The impact of intragenic CpG content on gene expression

Abstract

The development of vaccine components or recombinant therapeutics critically depends on sustained expression of the corresponding transgene. This study aimed to determine the contribution of intragenic CpG content to expression efficiency in transiently and stably transfected mammalian cells. Based upon a humanized version of green fluorescent protein (GFP) containing 60 CpGs within its coding sequence, a CpG-depleted variant of the GFP reporter was established by carefully modulating the codon usage. Interestingly, GFP reporter activity and detectable protein amounts in stably transfected CHO and 293 cells were significantly decreased upon CpG depletion and independent from promoter usage (CMV, EF1α). The reduction in protein expression associated with CpG depletion was likewise observed for other unrelated reporter genes and was clearly reflected by a decline in mRNA copy numbers rather than translational efficiency. Moreover, decreased mRNA levels were neither due to nuclear export restrictions nor alternative splicing or mRNA instability. Rather, the intragenic CpG content influenced de novo transcriptional activity thus implying a common transcription-based mechanism of gene regulation via CpGs. Increased high CpG transcription correlated with changed nucleosomal positions in vitro albeit histone density at the two genes did not change in vivo as monitored by ChIP.

Differences in codon bias and GC content contribute to the balanced expression of TLR7 and TLR9

“…On a smaller scale, optimizing codons of individual proteins can lead to increased protein expression, but the mechanism(s) underlying the increased protein remains controversial. Several groups have reported that the protein increase results from an increase in the rate of translation (26,27), whereas others have observed an increase in the rate of transcription (28,29). Furthermore, a recent study concluded that, although transcriptional control is the broadest means of controlling protein expression, a fraction of proteins are differentially regulated at the translational level (30).…”

Minimal doses of a sequence-optimized transgene mediate high-level and long-term EPO expression in vivo: challenging CpG-free gene design

“…The molecular mechanism underlying CpG-mediated transcriptional regulation is currently unknown, but has been reproducible for a diverse panel of genes and might be associated with sequence-specific differences in nucleosomal positions, as we have recently reported. 27 The negative effects of CpG depletion on mEPO expression in vivo, which were particularly evident at higher pDNA doses might have future implications for rational transgene design. Interestingly, our in vitro experiments have indicated that the expression of codonoptimized genes can be further augmented by inserting additional CpGs into the coding region.…”

“…27 For western blot analysis, 40 mg of total protein from both fractions were separated by 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Erythropoietin was detected using a specific mouse monoclonal antibody (1:500; R&D Systems) and a streptavidin alkaline-phosphatase-conjugated antibody (1:10 000; Roche) followed by staining with the chromogenic substrate NBT/BCIP (Roche).…”

“…Nuclear transcription rates of the various mEPO genes were determined with a non-radioactive nuclear run-on assay as described earlier 27 using biotin labeling, magnetic bead capture and analysis by fluorescence-based real-time PCR as outlined above.…”

“…11,25,26 Contrary to popular belief, we have recently reported that the depletion of CpG dinucleotides from a green fluorescent protein (GFP) gene results in a significant reduction of in vitro expression in transiently and stably transfected cells suggesting a methylationindependent effect of intragenic CpGs on gene expression. 27 In the present study, we have evaluated the influence of rational gene design on the expression level, longevity and bioactivity of the model gene murine erythropoietin (mEPO) in electroporated mice.…”