Activation of CMV promoter-controlled glycosyltransferase and β -galactosidase glycogenes by butyrate, tricostatin A, and 5-Aza-2′-deoxycytidine

Abstract

Cytomegalovirus (CMV) immediate early promoter is a powerful promoter frequently used for driving the expression of transgenes in mammalian cells. However, this promoter gradually becomes silenced in stably transfected cells. We employed Chinese Hamster Ovary (CHO) and human pancreatic cancer (Panc 1) cells stably tansfected with three glycogenes driven by a CMV promoter to study the activation of silenced glycogenes. We found that butyrate, tricostatin A (TSA), and 5-aza-2 -deoxycytidine (5-Aza-dC) can activate these CMV-driven glycogenes. The increase in mRNA and protein of a glycogene occurred 8-10 h after butyrate treatment, suggesting an indirect effect of butyrate in the activation of the transgene.The enhanced expression of the trangenes by butyrate and TSA, known inhibitors of histone deacetylase, was independent of the transgene or cell type. However, the transgene can be activated by these two agents in only a fraction of the cells derived from a single clone, suggesting that inactivation of histone deacetylase can only partially explain silencing of the transgenes. Combination treatment of one or both agents with 5-Aza-dC, a known inhibitor of DNA methylase, resulted in a synergistic activation of the transgene, suggesting a cross-talk between histone acetylation and DNA demethylation. Understanding the mechanisms of the inactivation and reactivation of CMV promoter-controlled transgenes should help develop an effective strategy to fully activate the CMV promoter-controlled therapeutic genes silenced by the host cells. Published in 2005.

Hemin Interactions and Alterations of the Subcellular Localization of Prion Protein

“…4, a and b, no alteration of NCAM was observed, whereas GFP bands increased with hemin treatment. GFP-GPI is expressed under a cytomegalovirus promoter whose activity can be enhanced by histone acetylation or demethylation (33). Given that hemin can regulate both histone acetylation and methylation (34), the increase of GFP-GPI on the cell surface could be due to increased expression, which was confirmed by a Western blot assay on total cell FIGURE 3.…”

Cell type-specific activation of the cytomegalovirus promoter by dimethylsulfoxide and 5-Aza-2'-deoxycytidine

“…However, it becomes inactivated over time after insertion into the host genome (Cameron, Bachman, Myohanen, Herman, & Baylin, 1999;Choi et al, 2005;Knust et al, 1989;Palmer et al, 1991;Verma & Somia, 1997). Finding ways to activate the silenced CMV promoter should help overcome this limitation and facilitate the use of this promoter for enhancing the expression of the transgenes and production of recombinant proteins (Singh et al, 2004).…”

“…However, many transgenes under the control of viral promoters, including the CMV promoter, have been found to be gradually silenced over a period of months in culture (Choi, Basma, Singh, & Cheng, 2005;Knust, Bruggemann, & Doerfler, 1989;Krishnan et al, 2006;Palmer, Rosman, Osborne, & Miller, 1991;Verma & Somia, 1997). The gene silencing mechanism of these promoters is not clear, although DNA methylation, histone hypoacetylation, activation of signaling molecules and altered levels of transcriptional factors have been suggested (Brooks, Harkins, Wang, Qian, Liu, & Rubanyi, 2004;Swindle & Klug, 2002).…”

Modulation of heparan sulfate biosynthesis by sodium butyrate in recombinant CHO cells

“…The Dual-10 cells are transfected with human NDST2 and mouse HS3st1 under the control of CMV promoters. It has been shown that butyrate affects transgenes regulated by CMV promoters (Choi et al 2005a). Therefore, butyrate may increase the expression of NDST2 and HS3st1, resulting in an increase in AT binding sites on HS.…”