4+/+ and Hoxc4−/− B cells. Spleen Hoxc4+/+ and Hoxc4−/− B cells were labeled with CFSE, cultured with LPS and IL-4, and harvested 2 and 3 d later for flow cytometry analysis. Cell division is indicated by progressive left shift of fluorescence histograms. Individual cell generations are enumerated above the graph. Data are from one representative of 3 experiments. (g) Staining of GCs in spleen sections prepared 10 d after immunization of Hoxc4+/+ and Hoxc4−/− mice. Scale bar: 200 µm. The original magnifications are indicated at the bottom of each set of panels. (h) Flow cytometric profiles of cells from Peyer’s patches from NP16-CGG immunized Hoxc4+/+ and Hoxc4−/− mice stained with PE-labeled anti-B220 mAb and FITC-PNA. (i) In vivo B cell proliferation. Three 10-week-old Hoxc4+/+ and Hoxc4−/− mice were immunized with NP16-CGG. After 10 d, the mice were injected with BrdU (1 mg) twice within 16 h and sacrificed 4 h after the last injection. Cells from spleen and Peyer’s patches were stained with PE-anti-mouse B220 mAb or this mAb together with FITC-PNA. Incorporated BrdU was detected with APC-anti-BrdU mAb and analyzed using by FACS. Data are from one representative of 3 pairs of Hoxc4+/+ and Hoxc4−/− mice.
HoxC4 deficiency impairs CSR and SHM
To determine the impact of HoxC4 deficiency on CSR, we stimulated spleen B cells with LPS (to induce switching to IgG2b and IgG3) or LPS or CD154 with IL-4 (to IgG1 and IgE), LPS or CD154 with IFN-γ (http://www.signaling-gateway.org/molecule/query?afcsid=A001238) (to IgG2a) and LPS or CD154 in the presence of transforming growth factor-β1 (TGF-β1, http://www.signaling-gateway.org/molecule/query?afcsid=A002271), IL-5, IL-4 and anti-δ-mAb-dextran (to IgA). After 4 d, in cultures stimulated with LPS or LPS and cytokines, surface IgG1+, IgG2a+, IgG2b+, IgG3+, IgA+ and IgE+ Hoxc4−/− B cells were decreased by 54%, 49%, 46%, 36%, 43% and 57%, respectively, as compared to Hoxc4+/+ B cell cultures (Supplementary Fig. 4a online); in cultures stimulated with CD154 and cytokines, surface IgG1+, IgG2a+ and IgE+ Hoxc4−/− B cells were decreased by 49%, 69% and 79%, respectively. Accordingly, aftre 7 d, IgG1, IgG2a, IgG2b, IgG3, IgA and IgE secreted by Hoxc4−/− B cells stimulated with LPS or LPS and cytokines were decreased by as much as 48%, 55%, 37%, 35%, 40% and 66%, respectively (Fig. 4a). Impaired CSR in Hoxc4−/− B cells was not due to altered proliferation, as after 2, 3 or 4 d of culture with LPS and IL-4 or CD154 and IL-4, Hoxc4−/− B cells completed the same number of divisions, as their Hoxc4+/+ counterparts (Fig. 3f, Fig.4b), or altered plasma cell differentiation, as after 4 d of culture with LPS, LPS and IL-4 or CD154 and IL-4, the number of CD138+B220lo plasma cells emerging from Hoxc4−/− B cells was comparable to that of their Hoxc4+/+ counterparts (Fig. 4c). Accordingly, the expression of transcription factors Blimp-1 and IRF-4, which are critical in plasma cell differentiation, were comparable in Hoxc4−/− and Hoxc4+/+ B cells, as determined by real-time qRT-PCR, 5 d after stimulation with LPS and IL-4 (data not shown). Further, reduced CSR in Hoxc4−/− B cells was not due to an impairment of germline IH-CH transcription, which is necessary for CSR. Real-time qRT-PCR showed that germline Iµ-Cµ, Iγ3-Cγ3, Iγ1-Cγ1, Iγ2b -Cγ2b, Iγ2a-Cγ2a, Iε-Cε and Iα-Cα transcripts in Hoxc4−/− B cells stimulated with LPS or LPS and cytokines for 3 d were comparable to those of their Hoxc4+/+ B cell counterparts (Fig. 5, Supplementary Fig. 3), while the post-recombination Iµ-CH transcripts, which are generated by CSR, were significantly decreased in Hoxc4−/− B cells, by as much as 89.2%. Thus, HoxC4-deficiency impairs CSR to all isotypes without affecting germline IH-CH transcription.
Impaired CSR in Hoxc4−/− B cells. (a) Spleen Hoxc4+/+ and Hoxc4−/− B cells were stimulated with LPS or CD154 in the presence of nil (for IgG2b and IgG3), IL-4 (for IgG1 and IgE), IFN-γ (for IgG2a), or TGF-β1/IL-4/IL-5/anti-δ mAb-dextran (for IgA). After 7 d, the supernatants from the cultures of Hoxc4+/+ B cells (empty symbols) and Hoxc4−/− B cell (full symbols) stimulated with LPS or LPS and cytokines were collected and analyzed for concentration of different Ig isotypes. Data are from 4 pairs of Hoxc4+/+ and Hoxc4−/− mice. (b) Hoxc4+/+ and Hoxc4−/− B cells were labeled with cell division-tracking fluorochrome CFSE and stimulated with LPS and IL-4 or CD154 and IL-4 to induce switching to IgG1. This showed that proliferation of Hoxc4−/− B cells was normal. After 4 d of culture, Hoxc4−/− and Hoxc4+/+ B cells completed the same number of divisions, but the percentage of surface IgG1+ B cells was significantly lower among Hoxc4−/− B cells. Data are from 2 independent experiments. (c) HoxC4 deficiency does not alter plasma cell differentiation. Hoxc4+/+ and Hoxc4−/− B cells were stimulated with LPS, LPS and IL-4 or CD154 and IL-4. After 4 d of culture, the cells were stained with PE-anti-mouse B220 mAb and FITC-anti-mouse CD138 mAb for flow cytometry analysis. Numbers in boxes indicate B220loCD138+ (plasma) cells as percentage of total cells analyzed. Data are from one representative of 3 independent experiments.
HoxC4 deficiency does not alter germline IH-CH transcripts but reduces the expression of post-recombination Iµ-CH transcripts. Spleen Hoxc4+/+ and Hoxc4−/− B cells were cultured with LPS or LPS and cytokines for 3 d and then harvested for RNA extraction. This was used as template in real-time qRT-PCR to measure the levels of germline Iµ-Cµ, Iγ3-Cγ3, Iγ1-Cγ1, Iγ2b-Cγ2b, Iγ2a -Cγ2a, Iε-Cεand Iα-Cα transcripts, and post-recombination Iµ-Cγ3, Iµ-Cγ1, Iµ-Cγ2b, Iµ-Cγ2a, Iµ-Cε and Iµ-Cα transcripts, as normalized to CD79b expression. Values are means ± s.e. (bars) of triplicate samples. Data are from one representative of 3 pairs of Hoxc4+/+ and Hoxc4−/− mice. The data from the remaining 2 pairs are depicted in Supplementary Fig. 3.
To determine the impact of HoxC4 deficiency on SHM, we analyzed the JH4-iEµ sequence downstream of rearranged VJ558DJH4 DNA in PNAhi B220+ GC B cells from Peyer’s patches of 3 pairs of 12-week-old non-immunized littermate Hoxc4−/− and Hoxc4+/+ mice. In these mice, the proportions of Peyer’s patch PNAhi B220+ GC B cells were comparable (Fig. 3h). Analysis of 324 and 319 JH4-iEµ intronic DNA sequences (720 bp) from Hoxc4−/− and Hoxc4+/+ mice showed a decrease in mutations in Hoxc4−/− mice by 59% (P < 0.00001) (Fig. 6a). The decreased mutation frequency was associated with a comparable reduction in mutations at dG:dC and dA:dT (Supplementary Fig. 4, online) and was not due to impaired transcription of the rearranged VJ558DJH genes, as shown by specific real-time qRT-PCR in Peyer’s patches B cells of these Hoxc4−/− mice, as compared to their Hoxc4+/+ littermates (Fig. 6b). Thus, HoxC4 deficiency significantly impairs SHM, without altering the spectrum of the residual mutations or VHDJH transcription.
Decreased somatic mutation frequency in the Ig H chain intronic JH4-iEµ DNA of Peyer’s patch PNAhiB220+ (GC) B cells from 3 12-week-old Hoxc4−/− mice as compare to their Hoxc4+/+ littermates. (a) Pie charts depict the proportions of sequences that carry 1, 2, 3, etc. mutations over the 720 bp JH4-iEµ DNA analyzed. The numbers of the sequences analyzed are at the center of the pies. (b) HoxC4 deficiency does not alter the level of VJ558DJH-Cµ transcripts. VJ558DJH-Cµ transcripts in Peyer’s patches B cells of these mice were measured by real-time qRT-PCR performed in triplicate samples using SYBR-green. In each sample, mRNA expression was normalized to CD79b expression. Data are means ± s.e. (bars) of triplicate samples from 3 independent pairs of Hoxc4+/+ and Hoxc4−/− mice. The analysis of the spectrum of mutations in Hoxc4+/+ and Hoxc4−/− mice is the subject of Supplementary Fig. 4.
HoxC4 deficiency impairs AID expression
CSR and SHM require transcription of the Igh locus and AID expression. The significant reduction of CSR and SHM, together with the normal amounts of mature VHDJH-Cµ and germline IH-CH transcripts in Hoxc4−/− B cells prompted us to hypothesize a modulation of AID expression by HoxC4. We stimulated spleen Hoxc4+/+ and Hoxc4−/− B cells with LPS and IL-4, or CD154 and IL-4 for 0, 12, 24, 48 and 72 h. Aicda mRNA expression could be detected by real-time qRT-PCR as early as 24 h and peaked within 48–72 h of stimulation in Hoxc4+/+ B cells. In Hoxc4−/− B cells, Aicda expression was reduced by more than 70%, after 72 h of stimulation (Fig. 7a). Decreased Aicda transcripts were associated with a significant decrease in AID protein (Fig. 7b), further suggesting that HoxC4 regulates Aicda expression.
(a,b) Decreased Aicda expression in Hoxc4−/− B cells. Spleen Hoxc4+/+ and Hoxc4−/− B cells were cultured in the presence of nil, LPS and IL-4, or CD154 and IL-4. After 0, 12, 24, 48 and 72 h of culture, cells were harvested for RNA or protein extraction. (a) RNA (2 µg) was reverse-transcribed to cDNA and used as template in real-time qRT-PCR, in which Aicda expression was normalized to CD79b expression. Data are means ± s.e. (bars) of 3 independent experiments. (b) AID and β-actin proteins were detected by immunoblotting. Data are representative of 3 independent experiments. (c) Depicted, not to scale, are portion of the human AICDA and mouse Aicda promoter region (AID-Pro), flanking 5’ region (5’R), cr1 and cr2 in the first intron, and five exons, within which the coding region is depicted in light blue. (d) The −349 to −1 (AID-Pro) region, which we tentatively defined as Aicda promoter based on its high conservation, but not the immediately adjacent 5’ region, nor the downstream cr1 or cr2 region promotes transcription. Human Ramos (undergoing spontaneous SHM) B cells and mouse CH12F3-2A (CSR inducible) B cells were transfected with the indicated pGL3-reporter gene vector to assess Aicda promoter activity. After transfection, CH12F3-2A cells were treated with LPS, IL-4 and TGF-β1 to induce CSR. Luciferase activity was measured after 16 h (Ramos) or 24 h (CH12F3-2A) of culture. Data are the means ± s.e. (bars) of 3 independent experiments.
Transcription factor binding to the Aicda promoter
To address the possibility that HoxC4 modulates Aicda expression by binding to a cis-regulatory element of this gene, we analyzed the sequence upstream of the putative transcriptional initiation site of the Aicda gene (Supplementary Fig. 5 online). In this, we identified eight motifs conserved in humans, chimps, mice, rats, dogs and cows (Supplementary Fig. 5 online, C1–C8). The first six motifs did not fulfill the minimal criteria for known transcription factor-binding sites by weight matrix search using Match™ (threshold score, 0.75). The last two (C7 and C8) consisted of a HoxC4 or Oct-binding 5'-ATTTGAAT-3' site (residues −29 to −22 in the human and mouse) (scores: 1.0 for HoxC4 and 0.93 for Oct), virtually identical to the conserved HoxC4-Oct motif we showed to be critical in inducing the human IGH 3' Eα enhancer elements24,25, and an upstream Sp-NF-κB 5'-GGGGAGGAGCC-3' site (residues −57 to −47 in the human and mouse)17 (scores: 0.93 for Sp1/Sp3 and 0.96 for NF-κB). This was first suggested to be a Pax5-binding site16, but it did not satisfy, in our analysis, the minimal requirement for such a binding site (score: 0.61). Both the putative HoxC4-Oct- and the Sp-NF-κB-binding sites are identical in all six species analyzed.
To determine the role of the –349 to –1 Aicda promoter region (AID-Pro) in the regulation of Aicda transcription, we constructed luciferase gene reporter pGL3 vectors consisting of the 349 bp AID-Pro and/or the 490 bp flanking 5'-region (5'R) (Fig. 7c) upstream of the luciferase gene, combined with nil, conserved region1 (cr1) and/or cr2, which lie in the first intron in the Aicda15,16. We used these vectors to transfect human Ramos B cells, which spontaneously express AICDA and undergo SHM, and mouse CH12F3-2A B cells, which express Aicda and undergo CSR upon stimulation by LPS, IL-4 and TGF-β1. We cultured these B cells and measured luciferase activity after 16 h (Ramos) or 24 h (CH12F3-2A). The pGL3-5'R-AID-Pro construct, containing both the Aicda promoter and its flanking 5'-region, displayed an 11–15-fold higher activity than the empty pGL3-nil vector, in Ramos and CH12F3-2A B cells (Fig. 7d). In both Ramos and CH12F3-2A B cells, neither cr1 nor cr2 displayed substantial enhancer activity. In addition, while the pGL3-AID-Pro construct, containing only Aicda promoter, promoted transcription as efficiently as the pGL3-5'R-AID-Pro construct, the pGL3-5'R construct, which included only the flanking 5' region, displayed only an (background) activity comparable to that of the empty pGL3 vector in both Ramos and stimulated CH12F3-2A B cells. These experiments show that the 349 bp AID-Pro region displays full promoter activity, while neither cr1 nor cr2 enhances Aicda promoter activity in human or mouse B cells.
To address the specificity of the two evolutionarily conserved 5'-ATTTGAAT-3' and 5'-GGGGAGGAGCC-3' motifs, we performed gel electrophoretic mobility shift assays (EMSAs), using WT and mutated oligonucleotide probes encompassing residues −65 to −14 of the human AICDA promoter sequence and containing both the HoxC4-Oct and the Sp-NF-κB sites (Sp-Hox), or an oligonucleotide probe encompassing residues −65 to −44 of the AICDA promoter sequence containing the Sp-NF-κB site (Sp) (Supplementary Fig. 6a online). Incubation of nuclear extracts from human 4B6 B cells, which spontaneously express AICDA and undergo CSR, or Ramos B cells with the Sp-Hox probe gave rise to four major protein–DNA complexes, A, A’ and B, B’ (Supplementary Fig. 6b,c). These were specific for the binding of HoxC4-Oct and Sp-NF-κB, respectively. The mutated oligonucleotides Sp-Hoxmut, Spmut-Hox, or Spmut-Hoxmut, in which the HoxC4-Oct site, the Sp-NF-κB site, or both of the two sites were disrupted, respectively, failed to compete efficiently the formation of complexes B and B’, A and A’, or all the four complexes. These results were confirmed by EMSAs involving the mutated oligonucleotides as radiolabeled probes; Sp-Hoxmut gave rise only complexes B and B’, Spmut-Hox yielded only A and A’, and Spmut-Hoxmut gave rise to none of the four complexes. Incubation of 4B6 or Ramos B cell nuclear extracts with the Sp probe gave rise to three major protein-DNA complexes, C, C’ and C”, specific for binding of Sp-NF-κB. The binding specificity of HoxC4, Oct1 and Oct2 to the 5'-ATTTGAAT-3' site, and the binding specificity of Sp1, Sp3 and NF-κB to the 5'-GGGGAGGAGCC-3' site were further proved by supershift or inhibition of formation of the respective protein-DNA complexes by specific mAb to HoxC4 and specific Abs to Oct1, Oct2, Sp1, Sp3 or the p52 subunit of NF-κB. No supershift or inhibition of protein-DNA complex involving the Sp-Hox or the Sp probe could be achieved using the Pax5-specific antibody. These experiments show that HoxC4, Oct1 and Oct2 bind specifically to the conserved 5'-ATTTGAAT-3' site, while Sp1, Sp3 and NF-κB, but not Pax5, bind specifically to the conserved 5'-GGGGAGGAGCC-3' site in the AICDA promoter.
Cis sites critical for Aicda promoter activation
To determine the contribution of the conserved HoxC4-Oct- and Sp-NF-κB-binding sites to the Aicda promoter activity, we constructed luciferase gene reporter vectors containing the mouse Aicda promoter sequence (residues −349 to −1), which was variously mutated or deleted at the HoxC4-Oct and/or the Sp-NF-κB site. In addition to the conserved HoxC4-Oct-binding 5'-ATTTGAAT-3' site, we identified a putative HoxC4-binding 5'-ATTT-3' site in the mouse and rat AID gene promoter (residues −155 to −158 of mouse Aicda), but not in the human, chimp or dog AID gene promoter (Supplementary Fig. 5 online). Deletion of this site (mut0) did not alter the Aicda promoter activity (Fig. 8a). By contrast, deletion of the Hoxc4-Oct motif (mut1) reduced promoter activity by 71%, 64% and 88% in LPS, IL-4 and TGF-b induced mouse CH12F3-2A B cells, human 4B6 and Ramos B cells, respectively. To determine the relative contribution of HoxC4- and Oct-binding to the promoter activity of the HoxC4-Oct motif as a whole, we mutated 5'-ATTTGAAT-3' to 5’-cTTTGAAT-3' (mut2), thereby disrupting the binding of HoxC4 but retaining Octbinding25, or to 5'-ATTTGccg-3' (mut3), thereby abrogating the binding of Oct1–Oct2 but not HoxC425. Mutation of the HoxC4 motif in the HoxC4-Oct site reduced transcription by 47%, 36%, or 55% in CH12F3-2A, 4B6 and Ramos B cells, respectively, while mutation of the Oct site reduced the promoter transcription by 28%, 21% and 55%, respectively. Thus, both the HoxC4 and the Oct motifs of the HoxC4-Oct site contribute to the Aicda promoter activity, as further confirmed by the up to 88% loss of Aicda promoter activity when the whole Hoxc4-Oct site was deleted (mut1). Further, mutation of the conserved Sp-NF-κB site to 5'-aaaaAGGAaa-3' (mut4) reduced promoter activity by 73%, 80% and 63% in CH12F3-2A, 4B6 and Ramos B cells, respectively. Accordingly, deletion of this site (mut5) resulted in 85%, 68% and 82% reduction in promoter activity in CH12F3-2A, 4B6 or Ramos B cells, respectively. Finally, deletion of the HoxC4-Oct site combined with mutation (mut6) or deletion (mut7) of the Sp-NF-κB site abrogated the Aicda promoter activity. These experiments show that the conserved HoxC4-Oct-binding site plays a major role in Aicda promoter activity and, together with the conserved Sp-NF-κB-binding site, is indispensable for full Aicda transcriptional activation.
The conserved HoxC4-Oct- and Sp-NF-κB-binding sites are essential for full Aicda promoter activity; HoxC4, Oct1, Oct2, OcaB, Pax5, Sp1, Sp3 and NF-κB (p52 subunit) are recruited to the Aicda promoter in B cells expressing AICDA/Aicda and undergoing CSR or SHM. (a) pGL3-Enhancer luciferase gene reporter constructs containing WT or mutant Aicda promoter, in which the HoxC4-Oct- and/or Sp-NF-κB-binding sites were deleted or disrupted by site-directed mutagenesis, were used to transfect CH12F3-2A, 4B6 or Ramos B cells. Luciferase activity was measured after 24 h (CH12F3-2A B cells) or 16 h (Ramos and 4B6 B cells) of culture. Data are means ± s.e. (bars) of 3 independent experiments. (b) AICDA/Aicda expression in human spontaneously switching 4B6 and Ramos B cells, human inducible switching 2E2 B cells stimulated with nil or anti-CD40 mAb and IL-4, mouse CH12F2-2A B cells stimulated with nil or LPS, IL-4 and TGF-β1, WT C57BL/6 mouse spleen B cells stimulated nil, LPS and IL-4, or CD154 and IL-4 were analyzed by semi-quantitative RT-PCR using serially two-fold diluted cDNA as a template. Data are representative of 3 independent experiments. (c) Cross-linked chromatin was precipitated from the human or mouse B cells of panel b using a mouse mAb specific to HoxC4, rabbit Abs specific to Oct1, Oct2, OcaB, Pax5, Sp1, Sp3 or p52, or preimmune control mouse or rabbit IgG. The precipitated DNA was specified by PCR using AICDA or Aicda promoter primers. Data are representative of 3 independent experiments.
To confirm the relevance of our EMSA and luciferase gene reporter experiments, we precipitated chromatin in human 4B6 and Ramos B cells using Abs to HoxC4, Oct1, Oct2, OcaB, Sp1, Sp3 or NF-κB (p52). In the DNA precipitated by all these Abs, we readily specified the AICDA promoter sequence (Fig. 8b,c). The specificity of these findings was further proved by our ability to readily detect AICDA or Aicda promoter DNA in ChIP assays involving human 2E2 B cells, which can be induced to express AID and undergo CSR by anti-CD40 mAb and cytokines, such as IL-4, mouse CH12F3-2A B cells, which can be induced to express AID and undergo CSR by LPS, IL-4 and TGF-β1, as well as spleen B cells from wild-type C57BL/6 mice activated by LPS and IL-4 or CD154 and IL-4. Induction of CSR in 2E2, CH12F3-2A B cells or primary mouse spleen B cells by these stimuli resulted in substantial Hoxc4 expression and recruitment of HoxC4, Oct1, Oct2, OcaB, Sp1, Sp3 and NF-κB to the AICDA and Aicda promoters. Because it has been suggested that Pax5 is (indirectly) recruited to the Aicda promoter16, we precipitated chromatin in these human and mouse B cells with an antibody to Pax5. In these immunoprecipitated DNA complexes, we readily detected the AICDA or Aicda promoter sequences, respectively. These findings show that HoxC4, Oct1, Oct2, OcaB, Sp1, Sp3 and NF-κB proteins are recruited to the AICDA and Aicda promoters in human and mouse B cells, respectively, which express AID and undergo CSR or SHM.
Enforced AID expression rescues CSR in Hoxc4−/− B cells
We then set up to demonstrate that the defective CSR in HoxC4-deficient B cells was actually due to impairment of AID expression and not other HoxC4-dependent activity. To this end, we enforced expression of AID in Hoxc4−/− B cells to restore CSR. We transduced LPS-activated spleen Hoxc4+/+ and Hoxc4−/− B cells with TAC-Aicda or TAC control retroviral vector, stimulated them with LPS and IL-4 for 72 h and 96 h before analyzing CSR. The TAC control retroviral construct encoding human IL-2 receptor (hIL2RA, TAC antigen) and the AID-expression TAC-Aicda retroviral construct encoding AID and hIL-2RA were described15 (Supplementary Fig. 7, online). Consistent with what we saw in untransduced Hoxc4+/+ and Hoxc4−/− B cells, CSR was greatly reduced in Hoxc4−/− B cells transduced with the TAC control retrovirus as compared to theirs transduced Hoxc4+/+ counterparts (Fig. 9a,b). In Hoxc4+/+ B cells, enforced expression of AID increased CSR to IgG1 by about 50%. TAC-Aicda retrovirus-transduction of Hoxc4−/− B cells increased Aicda expression. It did not modulate the expression of germline Iµ-Cµ and Iγ1-Cγ1 transcripts, but increased CSR to IgG1 to a level comparable to that of TAC-Aicda retrovirus-transduced Hoxc4+/+ B cells, as shown by the increased proportion of surface IgG+ B cells and more circle Iγ1-Cµ and post-recombination Iµ-Cγ1 transcripts. These experiments show that defective AID expression and CSR in Hoxc4−/− B cells are rescued by enforced AID expression further indicating that HoxC4 modulates CSR by regulating AID expression.
Enforced expression of AID rescues CSR in Hoxc4−/− B cells. Hoxc4+/+ and Hoxc4−/− B cells activated with LPS were transduced with the TAC control or AID-expression TAC-Aicda retrovirus and cultured in the presence of LPS and IL-4. Three or 4 d after transduction, B cells were harvested for analysis of surface expression of B220 and IgG1 (a) and expression of Aicda by real-time qRT-PCR and semi-quantitative RT-PCR, germline Iµ-Cµ and Iγ1-Cγ1 transcripts by real-time qRT-PCR, circle Iγ1-Cµ transcripts by semi-quantitative RT-PCR and post-recombination Iµ-Cγ1 transcripts by real-time qRT-PCR (b). Expression of these transcripts was normalized in each case to CD79b transcripts; expression of transcripts in Hoxc4−/− B cells were depicted as ratios to those in Hoxc4+/+ B cells. FACS data are from one representative of 5 independent experiments. Real-time qRT-PCR and semi-quantitative RT-PCR data are means ± s.e. (bars) of 3 independent experiments.
DISCUSSION
In B cells, AID expression is tightly regulated31–34, possibly in an activation-dependent manner in conditions under which CSR and SHM unfold. The specificity and the level of AID expression are likely controlled by a complex combination of different tissue specific transcription factors, both activators and repressors. Here we have provided evidence that by binding to the highly conserved 5'-ATTTGAAT-3' motif in the Aicda promoter, HoxC4 activates this promoter, thereby modulating AID expression, CSR and SHM. In this function, HoxC4 synergizes with Oct1–Oct2, which also binds to the 5'-ATTTGAAT-3' motif, in a fashion similar to that we reported for the HoxC4-Oct-mediated activation of the human 3'Eα hs1,2 enhancer element25. Accordingly, Hoxc4−/− mice or B cells display defective CSR and SHM, despite normal levels of mature VHDJH and germline IH-CH transcripts31. The efficient rescue of CSR by enforced expression of AID in Hoxc4−/− B cells indicates that induction of AID is the major pathway through which HoxC4 regulates CSR and, likely, SHM. Mutation of the 5'-ATTTGAAT-3' site to 5'-gcTTGAATT-3', which did not disrupt Oct-binding and introduced a putative Sp site, did not seemingly alter Aicda promoter activity in mouse B-lymphoma M12 and CH33 and human embryonic kidney fibroblast 293 cells16. In our experiments, mutation of 5'-ATTTGAAT-3' to disrupt HoxC4-binding, while preserving the Oct-binding activity, or to abrogate the binding of Oct1–Oct2 but not that of HoxC425, reduced Aicda transcription, thereby highlighting the role of these homeodomain transcription factors in AID expression and adding another dimension to the function of Oct1–Oct2 in B cell differentiation.
As we have shown in human B cells, HoxC4 plays an important and complex role in the regulation of the IgH locus24,25,35. The putatively dampening effect of HoxC4 on the baseline activity of the Iγ and Iε promoters would be effectively lifted and overridden by the strong activation of these promoters by CD40-signaling and CD154-induced HoxC4-mediated activation of the hs1,2 enhancer element24,25,35. The role of HoxC4 in the induction of AID expression is further strengthened by our demonstration that, like AID, HoxC4 is preferentially expressed in GC B cells of both humans24 and mice (these results), and CD154:CD40 engagement and cytokines, which induce the expression of AICDA and Aicda, also induce HOXC4 and Hoxc4 in human24 and mouse B cells, respectively (these results). Further, NF-κB-binding sites in both the human and mouse HOXC4 and Hoxc4 promoters underpin the upregulation of HoxC4 by CD40 signaling.
Consistent with the notion that the defect in CSR and SHM manifested by Hoxc4−/− B cells was due to a failure to induce Aicda expression, both Aicda transcripts and AID protein, as induced by LPS and IL-4 or CD154 and IL-4, were significantly decreased in Hoxc4−/− B cells. The tissue- and differentiation stage-specificity of HoxC4 expression would account to a great extent for the precise regulation of AID expression. In Hoxc4−/− mice, the decreased AID expression was reflected in vivo into the impairment of the maturation of the T-dependent antibody response. In these mice, although Aicda expression was significantly reduced, GC formation was seemingly normal. This is reminiscent of Aicda+/− mice36, which also show normal GCs in the presence of significantly decreased Aicda expression37,38 and contrasts with Aicda−/− mice, in which activated B cells accumulate and form giant GCs36.
The B cell lineage-specific Pax5 transcription factor has been suggested to play a role in Aicda expression by binding to the 5'-GGGGAGGAGCC-3' site in the Aicda promoter16. This cis-element, however, does not fulfill the requirements of a consensus Pax5-binding motif and did not bind Pax5 in our experiments and those by others17. Our finding on the lack of specificity of the conserved 5'-GGGGAGGAGCC-3' cis-element for Pax5 suggests that the recruitment of Pax5 to the Aicda promoter, as revealed by ChIP assays, occurred indirectly, through other DNA-binding transcription factors, perhaps, Sp1 or Sp3, in a fashion similar to the interaction between estrogen receptor and Sp1 on certain estrogen-responsive promoters39.
The B cell development-related E47 transcription factor has been suggested to contribute to the enhancement of the Aicda promoter activity by binding to E-boxes in the cr2 of the first intron of this gene21,22. E47-induced enhancement of the Aicda promoter activity would be modulated by the E2A inhibitor Id315. The presence of cr2 in human Ramos and mouse CH12F3-2A B cells (our findings), and in mouse BaF/3 pro-B cells or M12 B cells16 did not enhance luciferase reporter transcription, as driven by the Aicda promoter. This might reflect the dispensability of E2A transcription factors in AICDA and Aicda expression, CSR, and possibly, SHM, as shown by analysis of E2A-deficient B cells40,41, or a muted baseline activity of cr2, resulting from a preferential binding of Id2 and/or Id3 to this region. These experiments, however, cannot rule out the possibility that, like the IgH and Igκ intronic enhancers, which contain multiple E-box sites and show a high enhancer activity in GC B cells42, cr2 and E-proteins act together with HoxC4 to synergistically induce AID expression.
The HoxC4-mediated Aicda promoter activation is further enhanced by the upstream conserved 5'-GGGGAGGAGCC-3' site, which, as we have also shown, recruits Sp1, Sp3 and NF-κB. Sp1 and Sp3 bind directly to DNA through their zinc finger motifs and enhance gene transcription. These proteins are ubiquitously expressed and are directly involved not only in the regulation of basal transcription and expression of housekeeping genes, but also in developmentally controlled gene expression. In our experiments, the Sp-NF-κB site could partially mediate Aicda promoter activity in the absence of the HoxC4-Oct1–Oct2 site, and possibly accounted for the residual AID expression, CSR and SHM in Hoxc4−/− B cells and mice.
We showed that OcaB was also recruited to the AICDA and Aicda promoters in B cells undergoing CSR or SHM, likely through interaction with Oct1–Oct243. By clamping the Oct1–Oct2 POUH and POUS subdomains, OcaB would increase the affinity of these homeodomain proteins for DNA, thereby potentiating HoxC4- and Oct1–Oct2-mediated Aicda promoter activation. As we have shown, OcaB plays an important role in HoxC4 and Oct1–Oct2-mediated activation of the human 3’Eα enhancer hs1,2 element25. Accordingly, mice lacking OcaB show an impairment in CSR43. The present experiments unveil another role for Oct1–Oct2 and OcaB activity: the regulation of AID expression. Overall, our findings offer fundamental insights into the mechanisms of activation of the AICDA and Aicda promotera and induction of AID expression, CSR and SHM. The possibility that the induction of HoxC4 by stimuli other than CD40-signaling, LPS and cytokines, such as hormone (our unpublished data), modulates AID expression and, therefore, antibody diversification in health and disease should be addressed.
METHODS
Hoxc4−/− mice
In these Hoxc4−/− mice (A.M. Boulet and M.R. Capecchi, HHMI, University of Utah, Salt Lake City, UT), Hoxc4 was disrupted through insertion of a loxP site in exon 2 of this gene at the coding sequence for the amino-terminal end (between the third and the fourth codons) of the homeobox, which introduces a stop codon at the insertion site, yielding a nonfunctional truncated protein (lacking 95% of the homeodomain) (unpublished findings). We obtained this Hoxc4+/− frozen sperm and re-derived Hoxc4+/− mice by in vitro fertilization through the services of the UC Irvine transgenic mouse facility. These Hoxc4+/− mice are in the C57BL/6 background after backcrossing the 129Sv/Ev founder strain with C57BL/6 mice. Hoxc4−/− mice and their WT littermates have been bred under pathogen-free conditions. The Institutional Animal Care and Use Committee (IACUC) of University of California, Irvine, CA, approved all animal experiments.
B and T cells
The number of B cells (B220+) and T cells (CD3+), the proportion CD4+ T cells, dead B and T cells and the proportion of PNAhi B cells, plasma cells (B220lo CD138+) and NP-binding CD38hi IgG1+memory B cells were determined by FACS analysis using a FACSCalibur ™ flow cytometer (BD Biosciences). Single cell suspensions were prepared from spleens or Peyer’s patches of Hoxc4−/− and Hoxc4+/+ mice and stained with Phycoerythrin (PE)-labeled anti-mouse B220 mAb (clone RA3-6B2) (eBioscience Corp.), fluorescein isothiocyanate (FITC)-labeled anti-mouse CD3 mAb (clone 17A2) (BioLegend, Inc.), PerCP-anti-mouse CD4 mAb (clone GK1.5) (BioLegend, Inc.), 7-AAD (BD Biosciences), FITC-PNA, FITC-anti-mouse CD138 mAb (clone 281-2) or APC-anti-mouse IgG1 (clone X56) (BD Biosciences), PE-NP (Biosearch Technologies, Inc.) and PECy7-anti-mouse CD38 mAb (clone 90) (eBiosciences Corp.). Single B220+ cell suspensions were prepared from spleens or Peyer’s patches using the EasySep® Mouse B Cell Enrichment Kit (StemCell Technologies Inc.). For the preparation of PNAhi (GC) B cells, spleen or Peyer's patches B cells were stained with PE-anti-mouse CD45R (B220) mAb (clone RA3-6B2) (BD Biosciences) and FITC-PNA. Labeled lymphocytes were then sorted using a MoFlow™ cell sorter (Dako), yielding 95% pure PNAhi B220+ cells.
B cell lines
Ramos B cells were monitored for spontaneous SHM44. Monoclonal 4B6 and 2E2 B cell lines were derived from our CSR- and SHM-inducible human monoclonal IgM+IgD+ CL-01 B cell line45–50 by sequential subcloning and selection for spontaneous and inducible CSR, respectively. 4B6 B cells are IgM+IgD+ with an "early" GC phenotype and undergo spontaneous CSR to IgG, IgE, and IgA24. 2E2 B cells are IgM+IgD+ and undergo CSR to IgG, IgE, and IgA upon stimulation by an agonistic anti-huCD40 mAb and appropriate cytokines51. The mouse B lymphoma cell line CH12F3-2A was obtained from T. Honjo (Kyoto University, Japan). CH12F3-2A cells are surface IgM+ and switch to IgA upon the stimulation with CD154 or LPS in the presence of IL-4 and TGF-β152. All these monoclonal B cells were cultured in RPMI 1640 medium (Invitrogen Corp.) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), 2 mM L-glutamine, and 1x antibiotic-antimycotic mixture (100 units/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin B fungizone) (Invitrogen Corp.) (FBS-RPMI).
B cell cycle and proliferation
Cell cycle was analyzed by PI staining50. Proliferation was analyzed using the CellTrace™ CFSE Cell Proliferation Kit (Molecular Probes Co.). Cells were washed in serum-free HBSS (Invitrogen Corp.) and resuspended at 1 × 106 cells/ml. After adding an equal volume of 2.4 mM CFSE, cells were incubated at 37 °C for 12 min and then washed in FBS-RPMI. Cells were then diluted and cultured in the presence or absence of LPS (20 µg/ml) from E. coli (serotype 055:B5) (Sigma-Aldrich Co.) and recombinant mouse (rmo)IL-4 (5 ng/ml; R&D Systems, Inc.), harvested at various time points after activation and analyzed by FACS. For in vivo B cell proliferation, mice were immunized with NP16-CGG. After 10 d, they were injected intraperitoneally twice within 16 h with BrdU (1 mg) and sacrificed 4 h after the last injection. Cells from the spleen or Peyer's patches were stained with PE-anti-mouse B220 mAb (BD Biosciences) or this mAb together with FITC-PNA. Incorporated BrdU was stained with APC-anti-BrdU mAb using APC BrdU Flow Kit (BD Biosciences) and analyzed by FACS.
Analysis of in vitro CSR
Enriched spleen B cells were cultured at 1 × 106 cell/ml in FBS-RPMI with 0.05 mM β-mercaptoethanol. Cells were stimulated with LPS (20 µg/ml) or CD154-expressing membrane fragments of baculovirus-infected Sf21 insect cells (referred to CD154)53 and: with (i) nil for CSR to IgG3 and IgG2b; (ii) rmIL-4 (5 ng/ml) for CSR to IgG1 and IgE; (iii) IFN-γ (50 ng/ml; PeproTech Inc.) for CSR to IgG2a; and (iv) TGF-β1 (1 ng/ml; R&D Systems, Inc.), rmIL-5 (5 ng/ml; R&D Systems, Inc.), and anti-δmAb-dextran (3 ng/ml; provided by C. M. Snapper, Uniformed Services University of the Health Sciences, Bethesda, MD) for CSR to IgA. Cells were collected on day 4 for surface Ig analysis, after staining with FITC-anti-mouse IgG1 (clone A85-1), anti-mouse IgG2a (clone R19-15), anti-mouse IgG2b (clone R12-3) anti-mouse IgG3 (clone R40-82) or anti-mouse IgA (clone C10-3) rat mAb and PE-anti-mouse CD45R (B220) (clone RA3-6B2) rat mAb (BD Biosciences). Cells were fixed with 1% paraformaldehyde in PBS and analyzed by FACS. Specific ELISAs involving 96 well plates coated with polyclonal goat Ab F(ab)2 against the respective mouse isotype (SouthernBiotechnology Associates, Inc.) were used to measure IgG1, IgG2a, IgG2b, IgG3, IgA and IgE in culture supernatants of in vitro stimulated spleen Hoxc4+/+ and Hoxc4−/− B cells. Supernatants were serially twofold diluted from 1:5 to 1:640, and then added (100 µl per well) to the 96-well plates. Plates were incubated for 1 h at 25°C. After washing, biotin-labeled isotype-specific mAbs were added and then revealed using HRP-streptavidin, as described above. The concentration of the different Ig isotypes was determined by interpolation using a calibrated standard curve for each isotype. The assays were performed in triplicates.
Quantitative real-time RT-PCR (qRT-PCR) and semiquantitative RT-PCR
RNA was extracted using the RNeasy Mini Kit (Qiagen Inc.) according to the manufacturer’s protocol. Residual DNA was removed by treatment with DNase I (Invitrogen Corp.). First strand cDNAs were synthesized from equal amounts of total RNA (2 µg) using the SuperScript™ Preamplification System and oligo (dT) primer (Invitrogen Corp.). The expression of germline IH-CH, post-recombination Iµ-CH, mature VJ558DJH-Cµ, Hoxc4 and Aicda transcripts was quantified by real-time qRT-PCR54 using appropriate primers (Operon Corp.) (Supplementary Table 1 online). In some cases, circle Iγ1-Cµ transcripts, HOXC4, Hoxc4, AICDA and Aicda transcripts were analyzed by specific semiquantitative RT-PCR by performing serial twofold dilutions so that there was a virtually linear relationship between the amount of cDNA used and the intensity of the PCR product. Real-time qRT-PCR analysis was performed using an DNA Engine Opticon 2 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) to measure SYBR-green (DyNAmo HS SYBR Green, New England Biolabs, Inc.) incorporation with the following protocol: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 10 sec, 60 °C for 20 sec, 72 °C for 30 sec, 80 °C for 1 sec, and data acquisition at 80 °C, and 72 °C for 10 min. Melting curve analysis was performed from 72°–95 °C and samples were incubated for another 5 min at 72 °C. The ΔΔCt method was used for data analysis.
Analysis of somatic mutations in intronic JH4-iEµ DNA
Peyer’s patch B cells were obtained from non-intentionally immunized 12-week-old littermate Hoxc4−/− and Hoxc4+/+ C57BL/6 mice and used to analyze somatic-mutations in the intronic DNA downstream of rearranged VJ558DJH4 genes. Platinum® Pfx DNA polymerase (Invitrogen Corp.) was utilized for genomic DNA amplifications. The intronic IgH region downstream of rearranged VJ558DJH was amplified using nested PCR55 involving two VH J558 framework region (FR)3-specific forward primers, and two reverse primers specific for sequences downstream of JH4 (Supplementary Table 1).yielding an approximately 900 bp DNA if a JH4 rearrangement occurred. PCR conditions were 94°C for 45 sec, 58°C for 45 sec, 68°C for 1 min for 35 cycles. PCR products were cloned into the pCR-Blunt II-TOPO® vector (Invitrogen Corp.) and sequenced. Only sequences from rearrangements involving JH4-iEµ were analyzed. Sequences were analyzed using the MacVector™ 7.2.3 software (MacVector, Inc.).
Identification of putative transcription factor-binding sites
The putative transcription factor binding sites in the AID gene promoter sequence were identified by weight matrix search using Match™ (http://www.gene-regulation.com/cgi-bin/pub/programs/match/bin/match.cgi) (BIOBASE Corp.) which integrates TRANSFAC® 6.0 and uses its positional weight matrices for analysis. Scores indicate the degree of fitness of the putative binding site with the consensus sequence: score 1.0 = 100%; the cut-off score used was 0.75.
Aicda promoter luciferase gene reporter assays
The reporter constructs were consisted of the pGL3-Basic (experiments of Fig. 6d) or pGL3-Enhancer (experiments of Fig. 8a) Firefly luciferase gene reporter vector (Promega Corp.) and different Aicda locus DNA sequences. An 839-bp 5’R-AID-Pro, 490 bp 5’R or 349 bp AID-Pro sequences and first intron conserved regions (cr1 and cr2) were amplified by PCR from C57BL/6 mouse genomic DNA, and were inserted in upstream and/or downstream of the luciferase gene in pGL3 vector. Various mutant reporters were constructed by QuikChange™ Site-Directed Mutagenesis (Stratagene). Sequences of constructs were confirmed by at least two sequencing reactions. Co-transfection of the reporter construct and the constitutively active Renilla reniformis luciferase producing vector pRL-TK (Promega, Corp.) in human Ramos, 4B6 and mouse CH12F3-2A B cells was performed by electroporation24, 25, 35. Firefly and Renilla reniformis luciferase activities were measured using the Dual-Luciferase® Reporter Assay System (Promega, Corp.) according to manufacturer’s instructions.
Chromatin immunoprecipitation assays (ChIP)
B cells (5 × 107) were treated with 1% formaldehyde for 10 min at 25 °C to cross-link chromatin. After washing with cold PBS containing protease inhibitors (Roche, Basel, Switzerland), chromatin was separated using nuclear lysis buffer (10 mM Tris-HCl, 1 mM EDTA, 0.5 M NaCl, 1% Triton-X-100, 0.5% sodium deoxycholate, 0.5% Sarcosyl, pH 8.0) and resuspended in IP-1 buffer (20 mM Tris-HCl, pH 8.0 200 mM NaCl, 2 mM EDTA, 0.1% sodium deoxycholate, 0.1% SDS, protease inhibitors). Chromatin was sonicated to yield approximately 200–1000 bp DNA fragments, precleared with agarose beads bearing protein G (Santa Cruz Biotechnology, Inc.) and then incubated with mouse mAbs to HoxC4, or rabbit polyclonal Abs to Oct1, Oct2, OcaB, Pax5, Sp1, Sp3, or NF-κB (p52 subunit) at 4 °C. After overnight incubation, immune complexes were isolated using agarose-beads bearing protein G, eluted with Elution buffer (50 mM Tris-HCl, 0.5% SDS, 200 mM NaCl, 100 µg/ml Proteinase K, pH 8.0), and then incubated at 65 °C overnight to reverse formaldehyde cross-links. DNA was extracted by phenol/chloroform and precipitated by ethanol, and then resuspended in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). DNA sequences were specified by PCR using appropriate primers (Supplementary Table 1 online).
Retroviral transduction of B cells
The TAC and TAC-Aicda retroviral constructs15 containing the human IL2RA gene encoding CD25 (TAC antigen) or this gene together with the mouse Aicda coding sequence were obtained from C. Murre (University of California, San Diego). To generate retrovirus, the pCSretTAC-based constructs were transfected into the HEK-293T packaging cell line, using ProFection® Mammalian Transfection System (Promega Corp.). The retroviral constructs were used to transduce mouse spleen B cells as reported15.
Statistical analyses
The differences in frequency and spectrum of mutations in Hoxc4−/− and Hoxc4+/+ mice were analyzed using the χ2 test. The differences in Ig titers, CSR or mRNA expression were analyzed using paired t-tests.
ACKNOWLEDGEMENTS
We thank M.R. Capecchi and A.M. Boulet for kindly providing us with the Hoxc4+/− mouse frozen sperm, T. Fielder for re-deriving Hoxc4+/− mice, C.M. Snapper for the anti-δ-mAb-dextran, C. Murre for the Aicda retroviral construct, T. Honjo for CH12F3-2A cells and Z. Yu for statistic analysis. We are also grateful to A. Schaffer for discussion, S. Sabet and M. Kang for excellent technical assistance. This work was supported by NIH grants AI 045011, AI 079705 and AI 060573 to P.C., and Korea Research Foundation grant KRF-2005-214-C00131 to S.-R. P.