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

International Journal of Molecular Sciences

卷:21

Jacob Elmer¹  Reilly McCracken¹  CassandraM. Barrett²  KarmellaA. Haynes² 

[1] Department of Chemical Engineering, Villanova University, 217 White Hall, 800 East Lancaster Avenue, Villanova, PA 19085, USA;

[2] School of Biological and Health Systems Engineering, Arizona State University, 501 East Tyler Mall, Tempe, AZ 85287, USA;

关键词: myb;    c-myb;    transgene;    epigenetic silencing;    activator;    heterochromatin;    polycomb;   

DOI  :  10.3390/ijms21020530

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown