Enhancement of transgene expression by nuclear transcription factor Y and CCCTC‐binding factor

Abstract

If a transgene is effectively delivered to a cell, its expression may still be limited by epigenetic mechanisms that silence the transgene. Indeed, once the transgene reaches the nucleus, it may be bound by histone proteins and condensed into heterochromatin or associated with repressor proteins that block transcription. In this study, we sought to enhance transgene expression by adding binding motifs for several different epigenetic enzymes either upstream or downstream of two promoters (CMV and EF1α). Screening these plasmids revealed that luciferase expression was enhanced 10‐fold (10.4 ± 5.8) by the addition of a CCAAT box just upstream of the EF1α promoter to recruit nuclear transcription factor Y (NF‐Y), while inserting a CCCTC‐binding factor (CTCF) motif downstream of the EF1α promoter enhanced expression at least 14‐fold (14.03 ± 6.54). ChIP assays confirmed that NF‐Y and CTCF bound to the motifs that were added to each plasmid, but the presence of NF‐Y and CTCF did not significantly affect the levels of histone acetylation (H3K9ac) or methylation (H3K9me3). Overall, these results show that transgene expression from the EF1α promoter can be significantly increased with motifs that recruit NF‐Y or CTCF. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1581–1588, 2018

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

“…Here, we expand our previous work where we had identified cis-regulatory sequences that enhanced expression from plasmid-borne transgenes [12]. To regulate expression of chromosomally-inserted transgenes, we built site-specific fusion proteins with effector modules that represent diverse activities: transcriptional activation through cofactor recruitment, direct histone modification, and nucleosome repositioning and displacement.…”

“…Recruiting activators to a specific locus in order to reverse epigenetic silencing can be achieved either by including an activation-associated cis-regulatory DNA sequence within the construct itself, or through the targeting of engineered fusion proteins to the silenced transgene. Both natural and synthetic cis-regulatory motifs that recruit activators have been used [10][11][12][13] to help increase transgene expression as an alternative to viral promoters that are prone to methylation and silencing [1]. Previous screens by ourselves and other groups [11,14,15] have identified mammalian activation-associated cis-regulatory elements that recruit endogenous factors to increase the expression of epigenetically silenced transgenes, including motifs for nuclear factor Y, CTCF, and elongation factor alpha (EF1-α) [12,13].…”

Components from the human c-myb transcriptional regulation system reactivate epigenetically repressed transgenes

“…Plasmid construction, transfection of PC-3 cells, and luciferase assays were carried out as described previously (12) . Briefly, cloning of double-stranded oligos was used to insert motifs 222 bp upstream of the transcription start site of an EF1a promoter at XbaI/SphI.…”

“…Work from our group (57) and others (58,59) has shown that plasmid DNA becomes occupied by histones, which may contribute to transgene silencing in human cells. In a previous study, we used DNA sequences that were known targets of endogenous activation-associated proteins to reduce silencing of a luciferase reporter gene (12) . Here, we tested additional motifs ( Figure 1A) that are recognized by AAPs from the transcriptional activator group in our panel: MYB and p65.…”

“…Both natural and synthetic cis-regulatory motifs that recruit activators have been used (10)(11)(12)(13) to help increase transgene expression as an alternative to viral promoters that are prone to methylation and silencing (1) . Previous screens by ourselves and other groups (11,14,15) have identified mammalian activation-associated cis-regulatory elements that recruit endogenous factors to increase the expression of epigenetically silenced transgenes, including motifs for nuclear factor Y, CTCF, and elongation factor alpha (EF1-ɑ) (12,13) . The underlying regulatory mechanisms are not entirely understood, since in this case efficient screening for functional sequences has been prioritized over dissecting the mechanism of individual elements.…”