Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells

The human cytomegalovirus promoter (hCMV) is susceptible to gene silencing in CHO cells, most likely due to epigenetic events, such as DNA methylation and histone modifications. The core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene has been shown to prevent DNA methylation. A set of modified hCMV promoters was developed by inserting one or two copies of IE in either forward or reverse orientations either upstream of the hCMV enhancer, between the enhancer and core promoter (CP), or downstream of the CP. The modified hCMV with one copy of IE inserted between the enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability without compromising expression level when compared with the wild‐type (WT) hCMV. A third of 18 EGFP expressing clones generated using MR1 retained 70% of their starting expression level after 8 weeks of culture in the absence of selection pressure, while none of 18 WT hCMV generated clones had expression above 50%. MR1 also improved antibody expression stability of methotrexate (MTX) amplified CHO cell lines. Stably transfected pools generated using MR1 maintained 62% of their original monoclonal antibody titer after 8 weeks of culture in the absence of MTX, compared to only 37% for WT hCMV pools. Low levels of CpG methylation within both WT hCMV and MR1 were observed in all the analyzed cell lines and the methylation levels did not correlate to the expression stability, suggesting IE enhances expression stability by other mechanisms other than preventing methylation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:523–534, 2014     

Role of CpG context and content in evolutionary signatures of brain DNA methylation

DNA methylation is essential in brain function and behavior; therefore, understanding the role of DNA methylation in brain-based disorders begins with the study of DNA methylation profiles in normal brain. Determining the patterns and scale of methylation conservation and alteration in an evolutionary context enables the design of focused but effective methylation studies of disease states. We applied an enzymatic-based approach, Methylation Mapping Analysis by Paired-end Sequencing (Methyl-MAPS), which utilizes second-generation sequencing technology to provide an unbiased representation of genome-wide DNA methylation profiles of human and mouse brains. In this large-scale study, we assayed CpG methylation in cerebral cortex of neurologically and psychiatrically normal human postmortem specimens, as well as mouse forebrain specimens. Cross-species human-mouse DNA methylation conservation analysis shows that DNA methylation is not correlated with sequence conservation. Instead, greater DNA methylation conservation is correlated with increasing CpG density. In addition to CpG density, these data show that genomic context is a critical factor in DNA methylation conservation and alteration signatures throughout mammalian brain evolution. We identify key genomic features that can be targeted for identification of epigenetic loci that may be developmentally and evolutionarily conserved and wherein aberrations in DNA methylation patterns can confer risk for disease.     

Factors to preserve CpG-rich sequences in methylated CpG islands

Background

Mammalian CpG islands (CGIs) normally escape DNA methylation in all adult tissues and developmental stages. However, in our previous study we unexpectedly identified many methylated CGIs in human peripheral blood leukocytes. Methylated CpG dinucleotides convert to TpG dinucleotides through deaminization of their cytosine bases more frequently than hypomethylated CpG dinucleotides. Therefore, we wondered how methylated CGIs in germline or non-germline cells maintain their CpG-rich sequences. It is known that events such as germline hypomethylation, CpG selection, biased gene conversion (BGC), and frequent CpG fixation can contribute to the maintenance of CpG-rich sequences in methylated CGIs in germline or non-germline cells. However, it has not been investigated which of the processes maintain CpG-rich sequences of methylated CGIs in each genomic position.

Results

In this study, we comprehensively examined the contribution of the processes described above to the maintenance of CpG-rich sequences in methylated CGIs in germline and non-germline cells which were classified by genomic positions. Approximately 60–80% of CGIs with high methylation in H1 cell line (H1-HM) in all the genomic positions showed a low average CpG → TpG/CpA substitution rate. In contrast, fewer than half the numbers of CGIs with H1-HM in all the genomic positions showed a low average CpG → TpG/CpA substitution rate and low levels of methylation in sperm cells (SPM-LM). Furthermore, a small fraction of CGIs with a low average CpG → TpG/CpA substitution rate and high levels of methylation in sperm cells (SPM-HM) showed CpG selection.On the other hand, independent of the positions in genes, most CGIs with SPM-HM showed a slightly higher average TpG/CpA → CpG substitution rate compared with those with SPM-LM.

Conclusions

Relatively high numbers (approximately 60–80%) of CGIs with H1-HM in all the genomic positions preserve their CpG-rich sequences by a low CpG → TpG/CpA substitution rate caused mainly by their SPM-LM, and for those with SPM-HM partly by CpG selection and TpG/CpA → CpG fixation. BGC has little contribution to the maintenance of CpG-rich sequences of CGIs with SPM-HM which were classified by genomic positions.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1286-x) contains supplementary material, which is available to authorized users.     

人类基因组上CpG岛的定位和系统分析

为了研究CpG岛产生和消失机制以及位于基因启动子区域外的CpG岛保守性等问题,我们通过序列比对和进化保守性分析等方法,分析在人类和小鼠中保守的基因上的CpG岛。结果显示已有保守序列的突变以及序列插入删除是CpG岛产生和消失的主要原因,进一步分析发现52%的在小鼠基因组上保守序列完全缺失的CpG岛位于两个转座子之间,提示转座子所介导的序列插入是CpG岛形成和消失的重要原因。人类基因组上在启动子区域外的CpG岛中约有79%为新产生的CpG岛,显著高于启动子区域内新产生的CpG岛比例(41%)。GO分析表明与这些CpG岛相关的部分基因与神经系统发育显著相关,提示新产生的CpG岛参与神经发育过程。     

Genomic specification and epigenetic regulation of eukaryotic DNA replication origins

Identification of DNA replication origins (ORIs) at a genome-wide level in eukaryotes has proved to be difficult due to the high degree of degeneracy of their sequences. Recent structural and functional approaches, however, have circumvented this limitation and have provided reliable predictions of their genomic distribution in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and they have also significantly increased the number of characterized ORIs in animals. This article reviews recent evidence on how ORIs are specified and maintained in these systems and on their regulation and sensitivity to epigenetic signals. It also discusses the possible additional involvement of ORIs in processes other than DNA replication.     

Human-specific CpG “beacons” identify loci associated with human-specific traits and disease

Regulatory change has long been hypothesized to drive the delineation of the human phenotype from other closely related primates. Here we provide evidence that CpG dinucleotides play a special role in this process. CpGs enable epigenome variability via DNA methylation, and this epigenetic mark functions as a regulatory mechanism. Therefore, species-specific CpGs may influence species-specific regulation. We report non-polymorphic species-specific CpG dinucleotides (termed “CpG beacons”) as a distinct genomic feature associated with CpG island (CGI) evolution, human traits and disease. Using an inter-primate comparison, we identified 21 extreme CpG beacon clusters (≥ 20/kb peaks, empirical p < 1.0 × 10^?3) in humans, which include associations with four monogenic developmental and neurological disease related genes (Benjamini-Hochberg corrected p = 6.03 × 10^?3). We also demonstrate that beacon-mediated CpG density gain in CGIs correlates with reduced methylation in these species in orthologous CGIs over time, via human, chimpanzee and macaque MeDIP-seq. Therefore mapping into both the genomic and epigenomic space the identified CpG beacon clusters define points of intersection where a substantial two-way interaction between genetic sequence and epigenetic state has occurred. Taken together, our data support a model for CpG beacons to contribute to CGI evolution from genesis to tissue-specific to constitutively active CGIs.     

Features and trend of loss of promoter-associated CpG islands in the human and mouse genomes

CpG islands (CGIs) are often considered as gene markers, but the number of CGIs varies among mammalian genomes that have similar numbers of genes. In this study, we investigated the distribution of CGIs in the promoter regions of 3,197 human-mouse orthologous gene pairs and found that the mouse genome has notably fewer CGIs in the promoter regions and less pronounced CGI characteristics than does the human genome. We further inferred CGI's ancestral state using the dog genome as a reference and examined the nucleotide substitution pattern and the mutational direction in the conserved regions of human and mouse CGIs. The results reveal many losses of CGIs in both genomes but the loss rate in the mouse lineage is two to four times the rate in the human lineage. We found an intriguing feature of CGI loss, namely that the loss of a CGI usually starts from erosion at the both edges and gradually moves towards the center. We found functional bias in the genes that have lost promoter-associated CGIs in the human or mouse lineage. Finally, our analysis indicates that the association of CGIs with housekeeping genes is not as strong as previously estimated. Our study provides a detailed view of the evolution of promoter-associated CGIs in the human and mouse genomes and our findings are helpful for understanding the evolution of mammalian genomes and the role of CGIs in gene function.     

人类全基因组范围的CpG岛的预测与分析

CpG岛的甲基化是表观遗传中基因表达调控的重要机制。虽然目前已存在几个从DNA序列判别CpG岛的标准,但如何在标准中选择合适的参数仍是研究的焦点。文章通过分析比较两种经典CpG岛判定标准与三种预测方法,提出了改进的CpG岛预测方法——CpGISeeker。应用该预测方法,结合判定标准中的三个基本参数组合出的13组组合参数,在人类全基因组范围内进行了CpG岛预测,并统计分析了CpG岛的重复序列组成以及相对于基因转录起始位点的位置分布情况。分析结果表明CpGISeeker具有更精确判定CpG岛的特性;同时还提示,随着判定标准严格性的增加,CpG岛的重复序列含量降低,与基因转录起始位点的相关性提高。将CpG岛最小尺寸为500bp、GC含量为60%、CpG出现率达到0.65的组合参数作为标准,是目前预测CpG岛的最佳方式。