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[Coactivator p300-induced H3K27 acetylation mediates lipopolysaccharide-induced inflammatory mediator synthesis]
1Medical College of Hunan University of Medicine, Hunan University of Medicine, Huaihua 418000, China.
Objective:
To investigate the role of acetylated modification induced by coactivator p300 in lipopolysaccharide (LPS)- induced inflammatory mediator synthesis and its molecular mechanism.
Methods:
Agilent SurePrint G3 Mouse Gene Expression V2 microarray chip and Western blotting were used to screen the molecules whose expression levels in mouse macrophages (RAW246.7) were correlated with the stimulation intensity of LPS. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (chip-qPCR) were used to verify the binding of the molecules to the promoters of IL-6 and TNF-α genes. The effects of transfection of RAW246.7 cells with overexpression or interfering plasmids on IL-6 and TNF-α synthesis were evaluated with ELISA, and the binding level of the target molecules and acetylation level of H3K27 in the promoter region of IL-6 and TNF-α genes were analyzed by chromatin immunoprecipitation sequencing technique (chip-seq).
Results:
Gene microarray chip data and Western blotting both confirmed a strong correlation of p300 expression with the stimulation intensity of LPS. Immunocoprecipitation confirmed the binding between p300 and c-myb. The results of EMSA demonstrated that c-myb (P < 0.05), but not p300, could directly bind to the promoter region of IL-6 and TNF-α genes; p300 could bind to the promoters only in the presence of c-myb (P < 0.05). The expressions of p65, p300 and c-myb did not show interactions. Both p300 overexpression and LPS stimulation could increase the level of promoter-binding p300 and H3K27 acetylation level, thus promoting p65 binding and inflammatory gene transcription; such effects were obviously suppressed by interference of c-myb expression (P < 0.05). Interference of p65 resulted in inhibition of p65 binding to the promoters and gene transcription (P < 0.05) without affecting p300 binding or H3K27 acetylation level.
Conclusion:
LPS can stimulate the synthesis of p300, whose binding to the promoter region of inflammatory genes via c-myb facilitates the cohesion of p65 by inducing H3K27 acetylation, thus promoting the expression of the inflammatory genes.
Insights
Lipopolysaccharide (LPS) stimulates coactivator p300 synthesis, which binds inflammatory gene promoters via c-myb. This interaction promotes p65 binding and inflammatory gene expression through H3K27 acetylation.
Area of Science:
- Molecular Biology
- Immunology
- Epigenetics
Background:
- Lipopolysaccharide (LPS) triggers inflammatory responses.
- Coactivator p300 plays a role in inflammatory mediator synthesis.
- Acetylated modifications are implicated in inflammatory gene regulation.
Purpose of the Study:
- To investigate the role of p300-induced acetylation in LPS-induced inflammatory mediator synthesis.
- To elucidate the molecular mechanism underlying this process.
Main Methods:
- Gene expression profiling (microarray) and Western blotting to identify LPS-responsive molecules.
- Electrophoretic mobility shift assay (EMSA) and ChIP-qPCR to assess molecular binding to gene promoters.
- ELISA for measuring cytokine synthesis.
- ChIP-seq to analyze protein binding and histone acetylation at gene promoters.
Main Results:
- p300 expression strongly correlated with LPS stimulation intensity.
- c-myb directly binds IL-6 and TNF-α promoters; p300 binds in the presence of c-myb.
- p300 and LPS stimulation increased promoter-binding p300, H3K27 acetylation, and p65 binding, promoting inflammatory gene transcription.
- Interference with c-myb suppressed these effects; interference with p65 inhibited transcription without affecting p300 binding or acetylation.
Conclusions:
- LPS stimulates p300 synthesis.
- p300 binding to inflammatory gene promoters, facilitated by c-myb, promotes p65 cohesion via H3K27 acetylation.
- This mechanism drives the expression of inflammatory genes.
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