CpG domains downstream of TSSs promote high levels of gene expression

Abstract

CpG dinucleotides are known to play a crucial role in regulatory domains, affecting gene expression in their natural context. Here, we demonstrate that intragenic CpG frequency and distribution impacts transgene and genomic gene expression levels in mammalian cells. As shown for the Macrophage Inflammatory Protein 1α, de novo RNA synthesis correlates with the number of CpG dinucleotides, whereas RNA splicing, stability, nuclear export and translation are not affected by the sequence modification. Differences in chromatin accessibility in vivo and altered nucleosome positioning in vitro suggest that increased CpG levels destabilize the chromatin structure. Moreover, enriched CpG levels correlate with increased RNA polymerase II elongation rates in vivo. Interestingly, elevated CpG levels particularly at the 5′ end of the gene promote efficient transcription. We show that this is a genome-wide feature of highly expressed genes, by identifying a domain of ∼700 bp with high CpG content downstream of the transcription start site, correlating with high levels of transcription. We suggest that these 5′ CpG domains are required to distort the chromatin structure and to increase gene activity.

Codon usage is an important determinant of gene expression levels largely through its effects on transcription

“…Consistent with a transcriptional effect of codon usage, it was previously shown that mammalian genes with high GC contents, which means the use of more preferred codons, had higher expression levels than those with lower GC content; this observation was not a result of differences in mRNA degradation rates (50,51). More recently, codon usage was shown to contribute to the balanced expression of Toll-like receptors by affecting transcription rather than translation in mammals (52).…”

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

“…The latter can be due to various effects, such as increasing mRNA stability 58 or increasing de novo mRNA synthesis of transgenes. 19,25 For hgfp genes with modified CpG content in the open reading frame, we have previously shown, using T7 polymerase-driven transcription in the cytoplasm of mammalian cells, that modulation of the CpG content does not affect mRNA stability or translational efficiency. 18 To avoid the spreading of repressive epigenetic marks surrounding the integration locus into the proximal transgene and to prevent loss of the transgene through the outgrowth of transgene-lacking cells, transgene expression is optimally conducted under selection pressure.…”

“…[15][16][17] Further analyses attributed CpG-mediated expression enhancement in mammalian cells to an increased transcription rate, 18 which correlates with alterations in the chromatin structure and RNA polymerase II elongation rates. 19 Because cytosines in particular in promoter regions are the exclusive targets for methylation in vertebrates, 20 CpGs are often avoided in vector elements and also in transgene sequences to prevent silencing 21 and to provide prolonged transgene expression. 14,[22][23][24] In contrast, it was shown that CpG dinucleotides in the transgene open reading frame can provide improved and long-term transgene expression in mouse tissue.…”

“…29 Here, we provide a comparative analysis of highly expressed, CpG-rich, versus poorly expressed, CpGdepleted genes encoding humanized green fluorescent protein (hGFP), which was adopted from previous studies. 18,19 We addressed variable expression capacities of these CpG-modified hgfp genes for different standard promoters, cell lines, and gene delivery strategies. Plasmid DNA-mediated hGFP expression in Chinese hamster ovary (CHO) cells via the Flp-In system served as our model expression system to compare various sequence elements and give insight into the molecular regulation mechanisms on which the expression changes are based.…”

Efficient expression of a heterologous gene in plants depends on the nucleotide composition of mRNA’s 5'-region

“…Although it is known that many CG-rich sequences in eukaryotic genomes undergo inactivation by methylation, CpG-islands are transcriptionally active [3][4][5]. A twofold increase in the frequency of CpG dinucleotides in the regions of transcriptional initiation of human genes with a high level of transcription was demonstrated recently [6]. This indicates that the 5'-regions of genes with a high content of CpG dinucleotides may be important transcriptional regulatory elements at least in the human genome.…”