Abstract
In eukaryotic cells, transgene expression levels may be limited by an unfavourable chromatin structure at the integration site. Epigenetic regulators are DNA sequences which may protect transgenes from such position effect. We evaluated different epigenetic regulators for their ability to protect transgene expression at telomeres, which are commonly associated to low or inconsistent expression because of their repressive chromatin environment. Although to variable extents, matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE) and the chicken cHS4 insulator acted as barrier elements, protecting a telomeric-distal transgene from silencing. MARs also increased the probability of silent gene reactivation in time-course experiments. Additionally, all MARs improved the level of expression in non-silenced cells, unlike other elements. MARs were associated to histone marks usually linked to actively expressed genes, especially acetylation of histone H3 and H4, suggesting that they may prevent the spread of silencing chromatin by imposing acetylation marks on nearby nucleosomes. Alternatively, an UCOE was found to act by preventing deposition of repressive chromatin marks. We conclude that epigenetic DNA elements used to enhance and stabilize transgene expression all have specific epigenetic signature that might be at the basis of their mode of action.
Highlights
In the nucleus of eukaryotic cells, linear chromosomal DNA associates with histones and other proteins to form chromatin
In addition to epigenetic regulators, neutral sequences of various lengths were cloned as spacer controls, as the strength of telomeric silencing in HeLa cells was found to partly depend on the distance from the telomeres, to what was observed in yeast [12]
matrix attachment regions (MARs) greatly increased the proportion of cells expressing a telomeric-distal reporter gene, while they less frequently prevented the silencing of a telomericproximal transgene, suggesting a prominent barrier activity for this class of epigenetic regulators
Summary
In the nucleus of eukaryotic cells, linear chromosomal DNA associates with histones and other proteins to form chromatin. DNA expression and replication [1]. Numerous factors, including histone modifications, incorporation of histone variants and DNA methylation, affect the chromatin structure and the accessibility of DNA to the transcription and replication machineries. The histone tails can be decorated with a number of modifications. Some of them, such as acetylation of histone H3 and H4 or di/trimethylation of H3K4, are typically associated with active transcription and are referred to as euchromatin modifications. Modifications such as trimethylation of H3K9, H3K27 or H4K20 are commonly mapped on inactive genomic regions and termed heterochromatin modifications [1]
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