Abstract
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.
Highlights
The advancement of cell engineering requires robust and reliable control of endogenous and synthetic genetic material within living cells
We focus on reversal of silencing within Polycomb heterochromatin, which is known to accumulate at transgenes that are integrated into chromosomes [7–9] and is widely distributed across hundreds or thousands of endogenous mammalian genes that play critical roles in normal development and disease [9,35,36]
We have demonstrated that DNA enhancer elements and fusion proteins derived from endogenous mammalian systems can be used to enhance expression from transiently transfected DNA
Summary
The advancement of cell engineering requires robust and reliable control of endogenous and synthetic genetic material within living cells. A lack of tools for enhancing the expression of transgenes in mammalian cells currently limits effective gene regulation in different contexts. Unpredictable formation of heterochromatin around transgenic material in mammalian cells limits our ability to express foreign DNA for the production of therapeutic proteins and the development of engineered mammalian systems for biosensing and computing [1,2]. Nucleation of heterochromatin around transgenic material can be initiated and sustained by promoter methylation [1,5] and various histone modifications [2,4]. MyD88 pathway-mediated silencing of transgenes leads to an accumulation of repressive H3K9me on newly bound histones [2,6]. Silencing of transgenes may be Polycomb-mediated, where Polycomb repressive complexes deposit H3K27me on histones to establish a silenced state [7–9]. The frequency of undesired transgene silencing has led to the development of tools for mammalian cell engineering aimed at combating heterochromatin
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