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. 2010 Nov 26;285(48):37797-810.
doi: 10.1074/jbc.M110.169086. Epub 2010 Sep 20.

Estrogen receptors bind to and activate the HOXC4/HoxC4 promoter to potentiate HoxC4-mediated activation-induced cytosine deaminase induction, immunoglobulin class switch DNA recombination, and somatic hypermutation

Thach Mai  1 , Hong Zan, Jinsong Zhang, J Seth Hawkins, Zhenming Xu, Paolo Casali
Affiliations

Estrogen receptors bind to and activate the HOXC4/HoxC4 promoter to potentiate HoxC4-mediated activation-induced cytosine deaminase induction, immunoglobulin class switch DNA recombination, and somatic hypermutation

Thach Mai et al. J Biol Chem. .

Abstract

Estrogen enhances antibody and autoantibody responses through yet to be defined mechanisms. It has been suggested that estrogen up-regulates the expression of activation-induced cytosine deaminase (AID), which is critical for antibody class switch DNA recombination (CSR) and somatic hypermutation (SHM), through direct activation of this gene. AID, as we have shown, is induced by the HoxC4 homeodomain transcription factor, which binds to a conserved HoxC4/Oct site in the AICDA/Aicda promoter. Here we show that estrogen-estrogen receptor (ER) complexes do not directly activate the AID gene promoter in B cells undergoing CSR. Rather, they bind to three evolutionarily conserved and cooperative estrogen response elements (EREs) we identified in the HOXC4/HoxC4 promoter. By binding to these EREs, ERs synergized with CD154 or LPS and IL-4 signaling to up-regulate HoxC4 expression, thereby inducing AID and CSR without affecting B cell proliferation or plasmacytoid differentiation. Estrogen administration in vivo significantly potentiated CSR and SHM in the specific antibody response to the 4-hydroxy-3-nitrophenylacetyl hapten conjugated with chicken γ-globulin. Ablation of HoxC4 (HoxC4(-/-)) abrogated the estrogen-mediated enhancement of AID gene expression and decreased CSR and SHM. Thus, estrogen enhances AID expression by activating the HOXC4/HoxC4 promoter and inducing the critical AID gene activator, HoxC4.

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Figures

FIGURE 1.
FIGURE 1.
Estrogen enhances CD40 and IL-4-mediated HOXC4, AICDA expression, and CSR. Human 2E2 B cells were treated with E2 at the indicated concentrations in the presence or absence of anti-CD40 mAb and rhIL-4. A, E2 alone does not increase HOXC4 or AICDA mRNA expression. B, E2 enhanced in a dose-dependent manner HOXC4 and AICDA expression in 2E2 B cells stimulated by anti-CD40 mAb and rhIL-4. C, E2 enhanced anti-CD40 mAb and rhIL-4-induced CSR to IgG1, as measured by circle Iγ1-Cμ and mature VHDJH-Cγ1 transcripts. 2E2 B cells were cultured with the indicated concentration of E2 in the presence of nil or anti-CD40 mAb and rhIL-4 for 48 h. HOXC4, AICDA, circle Iγ1-Cμ, and mature VHDJH-Cγ1 transcripts were measured by real-time qRT-PCR using SYBR Green and normalized to GAPDH expression. Their expression level is depicted relative to that in 2E2 B cells cultured with anti-CD40 mAb and rhIL-4 in the absence of E2, set as 1. Data are from three independent experiments (mean ± S.E. (error bars)). D, E2 does not alter B cell viability. 2E2 B cells were cultured with indicated concentration of E2 in the presence of anti-CD40 mAb and rhIL-4 for 4 days and labeled with 7-AAD before flow cytometry analysis; the cells not stained by 7-AAD were viable. Data are from three independent experiments (mean ± S.E.). E, E2 does not alter B cell proliferation. 2E2B cells were labeled with CFSE, cultured with E2 at the indicated concentrations together with nil or anti-CD40 mAb and rhIL-4, and harvested 4 days later for flow cytometry analysis. Data are representative of three independent experiments.
FIGURE 2.
FIGURE 2.
HoxC4 deficiency impairs estrogen-mediated enhancement of AID expression and CSR to IgG1 and IgE, as induced by LPS and IL-4 or CD154 and IL-4. A, HoxC4+/+ and HoxC4−/− B cells were stimulated with LPS and rmIL-4 or with CD154 and rmIL-4 in the presence of nil or E2 (10 nm) for 4 days and then analyzed for surface IgG1+ or IgE+. Data are representative of three independent experiments. B and C, HoxC4 deficiency does not alter the levels of germ line Iμ-Cμ, Iγ1-Cγ1, and Iϵ-Cϵ transcripts but significantly impairs E2-mediated enhancement of circle Iγ1-Cμ and Iϵ-Cμ, postrecombination Iμ-Cγ1 and Iμ-Cϵ transcripts (B), and Aicda expression (C). HoxC4+/+ and HoxC4−/− B cells were stimulated with LPS and rmlL-4 or CD154 and rmlL-4 in the presence or absence of E2 (10 nm) for 48 h. Germline Iμ-Cμ, Iγ1-Cγ1 and Iϵ-Cϵ, circle Iγ1-Cμ and Iϵ-Cμ, post-recombination Iμ-Cγ1 and Iμ-Cϵ transcripts, and HoxC4 and Aicda transcripts were measured by qRT-PCR using SYBR-green; expression is normalized to CD79b levels and is presented as relative folds of enhancement by E2. Data are from three independent experiments (mean ± S.E. (error bars)). D, E2 enhances expression of HoxC4 and AID proteins. Left, HoxC4+/+ B cells stimulated with LPS and rmIL-4 for 0, 12, 24, or 48 h; right, HoxC4+/+ and HoxC4−/− B cells stimulated with nil or with LPS and rmIL-4 in the presence of E2 at the indicated concentrations for 48 h. HoxC4, AID, and β-actin proteins were detected by immunoblotting. Data are representative of three independent experiments. E, E2 does not alter expression of ERα. HoxC4+/+ and HoxC4−/− B cells were stimulated with LPS and rmIL-4 or CD154 and rmIL-4 in the presence or absence of E2 (10 nm) for 48 h; ERα and β-actin proteins were then detected by immunoblotting. Data are representative of three independent experiments. F, E2 does not alter B cell viability. HoxC4+/+ and HoxC4−/− B cells were stimulated with LPS and rmIL-4 or with CD154 and rmIL-4 in the presence of E2 at the indicated concentrations for 3 days and then stained with 7-AAD before flow cytometry analysis; the cells not stained by 7-AAD were viable. Data are from three independent experiments (mean ± S.E.). G, E2 does not alter B cell proliferation. HoxC4+/+ and HoxC4−/− B cells were labeled with CFSE and stimulated with LPS and rmIL-4 or CD154 and rmIL-4 in the presence of E2 at the indicated concentrations for 4 days. Data are representative of three independent experiments. H, E2 does not alter plasma cell differentiation. HoxC4+/+ and HoxC4−/− B cells were stimulated for 4 days with LPS and rmIL-4 or with CD154 and rmIL-4 in the presence or absence of E2 (10 nm). The numbers in outlined areas indicate the percentage of B220loCD138+ (plasma) cells among total cells. Data are representative of three independent experiments. NS, not significant.
FIGURE 3.
FIGURE 3.
Estrogen enhances HoxC4 and Aicda expression and CSR in HoxC4+/+ but not HoxC4−/− Mice. A, HoxC4+/+ and HoxC4−/− littermate mice were injected with Oil or E2-Oil once a day for 11 days; 1 day after the first Oil or E2-Oil injection, the mice were immunized with NP16-CGG. B, spleen B220+PNAhi GC B cells from these mice were analyzed for surface IgG1 expression. C and D, RNA extracted from spleen, lymph nodes and Peyer's patches was analyzed by real-time qRT-PCR for postrecombination Iμ-Cγ1, HoxC4, and Aicda transcripts; these were normalized to CD79b transcript level and depicted as relative to the expression of Iμ-Cγ1, HoxC4, and Aicda transcripts in Oil-injected HoxC4+/+ mice, set as 1. Data are from three independent experiments (mean ± S.E. (error bars)). E, proportion of spleen B220+PNAhi GC B cells as a percentage of total B220+ cells. F, surface expression of B220 and CD138 on spleen cells. The numbers in outlined areas indicate B220loCD138+ (plasma) cells as a percentage of total B220+ cells. G, the viability of spleen cells was analyzed by 7-AAD staining; the cells not stained by 7-AAD were viable. H, Mice were injected intraperitoneally twice within 16 h with BrdU and sacrificed 4 h after the last injection. The proliferation of spleen B cells was analyzed by the incorporation of BrdU, as stained with APC-anti-BrdU mAb using the BrdU Flow Kit (BD Biosciences). Data are representative of three independent experiments.
FIGURE 4.
FIGURE 4.
Estrogen increases Ig somatic mutations in HoxC4+/+ but Not HoxC4−/− Mice. V186.2DJH-Cγ1 transcripts were amplified from the B cells of the three sets of HoxC4+/+ and HoxC4−/− littermate female mice used in the experiments of Fig. 3 (two HoxC4+/+ and two HoxC4−/− littermates for each set, injected daily for 11 days with Oil or E2-Oil and immunized with NP16-CGG 1 day after the first Oil or E2-Oil injection). A, pie charts depict the proportion of sequences that carry 1, 2, 3, etc. mutations over the 294-bp V186.2 cDNA of V186.2DJH-Cγ1 transcripts analyzed. The numbers of sequences analyzed are at the centers of the pie charts. B, numbers and nature of independent mutational events scored. C, compilations, with the numbers indicating percentages of all mutations scored in the pool of the target sequences from different mice. Below the compilations, the ratio of mutations at dG/dC to those at dA/dT is indicated, as is the ratio of transition/transversion substitutions at dG/dC and dA/dT.
FIGURE 5.
FIGURE 5.
The conserved HoxC4-binding site (residues −29 to −22) but not the non-conserved putative ERE site (residues −1089 to −1075) is essential for E2-mediated enhancement of AICDA promoter activation. A, 2E2 B cells were transfected with pGL3-enhancer luciferase gene reporter constructs containing unmutated (unMut) or mutated (Mut 1, Mut 2, Mut 3, and Mut 4) AICDA upstream region. The transfected B cells were stimulated with nil, E2 (10 nm) alone, or anti-CD40 mAb and rhIL-4 in the presence or absence of E2 (10 nm), and luciferase activity was measured 24 h thereafter. Data are from three independent experiments (mean ± S.D. (error bars)). B, spleen B cells from HoxC4+/+ and HoxC4−/− female mice were preactivated with LPS and transfected with the pGL3-enhancer luciferase gene reporter construct containing the AICDA promoter using an Amaxa® mouse B cell Nucleofector® kit and a Nucleofector® device. Transfected B cells were stimulated with LPS and rmIL-4 in the presence or absence of E2 (10 nm), and luciferase activity was measured 24 h thereafter. Data are from three independent experiments (mean ± S.D.).
FIGURE 6.
FIGURE 6.
The promoter region of HOXC4/HoxC4 contains three conserved EREs. Alignment of the HOXC4/HoxC4 upstream region, promoter, 5′ non-coding region, and beginning of the coding region sequences in humans, mice, rats, and cows. The three conserved EREs are boxed in red; the conserved Oct-binding sites are boxed in green; the conserved NF-κB-binding sites are boxed in pink. The region utilized for the HOXC4 promoter luciferase gene reporter construct (Promoter construct) is indicated by a red frame. The blue arrow marks the putative initiation of transcription. The beginning of the coding region is indicated. Gray shading marks DNA sequences conserved among the four species.
FIGURE 7.
FIGURE 7.
ERs are recruited to the HOXC4/HoxC4 promoter and specifically bind to the three conserved EREs in HOXC4 promoter sequence. A, genomic DNA was precipitated from cross-linked chromatin of human 2E2 B cells or primary mouse B cells treated with nil, E2 (10 nm) alone, or E2 (10 nm) together with an agonistic anti-CD40 mAb (human B cells) or LPS (mouse B cells) and IL-4 for 3 days using a rabbit Ab specific for ERα or preimmune control rabbit IgG. The precipitated DNA was specified by PCR for HOXC4/HoxC4 or AICDA/Aicda promoter using specific primers. Data are representative of three independent experiments. B, genomic DNA was precipitated from cross-linked chromatin of primary mouse B cells treated with nil, E2 (10 nm) alone, LPS and rmIL-4, or E2 (10 nm) together with LPS and rmIL-4 for 3 days using rabbit IgG Abs specific for ERα or HoxC4 or preimmune control rabbit IgG. Shown is the enrichment of HoxC4 and Aicda promoter DNA relative to nil samples, as analyzed by real-time quantitative PCR. Data are from three independent experiments (mean ± S.E. (error bars)). C, oligonucleotide probes (ERE-1, ERE-2, and ERE-3) containing one of the three EREs in the HOXC4 promoter, respectively, as well as mutant oligonucleotides (ERE-1mut, ERE-2mut, and ERE-3mut), in which the EREs were deleted, were used as probes for EMSA. D–F, nuclear proteins from anti-CD40 mAb and rhIL-4-stimulated human 2E2 B cells treated with E2 (10 nm) specifically bound to oligonucleotide probes containing one of the EREs. D, efficient competition of the ERE oligonucleotide probes for the formation of the protein-DNA complexes with nuclear proteins of E2-treated anti-CD40 mAb and rhIL-4-stimulated 2E2 B cells was achieved by a 25- or 50-fold molar excess of unlabeled unmutated probe for each specific ERE. Data are representative of three independent experiments. E, the formation of the protein-DNA complexes was shifted by anti-ERα Ab. Rabbit IgG with irrelevant binding activity served as a negative control. F, nuclear proteins from anti-CD40 mAb and rhIL-4-stimulated human 2E2 B cells treated with E2 did not bind to the deletion mutant ERE oligonucleotide probes. Data are representative of three independent experiments. NS, not significant.
FIGURE 8.
FIGURE 8.
The three conserved EREs are essential and cooperative for full HOXC4 promoter activation. A, spleen B cells from HoxC4+/+ female mice were transfected with pGL3-enhancer luciferase gene reporter constructs containing unmutated (unMut) or mutated HOXC4 promoter in which all three of the EREs were deleted (ERE-1-2-3 Mut). The transfected B cells were stimulated with LPS and rmIL-4 in the presence or absence of E2 (10 nm), and luciferase activity was measured 24 h thereafter. Data are from three independent experiments (mean ± S.D. (error bars)). B, human 2E2 B cells were transfected with pGL3-enhancer luciferase gene reporter constructs containing unmutated or mutated HOXC4 promoter in which one (ERE-1 Mut, ERE-2 Mut, or ERE-3 Mut) or three (ERE-1-2-3 Mut) of the EREs were deleted. The transfected B cells were stimulated with nil, E2 (10 nm) alone, or anti-CD40 mAb and rhIL-4 in the presence or absence of E2 (10 nm), and luciferase activity was measured 24 h thereafter. Data are from three independent experiments (mean ± S.D.).
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