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Acetylation of p65 at lysine 314 is important for late NF-kappaB-dependent gene expression.

Rothgiesser KM, Fey M, Hottiger MO - BMC Genomics (2010)

Bottom Line: In this study, we describe that site-specific mutation of p65 at lysines 314 and 315 enhances gene expression of a subset of NF-kappaB target genes including Mmp10 and Mmp13.Chromatin immunoprecipitation (ChIP) experiments with an antibody raised against acetylated lysine 314 revealed that chromatin-bound p65 is indeed acetylated at lysine 314.Together, our results establish acetylation of K314 as an important regulatory modification of p65 and subsequently of NF-kappaB-dependent gene expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

ABSTRACT

Background: NF-kappaB regulates the expression of a large number of target genes involved in the immune and inflammatory response, apoptosis, cell proliferation, differentiation and survival. We have earlier reported that p65, a subunit of NF-kappaB, is acetylated in vitro and in vivo at three different lysines (K310, K314 and K315) by the histone acetyltransferase p300.

Results: In this study, we describe that site-specific mutation of p65 at lysines 314 and 315 enhances gene expression of a subset of NF-kappaB target genes including Mmp10 and Mmp13. Increased gene expression was mainly observed three hours after TNFalpha stimulation. Chromatin immunoprecipitation (ChIP) experiments with an antibody raised against acetylated lysine 314 revealed that chromatin-bound p65 is indeed acetylated at lysine 314.

Conclusions: Together, our results establish acetylation of K314 as an important regulatory modification of p65 and subsequently of NF-kappaB-dependent gene expression.

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Site-specific acetylation of p65 regulates the expression of distinct genes. Heat maps showing the gene expression profiles of KTR cells (A) or K314/315R cells (B) compared to wild type control at 3 hours after TNFα stimulation using whole mouse genome arrays. Each row represents a single gene, and each column represents a different cell line. Red or green represents up- or downregulation of genes relative to the mean on each row, respectively. Mean data from at least two biological replicates is displayed. Only genes upregulated in wild type cells compared to pTV control were taken into account (>2-fold, p-value < 0.05). From these genes, only the ones with significant changes in expression levels (> 1.8-fold or < 0.556-fold, p-value < 0.05) between wild type and mutant cells are shown.
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Figure 1: Site-specific acetylation of p65 regulates the expression of distinct genes. Heat maps showing the gene expression profiles of KTR cells (A) or K314/315R cells (B) compared to wild type control at 3 hours after TNFα stimulation using whole mouse genome arrays. Each row represents a single gene, and each column represents a different cell line. Red or green represents up- or downregulation of genes relative to the mean on each row, respectively. Mean data from at least two biological replicates is displayed. Only genes upregulated in wild type cells compared to pTV control were taken into account (>2-fold, p-value < 0.05). From these genes, only the ones with significant changes in expression levels (> 1.8-fold or < 0.556-fold, p-value < 0.05) between wild type and mutant cells are shown.

Mentions: We provided earlier evidence that acetylation of p65 at K310, 314 and 315 is important for the expression of a defined subset of genes [18]. These earlier studies provided a first glance of the functional relevance of p65 acetylation, since gene expression was measured only after 45 minutes of TNFα stimulation. In order to know if the requirement for site-specific acetylation is maintained for the same genes after longer exposure to TNFα, and to identify possible new genes regulated through p65 acetylation, we decided to extend our analysis to 3 hours of stimulation. For this, we used p65(-/-) mouse embryonic fibroblasts (MEFs) complemented with acetylation-deficient mutants, where the target lysines for acetylation were mutated to arginines. These cells were described and extensively characterized previously [18]. p65(-/-) cells complemented either with wild type p65, an empty plasmid as control (pTV), the acetylation-deficient double mutant K314/315R or the triple mutant KTR (K310/314/315R), were stimulated by TNFα for 3 hours and total RNA was isolated in three independent replicates from these cells. RNA was amplified, labeled and hybridized to the Agilent Whole Mouse Genome Array. After statistical analysis of the expression profiles, differentially expressed genes were identified (Fig. 1A-B, and Tables 1 and 2). We focused only on genes that required p65 for their proper induction, which were identified by comparing gene expression profiles from wild type and pTV cells. The majority of differentially expressed genes in KTR mutant were strongly downregulated compared to wild type cells, suggesting that acetylation of p65 at these residues is also an important modification for the expression of the extended NF-κB-dependent gene expression (Fig. 1A). In contrast, experiments with the double K314/315R mutant revealed that the majority of genes were slightly upregulated after 3 hours compared to wild type cells (Fig. 1B).


Acetylation of p65 at lysine 314 is important for late NF-kappaB-dependent gene expression.

Rothgiesser KM, Fey M, Hottiger MO - BMC Genomics (2010)

Site-specific acetylation of p65 regulates the expression of distinct genes. Heat maps showing the gene expression profiles of KTR cells (A) or K314/315R cells (B) compared to wild type control at 3 hours after TNFα stimulation using whole mouse genome arrays. Each row represents a single gene, and each column represents a different cell line. Red or green represents up- or downregulation of genes relative to the mean on each row, respectively. Mean data from at least two biological replicates is displayed. Only genes upregulated in wild type cells compared to pTV control were taken into account (>2-fold, p-value < 0.05). From these genes, only the ones with significant changes in expression levels (> 1.8-fold or < 0.556-fold, p-value < 0.05) between wild type and mutant cells are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2823688&req=5

Figure 1: Site-specific acetylation of p65 regulates the expression of distinct genes. Heat maps showing the gene expression profiles of KTR cells (A) or K314/315R cells (B) compared to wild type control at 3 hours after TNFα stimulation using whole mouse genome arrays. Each row represents a single gene, and each column represents a different cell line. Red or green represents up- or downregulation of genes relative to the mean on each row, respectively. Mean data from at least two biological replicates is displayed. Only genes upregulated in wild type cells compared to pTV control were taken into account (>2-fold, p-value < 0.05). From these genes, only the ones with significant changes in expression levels (> 1.8-fold or < 0.556-fold, p-value < 0.05) between wild type and mutant cells are shown.
Mentions: We provided earlier evidence that acetylation of p65 at K310, 314 and 315 is important for the expression of a defined subset of genes [18]. These earlier studies provided a first glance of the functional relevance of p65 acetylation, since gene expression was measured only after 45 minutes of TNFα stimulation. In order to know if the requirement for site-specific acetylation is maintained for the same genes after longer exposure to TNFα, and to identify possible new genes regulated through p65 acetylation, we decided to extend our analysis to 3 hours of stimulation. For this, we used p65(-/-) mouse embryonic fibroblasts (MEFs) complemented with acetylation-deficient mutants, where the target lysines for acetylation were mutated to arginines. These cells were described and extensively characterized previously [18]. p65(-/-) cells complemented either with wild type p65, an empty plasmid as control (pTV), the acetylation-deficient double mutant K314/315R or the triple mutant KTR (K310/314/315R), were stimulated by TNFα for 3 hours and total RNA was isolated in three independent replicates from these cells. RNA was amplified, labeled and hybridized to the Agilent Whole Mouse Genome Array. After statistical analysis of the expression profiles, differentially expressed genes were identified (Fig. 1A-B, and Tables 1 and 2). We focused only on genes that required p65 for their proper induction, which were identified by comparing gene expression profiles from wild type and pTV cells. The majority of differentially expressed genes in KTR mutant were strongly downregulated compared to wild type cells, suggesting that acetylation of p65 at these residues is also an important modification for the expression of the extended NF-κB-dependent gene expression (Fig. 1A). In contrast, experiments with the double K314/315R mutant revealed that the majority of genes were slightly upregulated after 3 hours compared to wild type cells (Fig. 1B).

Bottom Line: In this study, we describe that site-specific mutation of p65 at lysines 314 and 315 enhances gene expression of a subset of NF-kappaB target genes including Mmp10 and Mmp13.Chromatin immunoprecipitation (ChIP) experiments with an antibody raised against acetylated lysine 314 revealed that chromatin-bound p65 is indeed acetylated at lysine 314.Together, our results establish acetylation of K314 as an important regulatory modification of p65 and subsequently of NF-kappaB-dependent gene expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

ABSTRACT

Background: NF-kappaB regulates the expression of a large number of target genes involved in the immune and inflammatory response, apoptosis, cell proliferation, differentiation and survival. We have earlier reported that p65, a subunit of NF-kappaB, is acetylated in vitro and in vivo at three different lysines (K310, K314 and K315) by the histone acetyltransferase p300.

Results: In this study, we describe that site-specific mutation of p65 at lysines 314 and 315 enhances gene expression of a subset of NF-kappaB target genes including Mmp10 and Mmp13. Increased gene expression was mainly observed three hours after TNFalpha stimulation. Chromatin immunoprecipitation (ChIP) experiments with an antibody raised against acetylated lysine 314 revealed that chromatin-bound p65 is indeed acetylated at lysine 314.

Conclusions: Together, our results establish acetylation of K314 as an important regulatory modification of p65 and subsequently of NF-kappaB-dependent gene expression.

Show MeSH
Related in: MedlinePlus