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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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The effects of estrogen on AID protein in DT40. (A) Estrogen induces AID mRNA expression. AID-FM–tagged DT40 cells were treated with DMSO, 100 nM estrogen, and 10 nM estrogen with TNF-α, lysed, and analyzed for AID mRNA expression with pRT-PCR at various time-points. (B) Estrogen induces AID-FM fusion protein expression. Treatment as in A, but lysates were analyzed by quantitative Western blot. For each sample, FLAG and Tubulin expression was quantitated. The graph is derived from correlating the FLAG expression to Tubulin expression, and then determining the ratio of estrogen-induced FLAG expression to untreated DMSO samples. (C) Estrogen does not affect AID-FM fusion protein stability. Cells were incubated with CHX or MG-132 for 2 h, followed by estrogen treatment for 4 h. Protein levels were determined by quantitative Western blot. For all experiments, cells were grown in hormone depleted media for 48 h. Results are normalized to control treatments as indicated on each graph. Timelines of cell treatments are indicated below the graphs.
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fig4: The effects of estrogen on AID protein in DT40. (A) Estrogen induces AID mRNA expression. AID-FM–tagged DT40 cells were treated with DMSO, 100 nM estrogen, and 10 nM estrogen with TNF-α, lysed, and analyzed for AID mRNA expression with pRT-PCR at various time-points. (B) Estrogen induces AID-FM fusion protein expression. Treatment as in A, but lysates were analyzed by quantitative Western blot. For each sample, FLAG and Tubulin expression was quantitated. The graph is derived from correlating the FLAG expression to Tubulin expression, and then determining the ratio of estrogen-induced FLAG expression to untreated DMSO samples. (C) Estrogen does not affect AID-FM fusion protein stability. Cells were incubated with CHX or MG-132 for 2 h, followed by estrogen treatment for 4 h. Protein levels were determined by quantitative Western blot. For all experiments, cells were grown in hormone depleted media for 48 h. Results are normalized to control treatments as indicated on each graph. Timelines of cell treatments are indicated below the graphs.

Mentions: For us to determine if the effect of estrogen on AID would also extend to the protein level, we developed a quantitative approach for measuring AID protein. We generated a DT40 cell line (a cell line derived from a chicken B cell lymphoma that constitutively expresses AID and undergoes Ig diversification) to express a double tag (3xFLAG-2xTEV-3xc-Myc) fused to the C terminal exon of endogenous AID (AID FLAG-Myc–tagged AID protein [AID-FM]; Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20080521/DC1). The modified DT40 express a WT AID protein and an AID-FM fusion protein, both transcribed from the endogenous AID locus. Comparing the qRT-PCR induction kinetics with that of the AID-FM expression, we determined that the steady-state levels of AID protein correlated to those of the mRNA (Fig. 4, A and B). Using the translation inhibitor CHX (Fig. 4 C), we showed that estrogen was not able to increase AID-FM expression in the absence of translation. In the presence of the proteasome inhibitor MG-132 (Fig. 4 C), estrogen increased AID-FM production above that of the MG-132 alone, indicating that estrogen was not acting on the proteasome to increase AID-FM activity. The aforementioned co-stimulatory effects of TNF-α were also observed at the level of protein production (Fig. 4, E 10/TNF-α).


Estrogen directly activates AID transcription and function.

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

The effects of estrogen on AID protein in DT40. (A) Estrogen induces AID mRNA expression. AID-FM–tagged DT40 cells were treated with DMSO, 100 nM estrogen, and 10 nM estrogen with TNF-α, lysed, and analyzed for AID mRNA expression with pRT-PCR at various time-points. (B) Estrogen induces AID-FM fusion protein expression. Treatment as in A, but lysates were analyzed by quantitative Western blot. For each sample, FLAG and Tubulin expression was quantitated. The graph is derived from correlating the FLAG expression to Tubulin expression, and then determining the ratio of estrogen-induced FLAG expression to untreated DMSO samples. (C) Estrogen does not affect AID-FM fusion protein stability. Cells were incubated with CHX or MG-132 for 2 h, followed by estrogen treatment for 4 h. Protein levels were determined by quantitative Western blot. For all experiments, cells were grown in hormone depleted media for 48 h. Results are normalized to control treatments as indicated on each graph. Timelines of cell treatments are indicated below the graphs.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: The effects of estrogen on AID protein in DT40. (A) Estrogen induces AID mRNA expression. AID-FM–tagged DT40 cells were treated with DMSO, 100 nM estrogen, and 10 nM estrogen with TNF-α, lysed, and analyzed for AID mRNA expression with pRT-PCR at various time-points. (B) Estrogen induces AID-FM fusion protein expression. Treatment as in A, but lysates were analyzed by quantitative Western blot. For each sample, FLAG and Tubulin expression was quantitated. The graph is derived from correlating the FLAG expression to Tubulin expression, and then determining the ratio of estrogen-induced FLAG expression to untreated DMSO samples. (C) Estrogen does not affect AID-FM fusion protein stability. Cells were incubated with CHX or MG-132 for 2 h, followed by estrogen treatment for 4 h. Protein levels were determined by quantitative Western blot. For all experiments, cells were grown in hormone depleted media for 48 h. Results are normalized to control treatments as indicated on each graph. Timelines of cell treatments are indicated below the graphs.
Mentions: For us to determine if the effect of estrogen on AID would also extend to the protein level, we developed a quantitative approach for measuring AID protein. We generated a DT40 cell line (a cell line derived from a chicken B cell lymphoma that constitutively expresses AID and undergoes Ig diversification) to express a double tag (3xFLAG-2xTEV-3xc-Myc) fused to the C terminal exon of endogenous AID (AID FLAG-Myc–tagged AID protein [AID-FM]; Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20080521/DC1). The modified DT40 express a WT AID protein and an AID-FM fusion protein, both transcribed from the endogenous AID locus. Comparing the qRT-PCR induction kinetics with that of the AID-FM expression, we determined that the steady-state levels of AID protein correlated to those of the mRNA (Fig. 4, A and B). Using the translation inhibitor CHX (Fig. 4 C), we showed that estrogen was not able to increase AID-FM expression in the absence of translation. In the presence of the proteasome inhibitor MG-132 (Fig. 4 C), estrogen increased AID-FM production above that of the MG-132 alone, indicating that estrogen was not acting on the proteasome to increase AID-FM activity. The aforementioned co-stimulatory effects of TNF-α were also observed at the level of protein production (Fig. 4, E 10/TNF-α).

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