Dataset Information

Epigenetic Regulation of Intronic Transgenes in Arabidopsis.

ABSTRACT: Defense mechanisms of plant genomes can epigenetically inactivate repetitive sequences and exogenous transgenes. Loss of mutant phenotypes in intronic T-DNA insertion lines by interaction with another T-DNA locus, termed T-DNA suppression, has been observed in Arabidopsis thaliana, although the molecular basis of establishment and maintenance of T-DNA suppression is poorly understood. Here we show that maintenance of T-DNA suppression requires heterochromatinisation of T-DNA sequences and the nuclear proteins, INCREASED IN BONSAI METHYLATION 2 (IBM2) and ENHANCED DOWNY MILDEW 2 (EDM2), which prevent ectopic 3' end processing of mRNA in atypically long introns containing T-DNA sequences. Initiation of T-DNA suppression is mediated by the canonical RdDM pathway after hybridisation of two T-DNA strains, accompanied by DNA hypermethylation of T-DNA sequences in the F1 generation. Our results reveal the presence of a genome surveillance mechanism through genome hybridisation that masks repetitive DNAs intruding into transcription units.

SUBMITTER: Osabe K

PROVIDER: S-EPMC5364540 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Epigenetic Regulation of Intronic Transgenes in Arabidopsis.

Osabe Kenji K Harukawa Yoshiko Y Miura Saori S Saze Hidetoshi H

Scientific reports 20170324

Defense mechanisms of plant genomes can epigenetically inactivate repetitive sequences and exogenous transgenes. Loss of mutant phenotypes in intronic T-DNA insertion lines by interaction with another T-DNA locus, termed T-DNA suppression, has been observed in Arabidopsis thaliana, although the molecular basis of establishment and maintenance of T-DNA suppression is poorly understood. Here we show that maintenance of T-DNA suppression requires heterochromatinisation of T-DNA sequences and the nu ...[more]

PMID: 28338020

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Project description:Adeno-associated virus (AAV) is a powerful gene therapy vector that has been used in several FDA-approved therapies as well as in multiple clinical trials. This vector has high therapeutic versatility with the ability to deliver genetic payloads to a variety of human tissue types, yet there is currently a lack of transgene expression control once the virus is administered. There are also times when transgene expression is too low for the desired therapeutic outcome, necessitating high viral dose administration resulting in possible immunological complications. Herein, we validate a chemically controllable AAV transgene expression technology in vitro that utilizes bifunctional molecules known as chemical epigenetic modifiers (CEMs). These compounds employ endogenous epigenetic machinery to specifically enhance transgene expression of episomal DNA. A recombinant AAV (rAAV) was designed to both deliver the reporter transgene as well as deliver a synthetic zinc finger (ZFs) protein fused to FK506 binding protein (FKBP). These synthetic ZFs target a DNA-binding array sequence upstream of the promoter expressing the AAV transgene to specifically enhance AAV transgene expression in the presence of a CEM. The transcriptional activating compound CEM87 functions by recruiting the epigenetic transcription activator bromodomain-containing protein 4 (BRD4), increasing AAV transgene activity up to fivefold in a dose-dependent manner in HEK293T cells. The highest levels of transgene product activity are seen 24 h following CEM87 treatment. Additionally, the CEM87-mediated enhancement of different transgene products with either Luciferase or green fluorescent protein (GFP) was observed in multiple cell lines and enhancement of transgene expression was capsid serotype independent. The impact of CEM87 activity can be disrupted through drug removal or chemical recruitment site competition with FK506, thus demonstrating the reversibility of the impact of CEM87 on transgene expression. Collectively, this chemically controllable rAAV transgene technology provides temporal gene expression control that could increase the safety and efficiency of AAV-based research and therapies.

Dataset Information

Epigenetic Regulation of Intronic Transgenes in Arabidopsis.

Publications

Epigenetic Regulation of Intronic Transgenes in Arabidopsis.

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