title: A minimal ubiquitous chromatin opening element (UCOE) effectively prevents silencing of juxtaposed heterologous promoters by epigenetic remodeling in multipotent and pluripotent stem cells.
abstract: Epigenetic silencing of transgene expression represents a major obstacle for the efficient genetic modification of multipotent and pluripotent stem cells. We and others have demonstrated that a 1.5 kb methylation-free CpG island from the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) effectively prevents transgene silencing and variegation in cell lines, multipotent and pluripotent stem cells, and their differentiated progeny. However, the bidirectional promoter activity of this element may disturb expression of neighboring genes. Furthermore, the epigenetic basis underlying the anti-silencing effect of the UCOE on juxtaposed promoters has been only partially explored. In this study we removed the HNRPA2B1 moiety from the A2UCOE and demonstrate efficient anti-silencing properties also for a minimal 0.7 kb element containing merely the CBX3 promoter. This DNA element largely prevents silencing of viral and tissue-specific promoters in multipotent and pluripotent stem cells. The protective activity of CBX3 was associated with reduced promoter CpG-methylation, decreased levels of repressive and increased levels of active histone marks. Moreover, the anti-silencing effect of CBX3 was locally restricted and when linked to tissue-specific promoters did not activate transcription in off target cells. Thus, CBX3 is a highly attractive element for sustained, tissue-specific and copy-number dependent transgene expression in vitro and in vivo.
authors.affiliation: Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Hessen, 60596, Germany.
authors.forename: Uta
authors.initials: U
authors.lastname: Müller-Kuller
authors.affiliation: RG Reprogramming and Gene Therapy, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Lower Saxony, 30625, Germany Institute of Experimental Hematology, Hannover Medical School, Hannover, Lower Saxony, 30625, Germany.
authors.forename: Mania
authors.initials: M
authors.lastname: Ackermann
authors.affiliation: Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Hessen, 60596, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Kolodziej
authors.affiliation: Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Hessen, 60596, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Brendel
authors.affiliation: RG Reprogramming and Gene Therapy, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Lower Saxony, 30625, Germany Institute of Experimental Hematology, Hannover Medical School, Hannover, Lower Saxony, 30625, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Fritsch
authors.affiliation: RG Reprogramming and Gene Therapy, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Lower Saxony, 30625, Germany Institute of Experimental Hematology, Hannover Medical School, Hannover, Lower Saxony, 30625, Germany.
authors.forename: Nico
authors.initials: N
authors.lastname: Lachmann
authors.affiliation: Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Hessen, 60596, Germany.
authors.forename: Hana
authors.initials: H
authors.lastname: Kunkel
authors.affiliation: Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Hessen, 60596, Germany.
authors.forename: Jörn
authors.initials: J
authors.lastname: Lausen
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Lower Saxony, 30625, Germany Division of Pediatric Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: RG Reprogramming and Gene Therapy, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Lower Saxony, 30625, Germany Institute of Experimental Hematology, Hannover Medical School, Hannover, Lower Saxony, 30625, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Moritz
authors.affiliation: Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Hessen, 60596, Germany grez@gsh.uni-frankfurt.de.
authors.forename: Manuel
authors.initials: M
authors.lastname: Grez
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002469:Cell Separation; D002843:Chromatin / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D044127:Epigenesis, Genetic; D005434:Flow Cytometry; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D039902:Multipotent Stem Cells / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D019076:Transgenes
chemical_list: D002843:Chromatin