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The CpG-sites of the CBX3 ubiquitous chromatin opening element are critical structural determinants for the anti-silencing function.

The CpG-sites of the CBX3 ubiquitous chromatin opening element are critical structural determinants for the anti-silencing function.
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Kunkiel J, Gödecke N, Ackermann M, Hoffmann D, Schambach A, Lachmann N, Wirth D, Moritz T,


Kunkiel J, Gödecke N, Ackermann M, Hoffmann D, Schambach A, Lachmann N, Wirth D, Moritz T, (click to view)

Kunkiel J, Gödecke N, Ackermann M, Hoffmann D, Schambach A, Lachmann N, Wirth D, Moritz T,

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Scientific reports 2017 08 117(1) 7919 doi 10.1038/s41598-017-04212-8
Abstract

Suppression of therapeutic transgene expression from retroviral gene therapy vectors by epigenetic defence mechanisms represents a problem that is particularly encountered in pluripotent stem cells (PSCs) and their differentiated progeny. Transgene expression in these cells, however, can be stabilised by CpG-rich ubiquitous chromatin opening elements (UCOEs). In this context we recently demonstrated profound anti-silencing properties for the small (679 bp) CBX3-UCO element and we now confirmed this observation in the context of the defined murine chromosomal loci ROSA26 and TIGRE. Moreover, since the structural basis for the anti-silencing activity of UCOEs has remained poorly defined, we interrogated various CBX3 subfragments in the context of lentiviral vectors and murine PSCs. We demonstrated marked though distinct anti-silencing activity in the pluripotent state and during PSC-differentiation for several of the CBX3 subfragments. This activity was significantly correlated with CpG content as well as endogenous transcriptional activity. Interestingly, also a scrambled CBX3 version with preserved CpG-sites retained the anti-silencing activity despite the lack of endogenous promoter activity. Our data therefore highlight the importance of CpG-sites and transcriptional activity for UCOE functionality and suggest contributions from different mechanisms to the overall anti-silencing function of the CBX3 element.

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