Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.

2.50
Hdl Handle:
http://hdl.handle.net/10033/621130
Title:
Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.
Authors:
Gödecke, Natascha; Zha, Lisha; Spencer, Shawal; Behme, Sara; Riemer, Pamela; Rehli, Michael; Hauser, Hansjörg; Wirth, Dagmar
Abstract:
Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.
Affiliation:
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, D38124 Braunschweig, Germany.
Citation:
Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation. 2017, 45 (16):e147 Nucleic Acids Res.
Journal:
Nucleic acids research
Issue Date:
19-Sep-2017
URI:
http://hdl.handle.net/10033/621130
DOI:
10.1093/nar/gkx601
PubMed ID:
28934472
Type:
Article
Language:
en
ISSN:
1362-4962
Appears in Collections:
publications of the research group modell systems for infections and immunity (MSYS); publications of the scientific administration (GFW)

Full metadata record

DC FieldValue Language
dc.contributor.authorGödecke, Nataschaen
dc.contributor.authorZha, Lishaen
dc.contributor.authorSpencer, Shawalen
dc.contributor.authorBehme, Saraen
dc.contributor.authorRiemer, Pamelaen
dc.contributor.authorRehli, Michaelen
dc.contributor.authorHauser, Hansjörgen
dc.contributor.authorWirth, Dagmaren
dc.date.accessioned2017-10-09T13:56:28Z-
dc.date.available2017-10-09T13:56:28Z-
dc.date.issued2017-09-19-
dc.identifier.citationControlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation. 2017, 45 (16):e147 Nucleic Acids Res.en
dc.identifier.issn1362-4962-
dc.identifier.pmid28934472-
dc.identifier.doi10.1093/nar/gkx601-
dc.identifier.urihttp://hdl.handle.net/10033/621130-
dc.description.abstractFaithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.en
dc.language.isoenen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.titleControlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.en
dc.typeArticleen
dc.contributor.departmentHelmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, D38124 Braunschweig, Germany.en
dc.identifier.journalNucleic acids researchen

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