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Open AccessArticle

Components from the Human c-myb Transcriptional Regulation System Reactivate Epigenetically Repressed Transgenes

School of Biological and Health Systems Engineering, Arizona State University, 501 East Tyler Mall, Tempe, AZ 85287, USA
Department of Chemical Engineering, Villanova University, 217 White Hall, 800 East Lancaster Avenue, Villanova, PA 19085, USA
Wallace H. Coulter Department of Biomedical Engineering, Emory University, Atlanta, GA 30322, USA
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(2), 530;
Received: 11 November 2019 / Revised: 7 January 2020 / Accepted: 7 January 2020 / Published: 14 January 2020
(This article belongs to the Special Issue Advances in Epigenome Editing)
A persistent challenge for mammalian cell engineering is the undesirable epigenetic silencing of transgenes. Foreign DNA can be incorporated into closed chromatin before and after it has been integrated into a host cell’s genome. To identify elements that mitigate epigenetic silencing, we tested components from the c-myb and NF-kB transcriptional regulation systems in transiently transfected DNA and at chromosomally integrated transgenes in PC-3 and HEK 293 cells. DNA binding sites for MYB (c-myb) placed upstream of a minimal promoter enhanced expression from transiently transfected plasmid DNA. We targeted p65 and MYB fusion proteins to a chromosomal transgene, UAS-Tk-luciferase, that was silenced by ectopic Polycomb chromatin complexes. Transient expression of Gal4-MYB induced an activated state that resisted complete re-silencing. We used custom guide RNAs and dCas9-MYB to target MYB to different positions relative to the promoter and observed that transgene activation within ectopic Polycomb chromatin required proximity of dCas9-MYB to the transcriptional start site. Our report demonstrates the use of MYB in the context of the CRISPR-activation system, showing that DNA elements and fusion proteins derived from c-myb can mitigate epigenetic silencing to improve transgene expression in engineered cell lines. View Full-Text
Keywords: MYB; c-myb; transgene; epigenetic silencing; activator; heterochromatin; polycomb MYB; c-myb; transgene; epigenetic silencing; activator; heterochromatin; polycomb
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Barrett, C.M.; McCracken, R.; Elmer, J.; Haynes, K.A. Components from the Human c-myb Transcriptional Regulation System Reactivate Epigenetically Repressed Transgenes. Int. J. Mol. Sci. 2020, 21, 530.

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