Unknown,Transcriptomics,Genomics,Proteomics

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Acetylation of p65 at lysine 314 is important for late NF-k(kappa)B-dependent gene expression

ABSTRACT: We provided earlier evidence that acetylation of p65 at lysines 310, 314 and 315 is important for the expression of a defined subset of genes; acetylation at these residues regulates both positively and negatively gene expression, in a gene-specific manner. These earlier studies provided a first glance of the functional relevance of p65 acetylation, since gene expression was measured only after 45 minutes of TNFÎ± stimulation. In order to know if the requirement for site-specific acetylation is maintained for the same genes after longer exposure to TNFÎ±, and to identify possible new genes regulated through p65 acetylation, we decided to extent our analysis to 3 hours of stimulation. For this, we used p65(-/-) MEFs complemented with non-acetylatable mutants, where the target lysines for acetylation were mutated to arginines. 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 TNFÎ± 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.

ORGANISM(S): Mus musculus

SUBMITTER: Michael Hottiger

PROVIDER: E-GEOD-15196 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Specific histone modifications play important roles in chromatin functions such as activation or repression of gene transcription. These participation must occur as a dynamic process, however, most of histone modification state maps reported to date only provide static pictures linking certain modification with active or silenced states. This study focused on the global histone modification variation that occurs in response to transcriptional reprogramming produced by a physiological perturbation in yeast. We have performed genome-wide chromatin immunoprecipitation analysis for eight specific histone modifications before and after of a saline stress. The most striking change is a quick deacetylation of lysines 9 and 14 of H3 and lysine 8 of H4 associated to repression of genes. Genes that are activated increase the acetylation levels at these same sites, but this acetylation process of activated genes seems minor quantitatively to that of the deacetylation of repressed genes. The observed changes in tri-methylation of lysines 4, 36 and 79 of H3 and also di-methylation of lysine 79 of H3 are much more moderate than those of acetylation. Additionally, we have produced new genome-wide maps for six histone modifications at more than five times higher resolution of previous available data and analyzed for the first time in S. cerevisiae genome wide profiles of two more, acetylation of lysine 8 of H4 and di-methylation of lysine 79 of H3. In this research we have shown that dynamic of acetylation state of histones during activation or repression of transcription is a process much quicker than methylation and therefore the changes produced in the acetylation may affect methylation but the reverse path is not possible. The experiments described in this study compare ChIP with a histone modification antibody to a control ChIP with a core histone antibody. Budding yeast samples were analyzed in exponential growing conditions (YPD) or after 10 minutes of 0.4M NaCl stress. For each experiment 1 or 2 biological replicates were performed.

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Acetylation of p65 at lysine 314 is important for late NF-k(kappa)B-dependent gene expression

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