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

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.

Targeted regulation of plasmid DNA expression in eukaryotic cells with a methylated-DNA-binding activator

“…For instance, we have demonstrated that adding NF-Y and MYB transcriptional activator target DNA sequences upstream of a minimal promoter increased luciferase expression from transfected pDNA in prostate cancer PC-3 cells. [6,7] Fusion proteins can also be used as transgene-specific regulators to boost expression in mammalian cells. [8][9][10][11] Both approaches, enhancer elements and fusion protein regulators, require the addition of DNA target sequences into the pDNA, which may not always be possible or desirable.…”