Regulation of NF-kappaB activity by the class I histone deacetylases, HDAC1, HDAC2, HDAC3 corepressors and the spermidine/spermine N1 acetyltransferase 2 (SSAT2) coactivator.

摘要

The research described in this dissertation is focused on studying the role of individual histone deacetylases in regulation of NF-kappaB transcriptional activity using a RNA interfering system to repress the expression of hdac1, hdac2 or hdac3.;Our findings indicate that each of the individual HDACs play an important role in regulation of NF-kappaB transcriptional activity. Knockdown of HDAC1 enhances and prolongs NF-kappaB nuclear translocation and delays IkappaBalpha cytoplasmic reappearance after TNF stimulation. The extended duration of NF-kappaB in the nucleus, resulted from HDAC1 knockdown, causes enhanced cIAP2, IL-8, IL-6 and MCP1 expression but does not affect IkappaBalpha gene expression after TNF stimulation. In addition, not only does HDAC1 regulate NF-kappaB activity, but it also plays an essential role in cell cycle arrest, regulating p21 expression, as well as in apoptosis. Knockdown of HDAC1 increases p21 gene expression and p21 protein expression. Moreover HDAC1 knockdown enhances and speeds up the activation of caspase 3---an effector caspase activated in response to Doxorubicin stimulation. HDAC2 is yet another essential corepressor protein that has a substantial impact on regulation of NF-kappaB activity. Knockdown of HDAC2 also enhances and prolongs NF-kappaB residence in the nucleus after TNF treatment, but the effect is not as substantial as the impact that HDAC1 knockdown has on p65 nuclear translocation. Contrary to HDAC1 knockdown, the resynthesis of IkappaBalpha is unaffected in cells with reduced HDAC2 levels. Similar to HDAC1 knockdown, knockdown of HDAC2 enhances IL-8, IL-6, and MCP1 expression after TNF stimulation. However, reduced HDAC2 levels cause a decrease in IkappaBalpha expression and do not affect cIAP2 expression. HDAC3 also plays a essential role in regulation of NF-kappaB activity. Similar to HDAC2 knockdown, the reduction in HDAC3 levels enhances and prolongs NF-kappaB nuclear translocation but does not affect the cytoplasmic reappearance of IkappaBalpha after TNF treatment. Cells with HDAC1, HDAC2, and HDAC3 knockdown had increased levels of IL-8 and IL-6 genes. Similar to the knockdown of HDAC2, reduced HDAC3 levels cause a decrease in IkappaBalpha expression after TNF induction. On the other hand, only the knockdown of HDAC3 decreases cIAP2, and MCP1 levels.;The enhanced expression of NF-kappaB regulated genes correlated with increased binding of p65 and p65 phosphorylated on serine 536 to the promoters of its regulated genes. Most of the upregulated genes also had increased levels of CBP and IKKalpha associated with their promoters and had higher levels of Histone H3 acetylation and H3 phosphorylation on serine 10.;In conclusion all three HDACs regulate NF-kappaB activity in a unique way and at multiple levels, for example prolonging the duration of NF-kappaB in the nucleus, and modulating the expression of NF-kappaB dependent genes. In addition the outcome of the HDAC action depends on the gene: some genes are downregulated but others require the presence of HDACs for the initiation of transcription. Overall, a better understanding of the individual roles of these HDACs in the regulation of transcription factors, including NF-kappaB, will help in designing more effective cancer treatments than what is currently available.;Because of the similarity of SSAT2 to coactivators, we wanted to determine if SSAT2 could function as a coactivator for NF-kappaB. Coimmunoprecipitations confirmed the interaction between p65 and SSAT2. In transient transfection reporter gene assays, SSAT2 functions as a transcriptional coactivator for NF-kappaB and cooperates with CBP and P/CAF to enhance TNFalpha-induced NF-kappaB activity. Moreover, SSAT2 transiently associates with the promoters of the NF-kappaB-regulated cIAP2 and IL-8 genes in response to TNFalpha. Knockdown of SSAT2 decreases TNF-alpha induced NF-kappaB activity as measured by luciferase assays which results in decrease of MCP1 expression and increase of the expression of IL-6, IL-8 and cIAP2.;Based on our findings SSAT2 functions as a transcriptional coactivator. However ssat2 knockdown causes not only a decrease in gene expression of some NF-kappaB regulated genes like MCP1, but also an increase in other NF-kappaB regulated genes like cIAP2. Thus the function of SSAT2 in regulation of NF-kappaB activity might be more complex than it appears. (Abstract shortened by UMI.)