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  • br Mammalian chromosomal DNA binds to various proteins

    2021-09-18


    Mammalian chromosomal DNA binds to various proteins and the DNA-protein complexes form DNA packaging units, called nucleosomes. Histones are the chief protein components of nucleosomes and perform pivotal functions in chromosomal gene regulation. Moreover, many types of chemical modifications of histones, such as acetylation, methylation, phosphorylation, ubiquitination, and ADP-ribosylation, are believed to play important roles in the modulation of MRT67307 HCl function (histone code hypothesis) . Among them, acetylation is a well-characterized modification. Hyperacetylated and hypoacetylated histones are regarded as hallmarks of nucleosomes at active and inactive genes, respectively , , , . Acetylation may affect gene expression by modifying the chromatin conformation and/or the recruitment of regulatory factors. Nucleosomes are also formed on non-integrated plasmid DNAs delivered by nonviral vectors, indicating that plasmid DNAs bind histones in the nuclei , . In agreement with these findings, transgene expression was influenced by the introduction of DNA sequences that modulate histone positioning into plasmid DNAs , , , , . Thus, the binding of histones to plasmid DNA is a key factor for the intranuclear disposition of exogenous DNA and efficient transgene expression . Based on the dynamics of histones that bind to chromosomal DNA, those bound to plasmid DNA could also be chemically modified. This hypothesis suggests that transgene expression might be regulated by altering the histone modification patterns. For instance, the acetylation of histones that bind to plasmid DNA might upregulate transgene expression. Histone acetylation is controlled by two families of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs) , , . Inhibitors of HDACs are used for the enrichment of acetylated histones in cells. Treatments with HDAC inhibitors increased transgene expression from episomal/integrated plasmid DNAs and adenoviral DNA, as well as that in cell lines transduced by a lentivirus , , , . These reports strongly support the idea that the acetylation of histones that bind to plasmid DNA enhances transgene expression. However, the treatments with HDAC inhibitors result in the acetylation of histones bound to chromosomal DNA and affect genome-wide gene expression. Thus, plasmid DNA-specific histone acetylation is required. We previously described efficient transgene expression from plasmid DNA, using artificial transcription factors that bind to their recognition sites within the plasmid DNA , , . In this system, a fusion protein of the sequence-specific DNA binding domain of yeast () GAL4 and the transcription activation domains of viral and MRT67307 HCl mammalian transcription factors were used. The reporter plasmid DNA contains five tandem copies of the 17-bp GAL4 DNA binding site (G5) in both the upstream and downstream regions of the luciferase gene expression cassette. The G5 sequences are also present in both the upstream and downstream regions of the GAL4-transcription factor expression cassette in the activator plasmid DNA. As a result, the expression of both the reporter and activator genes was strongly promoted by the plasmid-specific transcription factors. We noticed that the replacement of the transcription factor with HAT achieved plasmid DNA-specific histone acetylation by the artificial HAT, and consequently increased transgene expression. In this study, we constructed an activator plasmid DNA containing the gene encoding a fusion protein of GAL4 and the HAT domain of mouse CREB-binding protein (CREBBP) (amino acid residues 1092–1764) . We introduced the activator GAL4-HAT and the reporter luciferase plasmid DNAs into mouse Hepa1-6 cells by lipofection. The expression of the activator plasmid DNA enriched the acetylated histones bound to the reporter plasmid and efficiently acted as an activator. These results indicated that this strategy is useful to promote transgene expression.