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Estrogen directly activates AID transcription and function.

Pauklin S, Sernández IV, Bachmann G, Ramiro AR, Petersen-Mahrt SK - J. Exp. Med. (2009)

Bottom Line: Enhanced translocations of the c-myc oncogene showed that the genotoxicity of estrogen via AID production was not limited to the Ig locus.Outside of the immune system (e.g., breast and ovaries), estrogen induced AID expression by >20-fold.The estrogen response was also partially conserved within the DNA deaminase family (APOBEC3B, -3F, and -3G), and could be inhibited by tamoxifen, an estrogen antagonist.

View Article: PubMed Central - PubMed

Affiliation: DNA Editing Laboratory, Cancer Research UK, Clare Hall Laboratories, South Mimms, EN6 3LD, England, UK.

ABSTRACT
The immunological targets of estrogen at the molecular, humoral, and cellular level have been well documented, as has estrogen's role in establishing a gender bias in autoimmunity and cancer. During a healthy immune response, activation-induced deaminase (AID) deaminates cytosines at immunoglobulin (Ig) loci, initiating somatic hypermutation (SHM) and class switch recombination (CSR). Protein levels of nuclear AID are tightly controlled, as unregulated expression can lead to alterations in the immune response. Furthermore, hyperactivation of AID outside the immune system leads to oncogenesis. Here, we demonstrate that the estrogen-estrogen receptor complex binds to the AID promoter, enhancing AID messenger RNA expression, leading to a direct increase in AID protein production and alterations in SHM and CSR at the Ig locus. Enhanced translocations of the c-myc oncogene showed that the genotoxicity of estrogen via AID production was not limited to the Ig locus. Outside of the immune system (e.g., breast and ovaries), estrogen induced AID expression by >20-fold. The estrogen response was also partially conserved within the DNA deaminase family (APOBEC3B, -3F, and -3G), and could be inhibited by tamoxifen, an estrogen antagonist. We therefore suggest that estrogen-induced autoimmunity and oncogenesis may be derived through AID-dependent DNA instability.

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Human AID promoter analysis for hormone response elements. (A) Schematic representation of potential EREs (square) and NF-κB sites (circle) and their respective locations in the human promoter. The indicated promoter regions (marked A–E) were inserted into a luciferase reporter construct with a minimal promoter. The vectors were transfected into SiHa cells, incubated for 24 h, treated for 4 h with hormones or TNF-α, and analyzed for luciferase activity. (B) Relative luciferase activity after estrogen treatment. Cells were transfected with constructs containing AID promoter fragments and treated with estrogen for 4 h. (C) Effect of TNF-α and estrogen on the human AID promoter. Expression construct with an AID promoter region containing NF-κB sites and putative ERE (Fragment C) were transfected into cells, followed by TNF-α and/or estrogen treatment for 4 h. (D) Estrogen can act independently from NF-κB. Cells were cotransfected with Fragment C and an IκBα-mt expression vector. After 24 h, cells were treated with TNF-α and/or 100 nM estrogen for 4 h. Timelines of cell treatments are indicated below the graphs. NT, not treated.
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fig2: Human AID promoter analysis for hormone response elements. (A) Schematic representation of potential EREs (square) and NF-κB sites (circle) and their respective locations in the human promoter. The indicated promoter regions (marked A–E) were inserted into a luciferase reporter construct with a minimal promoter. The vectors were transfected into SiHa cells, incubated for 24 h, treated for 4 h with hormones or TNF-α, and analyzed for luciferase activity. (B) Relative luciferase activity after estrogen treatment. Cells were transfected with constructs containing AID promoter fragments and treated with estrogen for 4 h. (C) Effect of TNF-α and estrogen on the human AID promoter. Expression construct with an AID promoter region containing NF-κB sites and putative ERE (Fragment C) were transfected into cells, followed by TNF-α and/or estrogen treatment for 4 h. (D) Estrogen can act independently from NF-κB. Cells were cotransfected with Fragment C and an IκBα-mt expression vector. After 24 h, cells were treated with TNF-α and/or 100 nM estrogen for 4 h. Timelines of cell treatments are indicated below the graphs. NT, not treated.

Mentions: The rapid effect of the hormones on AID message via transcription suggested that the AID gene is a direct target for hormonal regulation. Using bioinformatic analysis (Fig. 2 A), we were able to identify putative EREs in the context of other response elements, such as NF-κB. We dissected the 1.5 kb upstream and the 2 kb downstream of the ATG regions for hormone-responsive elements in a heterologous transcription assay. The potential response regions were placed into a luciferase reporter construct and transfected into human SiHa cells, followed by treatment with the indicated hormone (or cotransfected with expression plasmids), and then analyzed for luciferase activity. As we were primarily interested in the effect of hormones on expression, we used relative change as a readout rather than absolute values, which provided a more direct evaluation of the hormone treatment but potentially obscured the individual effect of the various DNA elements. When compared with DMSO treatment, estrogen responsiveness was most significant with Fragment C, indicating that this contained the predominant estrogen-responsive DNA element. Comparable to the mRNA production of AID in B cells, Fragment C also responded in a dose-dependent manner to estrogen (Fig. 2 C). Aside from the putative ERE, Fragment C also harbored the two published NF-κB binding sites (24). As indicated by the qRT-PCR analysis in Fig. 1 A, estrogen and the LPS/IL-4–induced NF-κB stress-response pathway could act synergistically on AID mRNA production. To more directly stimulate the NF-κB pathway in SiHa cells, we used the cell-autonomous activator TNF-α (Fig. 2 C). Interestingly, aside from the synergy (e.g., 10−9 M), the two maxima of the dose response were offset (TNF-α treated, 10−9 M; untreated, 10−7 M), indicating a higher complexity of the two interacting pathways. To demonstrate independence of the two pathways, we analyzed the response of Fragment C to treatment with TNF-α and estrogen upon cotransfection of the dominant-negative mutant of IκBα (IκBα S32A/S36A dominant mutant [IκBα-mt]), which is known to inhibit the release of NF-κB from the cytoplasm into the nucleus after stimulation. As shown in Fig. 2 D, the TNF-α activation was inhibited in the presence of IκBα-mt, yet estrogen was able to independently activate the transcription. This indicated that in the AID promoter, the NF-κB site and its proximal ERE could act independently as well as synergistically.


Estrogen directly activates AID transcription and function.

Pauklin S, Sernández IV, Bachmann G, Ramiro AR, Petersen-Mahrt SK - J. Exp. Med. (2009)

Human AID promoter analysis for hormone response elements. (A) Schematic representation of potential EREs (square) and NF-κB sites (circle) and their respective locations in the human promoter. The indicated promoter regions (marked A–E) were inserted into a luciferase reporter construct with a minimal promoter. The vectors were transfected into SiHa cells, incubated for 24 h, treated for 4 h with hormones or TNF-α, and analyzed for luciferase activity. (B) Relative luciferase activity after estrogen treatment. Cells were transfected with constructs containing AID promoter fragments and treated with estrogen for 4 h. (C) Effect of TNF-α and estrogen on the human AID promoter. Expression construct with an AID promoter region containing NF-κB sites and putative ERE (Fragment C) were transfected into cells, followed by TNF-α and/or estrogen treatment for 4 h. (D) Estrogen can act independently from NF-κB. Cells were cotransfected with Fragment C and an IκBα-mt expression vector. After 24 h, cells were treated with TNF-α and/or 100 nM estrogen for 4 h. Timelines of cell treatments are indicated below the graphs. NT, not treated.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2626679&req=5

fig2: Human AID promoter analysis for hormone response elements. (A) Schematic representation of potential EREs (square) and NF-κB sites (circle) and their respective locations in the human promoter. The indicated promoter regions (marked A–E) were inserted into a luciferase reporter construct with a minimal promoter. The vectors were transfected into SiHa cells, incubated for 24 h, treated for 4 h with hormones or TNF-α, and analyzed for luciferase activity. (B) Relative luciferase activity after estrogen treatment. Cells were transfected with constructs containing AID promoter fragments and treated with estrogen for 4 h. (C) Effect of TNF-α and estrogen on the human AID promoter. Expression construct with an AID promoter region containing NF-κB sites and putative ERE (Fragment C) were transfected into cells, followed by TNF-α and/or estrogen treatment for 4 h. (D) Estrogen can act independently from NF-κB. Cells were cotransfected with Fragment C and an IκBα-mt expression vector. After 24 h, cells were treated with TNF-α and/or 100 nM estrogen for 4 h. Timelines of cell treatments are indicated below the graphs. NT, not treated.
Mentions: The rapid effect of the hormones on AID message via transcription suggested that the AID gene is a direct target for hormonal regulation. Using bioinformatic analysis (Fig. 2 A), we were able to identify putative EREs in the context of other response elements, such as NF-κB. We dissected the 1.5 kb upstream and the 2 kb downstream of the ATG regions for hormone-responsive elements in a heterologous transcription assay. The potential response regions were placed into a luciferase reporter construct and transfected into human SiHa cells, followed by treatment with the indicated hormone (or cotransfected with expression plasmids), and then analyzed for luciferase activity. As we were primarily interested in the effect of hormones on expression, we used relative change as a readout rather than absolute values, which provided a more direct evaluation of the hormone treatment but potentially obscured the individual effect of the various DNA elements. When compared with DMSO treatment, estrogen responsiveness was most significant with Fragment C, indicating that this contained the predominant estrogen-responsive DNA element. Comparable to the mRNA production of AID in B cells, Fragment C also responded in a dose-dependent manner to estrogen (Fig. 2 C). Aside from the putative ERE, Fragment C also harbored the two published NF-κB binding sites (24). As indicated by the qRT-PCR analysis in Fig. 1 A, estrogen and the LPS/IL-4–induced NF-κB stress-response pathway could act synergistically on AID mRNA production. To more directly stimulate the NF-κB pathway in SiHa cells, we used the cell-autonomous activator TNF-α (Fig. 2 C). Interestingly, aside from the synergy (e.g., 10−9 M), the two maxima of the dose response were offset (TNF-α treated, 10−9 M; untreated, 10−7 M), indicating a higher complexity of the two interacting pathways. To demonstrate independence of the two pathways, we analyzed the response of Fragment C to treatment with TNF-α and estrogen upon cotransfection of the dominant-negative mutant of IκBα (IκBα S32A/S36A dominant mutant [IκBα-mt]), which is known to inhibit the release of NF-κB from the cytoplasm into the nucleus after stimulation. As shown in Fig. 2 D, the TNF-α activation was inhibited in the presence of IκBα-mt, yet estrogen was able to independently activate the transcription. This indicated that in the AID promoter, the NF-κB site and its proximal ERE could act independently as well as synergistically.

Bottom Line: Enhanced translocations of the c-myc oncogene showed that the genotoxicity of estrogen via AID production was not limited to the Ig locus.Outside of the immune system (e.g., breast and ovaries), estrogen induced AID expression by >20-fold.The estrogen response was also partially conserved within the DNA deaminase family (APOBEC3B, -3F, and -3G), and could be inhibited by tamoxifen, an estrogen antagonist.

View Article: PubMed Central - PubMed

Affiliation: DNA Editing Laboratory, Cancer Research UK, Clare Hall Laboratories, South Mimms, EN6 3LD, England, UK.

ABSTRACT
The immunological targets of estrogen at the molecular, humoral, and cellular level have been well documented, as has estrogen's role in establishing a gender bias in autoimmunity and cancer. During a healthy immune response, activation-induced deaminase (AID) deaminates cytosines at immunoglobulin (Ig) loci, initiating somatic hypermutation (SHM) and class switch recombination (CSR). Protein levels of nuclear AID are tightly controlled, as unregulated expression can lead to alterations in the immune response. Furthermore, hyperactivation of AID outside the immune system leads to oncogenesis. Here, we demonstrate that the estrogen-estrogen receptor complex binds to the AID promoter, enhancing AID messenger RNA expression, leading to a direct increase in AID protein production and alterations in SHM and CSR at the Ig locus. Enhanced translocations of the c-myc oncogene showed that the genotoxicity of estrogen via AID production was not limited to the Ig locus. Outside of the immune system (e.g., breast and ovaries), estrogen induced AID expression by >20-fold. The estrogen response was also partially conserved within the DNA deaminase family (APOBEC3B, -3F, and -3G), and could be inhibited by tamoxifen, an estrogen antagonist. We therefore suggest that estrogen-induced autoimmunity and oncogenesis may be derived through AID-dependent DNA instability.

Show MeSH
Related in: MedlinePlus