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Open AccessArticle

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

1
School of Biological and Health Systems Engineering, Arizona State University, 501 East Tyler Mall, Tempe, AZ 85287, USA
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Department of Chemical Engineering, Villanova University, 217 White Hall, 800 East Lancaster Avenue, Villanova, PA 19085, USA
3
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; https://doi.org/10.3390/ijms21020530
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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