PU.1, a member of the Ets family transcription factors, is expressed restrictively in hematopoietic cells including monocytes and macrophages, and plays critical roles in the inflammatory responses and the development of hematopoietic cells. CREB-binding protein (CBP) regulates transcription by acetylating not only histones but also certain transcription factors. Here, we demonstrated that a specific inhibitor of histone deacetylases, trichostatin A (TSA) inhibits PU.1 transcriptional activity in monocytes and further showed that deletion of a histone acetyltransferase (HAT) domain of CBP resulted in synergistic cooperativity between CBP and PU.1. When human monocytic cells THP-1 were treated with TSA, our immunoprecipitaion and western blot assay showed that TSA enhanced PU.1 acetylation. Next, we investigated the effect of TSA on the transcriptional regulation of PU.1-dependent gene promoters such as the human prointerleukin 1β (IL1B) gene and the human granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene in transient transfection studies. Two distinct luciferase reporter plasmids (Luc) for the IL1B gene promoter and the GM-CSFRα gene promoter, IL1B-Luc and GM-CSFRα-Luc were used. When these plasmids were transiently transfected into THP-1 cells, TSA suppressed LPS-induced activities for the IL1B promoter and the GM-CSFRα promoter in a dose-dependent manner. In contrast, when NF-κB luciferase reporter, NF-κB-Luc was transfected into THP-1 cells, TSA synergistically increased LPS-induced NF-κB activities. Moreover, when a PU.1 expression vector, pECEPU.1 was cotransfected into PU.1-deficient murine thymocytes EL4 along with either IL1B-Luc or GM-CSFRα-Luc. The PU.1-induced promoter activities were strongly suppressed through TSA treatment. FACS analysis further indicated that TSA suppressed LPS-induced expression of IL-1β and GM-CSFRα proteins. In addition, our EMSA data showed that TSA treatment did not affect DNA binding activity of PU.1 to the IL1B promoter. PU.1 has been shown to interact physically with CBP to transactivate their target genes. In our study, expression vectors for CBP wild-type or with a deletion of its HAT domain was cotransfected into EL4 cells along with IL1B-Luc and pECEPU.1. The HAT activity-deficient mutant showed synergistic transcriptional activity with PU.1 more strongly than the wild-type CBP. In this regard, our GST-pulldown assay showed that deletion of CBP HAT domain did not change binding affinity of CBP for PU.1. Our results propose a novel molecular mechanism by which PU.1-dependent genes is negatively regulated by HAT-induced acetylation in monocytes.
Disclosure: No relevant conflicts of interest to declare.