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Effective transgene expression in mammalian cells relies on successful delivery, cytoplasmic trafficking, and nuclear translocation of the delivered vector, but delivery is impeded by several formidable physicochemical barriers on the surface of and within the target cell. Although methods to overcome cellular exclusion and endosomal entrapment have been studied extensively, strategies to overcome inefficient nuclear entry and subsequent intranuclear barriers to effective transient gene expression have only been sparsely explored. In particular, the role of nuclear packaging of DNA with histone proteins, which governs endogenous gene expression, has not been extensively elucidated in the case of exogenously delivered plasmids. In this work, a parallel screen of small molecule inhibitors of chromatin-modifying enzymes resulted in the identification of class I/II HDACs, sirtuins, LSD1, HATs, and the methyltransferases EZH2 and MLL as targets whose inhibition led to the enhancement of transgene expression following polymer-mediated delivery of plasmid DNA. Quantitative PCR studies revealed that HDAC inhibition enhances the amount of plasmid DNA delivered to the nucleus in UMUC3 human bladder cancer cells. Native chromatin immunoprecipitation (N-ChIP)-qPCR experiments in CHO-K1 cells indicated that plasmids indeed interact with intracellular core Histone H3, and inhibitors of HDAC and LSD1 proteins are able to modulate this interaction. Pair-wise treatments of effective inhibitors led to synergistic enhancement of transgene expression to varying extents in both cell types. Our results demonstrate that the ability to modulate enzymes that play a role in epigenetic processes can enhance the efficacy of non-viral gene delivery, resulting in significant implications for gene therapy and industrial biotechnology.Inhibition of chromatin-modifying enzymes enhances transgene expression.Combinatorial enzyme inhibition further affects transient expression.HDAC inhibition increases nuclear entry of plasmid in UMUC3 bladder cancer cells.Exogenous plasmids interact with endogenous core histone H3.Inhibition of HDAC and/or LSD1 modulates plasmid/histone H3 interactions.