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Functional relevance of novel p300-mediated lysine 314 and 315 acetylation of RelA/p65.

Buerki C, Rothgiesser KM, Valovka T, Owen HR, Rehrauer H, Fey M, Lane WS, Hottiger MO - Nucleic Acids Res. (2008)

Bottom Line: Additionally, we confirmed the acetylation on lysine 310 shown previously.Specific genes were either stimulated or repressed by the acetylation-deficient mutants when compared to RelA/p65 wild type.These results support the hypothesis that site-specific p300-mediated acetylation of RelA/p65 regulates the specificity of NF-kappaB dependent gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Biochemistry and Molecular Biology, 8057 Zurich, Switzerland.

ABSTRACT
Nuclear factor kappaB (NF-kappaB) plays an important role in the transcriptional regulation of genes involved in immunity and cell survival. We show here in vitro and in vivo acetylation of RelA/p65 by p300 on lysine 314 and 315, two novel acetylation sites. Additionally, we confirmed the acetylation on lysine 310 shown previously. Genetic complementation of RelA/p65-/- cells with wild type and non-acetylatable mutants of RelA/p65 (K314R and K315R) revealed that neither shuttling, DNA binding nor the induction of anti-apoptotic genes by tumor necrosis factor alpha was affected by acetylation on these residues. Microarray analysis of these cells treated with TNFalpha identified specific sets of genes differently regulated by wild type or acetylation-deficient mutants of RelA/p65. Specific genes were either stimulated or repressed by the acetylation-deficient mutants when compared to RelA/p65 wild type. These results support the hypothesis that site-specific p300-mediated acetylation of RelA/p65 regulates the specificity of NF-kappaB dependent gene expression.

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Endogenous RelA/p65 is acetylated at lysine 310 in vivo in response to TNFα. (A) Western blot analysis with anti-RelA/p65 antibody of whole cell extracts from RelA/p65–/– MEFs complemented with RelA/p65 wild type (wt) and the different substitution mutants of RelA/p65 (K310R, K314/315R and KTR). Mock transduced (pTV) and NIH 3T3 cells were used as controls. The membrane was reprobed with anti-tubulin antibody as loading control. (B) Nuclear extracts of the complemented cell lines untreated or treated with TNFα (30 ng/ml) for 30 min were subjected to western blot analysis using anti-RelA/p65 antibody. The anti-PARP western blot was performed to check extract fractionation. (C) In the left panel, nuclear extracts of the complemented cell lines treated with TNFα and HDACi (TSA/NAM) were subjected to immunoprecipitation analysis using anti-RelA/p65 antibodies. Western blot analysis with the anti-RelA/p65ac310 antibody was performed. The membranes were reprobed with anti-RelA/p65 antibody. In the right panel, RelA/p65 was immunoprecipitated from whole cell extracts of the complemented cell lines treated –/+ TNFα. The immunocomplexes were analyzed by western blot using anti-RelA/p65ac310 antibody and the membrane was reprobed with anti-RelA/p65 antibody. (D) Endogenous interaction analysis of RelA/p65 with p300 in TNFα-treated complemented cell lines. Endogenous p300 was immunoprecipitated from whole cell extract of complemented cell lines after TNFα stimulation for 30 min using anti-p300 antibody. Immunocomplexes were resolved on SDS-PAGE and subsequently analyzed by western blot using anti-RelA/p65, anti-p300 and anti-tubulin antibodies. Left panel: 5% inputs are shown, middle panel: IgG control immunoprecipitation; right panel: anti-p300 immunocomplexes.
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Figure 4: Endogenous RelA/p65 is acetylated at lysine 310 in vivo in response to TNFα. (A) Western blot analysis with anti-RelA/p65 antibody of whole cell extracts from RelA/p65–/– MEFs complemented with RelA/p65 wild type (wt) and the different substitution mutants of RelA/p65 (K310R, K314/315R and KTR). Mock transduced (pTV) and NIH 3T3 cells were used as controls. The membrane was reprobed with anti-tubulin antibody as loading control. (B) Nuclear extracts of the complemented cell lines untreated or treated with TNFα (30 ng/ml) for 30 min were subjected to western blot analysis using anti-RelA/p65 antibody. The anti-PARP western blot was performed to check extract fractionation. (C) In the left panel, nuclear extracts of the complemented cell lines treated with TNFα and HDACi (TSA/NAM) were subjected to immunoprecipitation analysis using anti-RelA/p65 antibodies. Western blot analysis with the anti-RelA/p65ac310 antibody was performed. The membranes were reprobed with anti-RelA/p65 antibody. In the right panel, RelA/p65 was immunoprecipitated from whole cell extracts of the complemented cell lines treated –/+ TNFα. The immunocomplexes were analyzed by western blot using anti-RelA/p65ac310 antibody and the membrane was reprobed with anti-RelA/p65 antibody. (D) Endogenous interaction analysis of RelA/p65 with p300 in TNFα-treated complemented cell lines. Endogenous p300 was immunoprecipitated from whole cell extract of complemented cell lines after TNFα stimulation for 30 min using anti-p300 antibody. Immunocomplexes were resolved on SDS-PAGE and subsequently analyzed by western blot using anti-RelA/p65, anti-p300 and anti-tubulin antibodies. Left panel: 5% inputs are shown, middle panel: IgG control immunoprecipitation; right panel: anti-p300 immunocomplexes.

Mentions: To investigate the role of RelA/p65 acetylation of K310, K314 and K315 in vivo, RelA/p65–/– NIH 3T3 mouse embryonic fibroblasts (MEF) were genetically complemented using lentiviruses encoding myc-RelA/p65 wild type, myc-RelA/p65K310R, myc-RelA/p65K314/315R or myc-RelA/p65KTR. After appropriate selection cells were kept in pools and the expression of recombinant proteins was analyzed by western blotting using an anti-RelA/p65 antibody (Figure 4A). The cells transduced with control virus encoding the resistance gene [mock infected (pTV)] and non-transduced wild type NIH 3T3 cells expressing endogenous RelA/p65 protein were included as controls. The expression levels of the recombinant wild type and mutated RelA/p65 proteins were comparable to that observed for endogenous RelA/p65 in NIH 3T3 cells. Furthermore, cell growth analysis revealed that the proliferation rate was comparable between the tested cell pools under normal growth conditions (data not shown).Figure 4.


Functional relevance of novel p300-mediated lysine 314 and 315 acetylation of RelA/p65.

Buerki C, Rothgiesser KM, Valovka T, Owen HR, Rehrauer H, Fey M, Lane WS, Hottiger MO - Nucleic Acids Res. (2008)

Endogenous RelA/p65 is acetylated at lysine 310 in vivo in response to TNFα. (A) Western blot analysis with anti-RelA/p65 antibody of whole cell extracts from RelA/p65–/– MEFs complemented with RelA/p65 wild type (wt) and the different substitution mutants of RelA/p65 (K310R, K314/315R and KTR). Mock transduced (pTV) and NIH 3T3 cells were used as controls. The membrane was reprobed with anti-tubulin antibody as loading control. (B) Nuclear extracts of the complemented cell lines untreated or treated with TNFα (30 ng/ml) for 30 min were subjected to western blot analysis using anti-RelA/p65 antibody. The anti-PARP western blot was performed to check extract fractionation. (C) In the left panel, nuclear extracts of the complemented cell lines treated with TNFα and HDACi (TSA/NAM) were subjected to immunoprecipitation analysis using anti-RelA/p65 antibodies. Western blot analysis with the anti-RelA/p65ac310 antibody was performed. The membranes were reprobed with anti-RelA/p65 antibody. In the right panel, RelA/p65 was immunoprecipitated from whole cell extracts of the complemented cell lines treated –/+ TNFα. The immunocomplexes were analyzed by western blot using anti-RelA/p65ac310 antibody and the membrane was reprobed with anti-RelA/p65 antibody. (D) Endogenous interaction analysis of RelA/p65 with p300 in TNFα-treated complemented cell lines. Endogenous p300 was immunoprecipitated from whole cell extract of complemented cell lines after TNFα stimulation for 30 min using anti-p300 antibody. Immunocomplexes were resolved on SDS-PAGE and subsequently analyzed by western blot using anti-RelA/p65, anti-p300 and anti-tubulin antibodies. Left panel: 5% inputs are shown, middle panel: IgG control immunoprecipitation; right panel: anti-p300 immunocomplexes.
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Figure 4: Endogenous RelA/p65 is acetylated at lysine 310 in vivo in response to TNFα. (A) Western blot analysis with anti-RelA/p65 antibody of whole cell extracts from RelA/p65–/– MEFs complemented with RelA/p65 wild type (wt) and the different substitution mutants of RelA/p65 (K310R, K314/315R and KTR). Mock transduced (pTV) and NIH 3T3 cells were used as controls. The membrane was reprobed with anti-tubulin antibody as loading control. (B) Nuclear extracts of the complemented cell lines untreated or treated with TNFα (30 ng/ml) for 30 min were subjected to western blot analysis using anti-RelA/p65 antibody. The anti-PARP western blot was performed to check extract fractionation. (C) In the left panel, nuclear extracts of the complemented cell lines treated with TNFα and HDACi (TSA/NAM) were subjected to immunoprecipitation analysis using anti-RelA/p65 antibodies. Western blot analysis with the anti-RelA/p65ac310 antibody was performed. The membranes were reprobed with anti-RelA/p65 antibody. In the right panel, RelA/p65 was immunoprecipitated from whole cell extracts of the complemented cell lines treated –/+ TNFα. The immunocomplexes were analyzed by western blot using anti-RelA/p65ac310 antibody and the membrane was reprobed with anti-RelA/p65 antibody. (D) Endogenous interaction analysis of RelA/p65 with p300 in TNFα-treated complemented cell lines. Endogenous p300 was immunoprecipitated from whole cell extract of complemented cell lines after TNFα stimulation for 30 min using anti-p300 antibody. Immunocomplexes were resolved on SDS-PAGE and subsequently analyzed by western blot using anti-RelA/p65, anti-p300 and anti-tubulin antibodies. Left panel: 5% inputs are shown, middle panel: IgG control immunoprecipitation; right panel: anti-p300 immunocomplexes.
Mentions: To investigate the role of RelA/p65 acetylation of K310, K314 and K315 in vivo, RelA/p65–/– NIH 3T3 mouse embryonic fibroblasts (MEF) were genetically complemented using lentiviruses encoding myc-RelA/p65 wild type, myc-RelA/p65K310R, myc-RelA/p65K314/315R or myc-RelA/p65KTR. After appropriate selection cells were kept in pools and the expression of recombinant proteins was analyzed by western blotting using an anti-RelA/p65 antibody (Figure 4A). The cells transduced with control virus encoding the resistance gene [mock infected (pTV)] and non-transduced wild type NIH 3T3 cells expressing endogenous RelA/p65 protein were included as controls. The expression levels of the recombinant wild type and mutated RelA/p65 proteins were comparable to that observed for endogenous RelA/p65 in NIH 3T3 cells. Furthermore, cell growth analysis revealed that the proliferation rate was comparable between the tested cell pools under normal growth conditions (data not shown).Figure 4.

Bottom Line: Additionally, we confirmed the acetylation on lysine 310 shown previously.Specific genes were either stimulated or repressed by the acetylation-deficient mutants when compared to RelA/p65 wild type.These results support the hypothesis that site-specific p300-mediated acetylation of RelA/p65 regulates the specificity of NF-kappaB dependent gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Biochemistry and Molecular Biology, 8057 Zurich, Switzerland.

ABSTRACT
Nuclear factor kappaB (NF-kappaB) plays an important role in the transcriptional regulation of genes involved in immunity and cell survival. We show here in vitro and in vivo acetylation of RelA/p65 by p300 on lysine 314 and 315, two novel acetylation sites. Additionally, we confirmed the acetylation on lysine 310 shown previously. Genetic complementation of RelA/p65-/- cells with wild type and non-acetylatable mutants of RelA/p65 (K314R and K315R) revealed that neither shuttling, DNA binding nor the induction of anti-apoptotic genes by tumor necrosis factor alpha was affected by acetylation on these residues. Microarray analysis of these cells treated with TNFalpha identified specific sets of genes differently regulated by wild type or acetylation-deficient mutants of RelA/p65. Specific genes were either stimulated or repressed by the acetylation-deficient mutants when compared to RelA/p65 wild type. These results support the hypothesis that site-specific p300-mediated acetylation of RelA/p65 regulates the specificity of NF-kappaB dependent gene expression.

Show MeSH
Related in: MedlinePlus