PT  - JOURNAL ARTICLE
AU  - Cassandra M. Barrett
AU  - Reilly McCracken
AU  - Jacob Elmer
AU  - Karmella A. Haynes
TI  - Use of MYB as a new synthetic activator to enhance transgene expression within repressed Polycomb chromatin
AID  - 10.1101/487736
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 487736
4099  - http://biorxiv.org/content/early/2018/12/05/487736.short
4100  - http://biorxiv.org/content/early/2018/12/05/487736.full
AB  - Background Epigenetic silencing of transgenes through chromatin packaging has been a persistent issue for the development of transgenic mammalian cell lines. Endogenous mechanisms are known to induce a closed chromatin state around foreign DNA before and after it has been integrated into a host cell’s genome. Scientists are interested in reversing this silencing, but a lack of a priori knowledge of the chromatin features at transgenes hinders the rational design and application of effective strategies for transcriptional activation.Results Here, we systematically tested activation-associated DNA elements and proteins in transfected plasmid DNA and at epigenetically-silenced chromosomal transgenes. We demonstrated that placing DNA elements that are targeted by MYB (c-myb) and p65 upstream of a minimal promoter enhance expression from transfected plasmid DNA. To regulate the expression of chromosomally-integrated transgenes, we used proteins fused to the Gal4 DNA binding domain or dCas9/sgRNA. Three activation-associated peptides, p65, VP64, and MYB, sustained reactivation of transgene expression over 15 cell divisions in an immortalized human cell line (HEK293). Activity of the MYB fusion was inhibited by celastrol, a drug that blocks interactions between MYB and the p300/CBP histone acetyltransferase complex. Single-site targeting via dCas9-MYB was sufficient to activate transgenes within ectopic Polycomb heterochromatin and at a different site that had undergone position effect silencing.Conclusion Here we demonstrate the utility and flexibility of cis-regulatory elements and fusion proteins derived from natural gene regulation systems to enhance expression from epigenetically silenced transgenes. DNA motifs for p65 and MYB can be added to the transgene itself, or the activating proteins can be targeted to transgenes without enhancers to stimulate gene activation. This work has implications for determining the most appropriate strategy to enhance gene expression specifically in Polycomb-repressed chromatin.AAPactivation associated peptideCMVcytomegalovirusCRchromatin remodelerGal4Gal4 DNA binding domainHAThistone acetyltransferaseNLSnuclear localization signalORFopen reading framePFpioneer factorPolIIRNA polymerase IIPRCPolycomb repressive complexTADtranscriptional activation domainUASupstream activation sequence