Regulation of NF-kappaB2 by TSA: Role of Acetylation

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DESCRIPTION (provided by applicant): Histone deacetylase (HDAC) inhibitor trichostatin A (TSA) exerts potent anti-tumor effect. Our findings suggest that Nuclear Factor (B (NF-(B)is a primary transcription factor target of TSA. NF-kappaB is important in many aspects of tumor development and treatment. Our long-term goal is to elucidate the molecular mechanisms by which HDAC inhibitor TSA down-regulates NF-kappaB and NF-kappaB controlled target gene expression. The specific hypothesis of this proposal is that TSA-induced p100 processing and p52 acetylation plays a causal role in the regulation of NF-kappaB activity and NF-kappaB controlled cyclin D1 gene transcription. We base that hypothesis on the observations that 1) TSA promotes NF-kappaB2/p100 processing, 2) TSA enhances p52 level and acetylation, 3) Accumulation of acetylated p52 coincides with disruption of NF-kappaB DNA binding and cyclin D1 downregulation. Based on these observations, our specific aims are to: 1. Determine the role of p100 acetylation in HDAC inhibitor TSA induced NF-kappaB2/p100 processing. We will use p100 acetylation mutants to test the hypothesis that p100 processing into p52 requires acetylation at certain sites. 2. Identify the acetylation sites on p52 and ascertain their causal role in regulating NF-kappaB DNA binding upon TSA treatment. The Electrophoretic-Mobility Shift Assay (EMSA) of nuclear protein from cells transfected with p52 acetylation mutants or wild type p52 should reveal which acetylation sites are required for TSA disruption of NF-kappaB DNA binding. 3. Determine the possible causal role of p52 acetylation in TSA down regulated cyclin D1 gene expression. If p52 acetylation at certain sites contributes to the down regulation of cyclin D1 expression by TSA, then transfection of p52 mutated at those sites will fail to down regulate cyclin D1 RNA and protein expression. In addition, we will determine whether p52 acetylation affects the binding of transcription factors Sp-1, AP-1, CREB to cyclin D1 promoter using EMSA and chromatin immunoprecipitation (ChIP).