Evidence of the capability of the CMV enhancer to activate in trans gene expression in mammalian cells.
Studies have reported that an enhancer can act in trans when artificially, noncovalently bridged to the promoter by a protein-linked biotin:streptavidin complex, or when an enhancer and a promoter are located on separate concatenated plasmids. To investigate such transactivation in mammalian cells, we constructed CMV promoter-enhancer mutants driving the expression of the EGFP reporter gene and transfected cultured cells with various combinations of the mutant PCR products; results were analyzed using fluorescence microscopy and flow cytometry. Our results show that the CMV enhancer can stimulate transcription in trans, even in the absence of physical association of the enhancer and promoter. Furthermore, we show that the transactivation of the CMV enhancer can be strengthened by the histone deacetylase inhibitor sodium butyrate. Finally, we provide evidence that the CMV enhancer can influence, in trans, the activity of heterologous promoters. Although different mechanisms may lead to transcriptional activation when the CMV enhancer is not covalently linked to the promoter, our results suggest that the main mechanism resembles the process of transfection and may be important for gene regulation. These findings may have implications in understanding the processes that underlie gene therapy because of the potential alteration of endogenous gene expression.
D'Aiuto
Leonardo
L
Department of Surgery, Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219, USA. daiutol@msx.upmc.edu
De Marco
Richard
R
Edward
Nwanegbo
N
Rizzo
Annalisa
A
Chaillet
J Richard
JR
Montecalvo
Angela
A
Lotze
Michael T
MT
Gambotto
Andrea
A
D000818:Animals; D020148:Butyric Acid / Q000494:pharmacology; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004742:Enhancer Elements, Genetic; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006655:Histone Deacetylases / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009154:Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D018076:DNA, Complementary; D020148:Butyric Acid; D049452:Green Fluorescent Proteins; D006655:Histone Deacetylases