Interplay of Promoter Usage and Intragenic CpG Content: Impact on GFP Reporter Gene Expression

Abstract

Successful therapeutic protein production in vitro and in vivo requires efficient and long-term transgene expression supported by optimized vector and transgene cis-regulatory sequence elements. This study provides a comparative analysis of CpG-rich, highly expressed, versus CpG-depleted, poorly expressed green fluorescent protein (GFP) reporter transgenes, transcribed by various promoters in two different cell systems. Long-term GFP expression from a defined locus in stable Chinese hamster ovary cells was clearly influenced by the combination of transgene CpG content and promoter usage, as shown by differential silencing effects on selection pressure removal among the cytomegalovirus (CMV) promoter and elongation factor (EF)-1α promoter. Whereas a high intragenic CpG content promoted local DNA methylation, CpG depletion rather accelerated transgene loss and increased the local chromatin density. On lentiviral transfer of various expression modules into epigenetically sensitive P19 embryonic pluripotent carcinoma cells, CMV promoter usage led to rapid gene silencing irrespective of the intragenic CpG content. In contrast, EF-1α promoter-controlled constructs showed delayed silencing activity and high-level transgene expression, in particular when the CpG-rich GFP reporter was used. Notably, GFP silencing in P19 cells could be prevented completely by the bidirectional, dual divergently transcribed A2UCOE (ubiquitously acting chromatin-opening element derived from the human HNRPA2B1-CBX3 locus) promoter. Because the level of GFP expression by the A2UCOE promoter was entirely unaffected by the intragenic CpG level, we suggest that A2UCOE can overcome chromatin compaction resulting from intragenic CpG depletion due to its ascribed chromatin-opening abilities. Our analyses provide insights into the interplay of the intragenic CpG content with promoter sequences and regulatory sequence elements, thus contributing toward the design of therapeutic transgene expression cassettes for future gene therapy applications.

An improved Tet-on system in microRNA overexpression and CRISPR/Cas9-mediated gene editing

“…CMV promoter was an exception in that it showed variability from one cell type to another. Such variation might be due to different levels of promoter silencing by DNA methylation in different cell types [33–35].…”

DNA Vaccines—How Far From Clinical Use?

“…Conventionally, viral promoters like the human immediate/early CMV (cytomegalovirus) promoter which are ubiquitously active at high level have been employed to drive transgene expression [ 49 ]. However, especially with regard to long term expression, viral promotors are often subjected to methylation-mediated inactivation, whereas eukaryotic promoters and hybrids of eukaryotic/viral promoters remain active [ 50 ]. Furthermore, the use of cell type-specific promotors may allow to restrict antigen expression to APC.…”

Nucleic Acid-Based Approaches for Tumor Therapy

“…Expression units of pDNA-based vaccines are often under transcriptional control of virus-derived promotors characterized by ubiquitous activity at high level, like the human intermediate/early CMV or the SV40 promoter [ 114 ]. Since viral promoters may be subjected to methylation-mediated inactivation, both eukaryotic promoters, like the human elongation factor (EF)1α or beta-actin gene promoter, as well as viral/eukaryotic hybrid (e.g., CMV/beta-actin) promoters have been introduced that allow long term transgene expression [ 115 ]. These types of promoters are still widely used in preclinical and clinical studies.…”