CRISPR/Cas9‐mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long‐term stability

CHO cells are the preferred host for the production of complex pharmaceutical proteins in the biopharmaceutical industry, and genome engineering of CHO cells would benefit product yield and stability. Here, we demonstrated the efficacy of a Dnmt3a‐deficient CHO cell line created by CRISPR/Cas9 genome editing technology through gene disruptions in Dnmt3a, which encode the proteins involved in DNA methyltransferases. The transgenes, which were driven by the 2 commonly used CMV and EF1α promoters, were evaluated for their expression level and stability. The methylation levels of CpG sites in the promoter regions and the global DNA were compared in the transfected cells. The Dnmt3a‐deficent CHO cell line based on Dnmt3a KO displayed an enhanced long‐term stability of transgene expression under the control of the CMV promoter in transfected cells in over 60 passages. Under the CMV promoter, the Dnmt3a‐deficent cell line with a high transgene expression displayed a low methylation rate in the promoter region and global DNA. Under the EF1α promoter, the Dnmt3a‐deficient and normal cell lines with low transgene expression exhibited high DNA methylation rates. These findings provide insight into cell line modification and design for improved recombinant protein production in CHO and other mammalian cells.     

Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells

Nonviral episomal vectors present attractive alternative vehicles for gene therapy applications. Previously, we have established a new type of nonviral episomal vector-mediated by the characteristic motifs of matrix attachment regions (MARs), which is driven by the cytomegalovirus (CMV) promoter. However, the CMV promoter is intrinsically susceptible to silencing, resulting in declined productivity during long-term culture. In this study, Chinese hamster ovary (CHO) cells and DNA methyltransferase-deficient (Dnmt3a-deficient) CHO cells were transfected with plasmid-mediated by MAR, or CHO cells were treated with the DNA methylation inhibitor 5-Aza-2′-deoxycytidine. Flow cytometry, plasmid rescue experiments, fluorescence in-situ hybridization (FISH), and bisulfite sequencing were performed to observe transgene expression, its state of existence, and the CpG methylation level of the CMV promoter. The results indicated that all DNA methylation inhibitor and methyltransferase deficient cells could increase transgene expression levels and stability in the presence or absence of selection pressure after a 60-generation culture. Plasmid rescue assay and FISH analysis showed that the vector still existed episomally after long-time culture. Moreover, a relatively lower CMV promoter methylation level was observed in Dnmt3a-deficient cell lines and CHO cells treated with 5-Aza-2′-deoxycytidine. In addition, Dnmt3a-deficient cells were superior to the DNA methylation inhibitor treatment regarding the transgene expression and long-term stability. Our study provides the first evidence that lower DNA methyltransferase can enhance expression level and stability of transgenes mediated by episomal vectors in transfected CHO cells.     

Epigenetic regulation of human gamma-glutamyl hydrolase activity in acute lymphoblastic leukemia cells

Gamma-glutamyl hydrolase (GGH) catalyzes degradation of the active polyglutamates of natural folates and the antifolate methotrexate (MTX). We found that GGH activity is directly related to GGH messenger RNA expression in acute lymphoblastic leukemia (ALL) cells of patients with a wild-type germline GGH genotype. We identified two CpG islands (CpG1 and CpG2) in the region extending from the GGH promoter through the first exon and into intron 1 and showed that methylation of both CpG islands in the GGH promoter (seen in leukemia cells from approximately 15% of patients with nonhyperdiploid B-lineage ALL) is associated with significantly reduced GGH mRNA expression and catalytic activity and with significantly higher accumulation of MTX polyglutamates (MTXPG(4-7)) in ALL cells. Furthermore, methylation of CpG1 was leukemia-cell specific and had a pronounced effect on GGH expression, whereas methylation of CpG2 was common in leukemia cells and normal leukocytes but did not significantly alter GGH expression. These findings indicate that GGH activity in human leukemia cells is regulated by epigenetic changes, in addition to previously recognized genetic polymorphisms and karyotypic abnormalities, which collectively determine interindividual differences in GGH activity and influence MTXPG accumulation in leukemia cells.     

The use of engineered E1A genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines.

Vectors expressing adenovirus 5 E1A or a domain 2 mutant E1A were introduced into CHO-K1 cells in order to transactivate the hCMV-MIE promoter in transient and stable transfections. Expression from the hCMV promoter was efficiently activated by both wild-type and mutant E1A in contrast to other viral promoters such as the SV40 early promoter which are repressed by E1A. E1A genes expressed from a strong promoter were inhibitory to the growth of CHO cells. Nevertheless, by the use of a weaker promoter, it was possible to isolate stably transfected cell lines containing a level of E1A compatible with both continued cell growth and significant transactivation of the hCMV promoter. By this means we have generated cell lines secreting tissue inhibitor of metalloproteinases (TIMP) at levels approaching those previously attained using gene amplification. CHO cell lines constitutively expressing wild-type and mutant E1A genes have been derived which can serve as new host cell lines for transient expression and efficient stable expression without gene amplification.     

Promoter methylation and transgene copy numbers predict unstable protein production in recombinant Chinese hamster ovary cell lines

Mammalian cell lines for recombinant protein production need to maintain productivity over extended cultivation times. Long-term stability studies are time and resource intensive, but are widely performed to identify and eliminate unstable candidates during cell line development. Production instability of manufacturing cell lines can be associated with methylation and silencing of the heterologous promoter. We have identified CpG dinucleotides within the human cytomegalovirus major immediate early promoter/enhancer (hCMV-MIE) that are frequently methylated in unstable antibody-producing Chinese hamster ovary (CHO) cell lines. We have established methylation-specific real-time qPCR for the rapid and sensitive measurement of hCMV-MIE methylation in multiple cell lines and provide evidence that hCMV-MIE methylation and transgene copy numbers can be used as early markers to predict production instability of recombinant CHO cell lines. These markers should provide the opportunity to enrich stable producers early in cell line development and allow developers to put more emphasis on other criteria, such as product quality and bioprocess robustness.     

The human cytomegalovirus major immediate-early distal enhancer region is required for efficient viral replication and immediate-early gene expression

The human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 p72 and IE2 p86, are activated by a complex enhancer region (base positions -65 to -550) that operates in a cell type- and differentiation-dependent manner. The expression of MIE genes is required for HCMV replication. Previous studies analyzing functions of MIE promoter-enhancer segments suggest that the distal enhancer region variably modifies MIE promoter activity, depending on cell type, stimuli, or state of differentiation. To further understand the mechanism by which the MIE promoter is regulated, we constructed and analyzed several different recombinant HCMVs that lack the distal enhancer region (-300 to -582, -640, or -1108). In human fibroblasts, the HCMVs without the distal enhancer replicate normally at high multiplicity of infection (MOI) but replicate poorly at low MOI in comparison to wild-type virus (WT) or HCMVs that lack the neighboring upstream unique region and modulator (-582 or -640 to -1108). The growth aberrancy was normalized after restoring the distal enhancer in a virus lacking this region. For HCMVs without a distal enhancer, the impairment in replication at low MOI corresponds to a deficiency in production of MIE RNAs compared to WT or virus lacking the unique region and modulator. An underproduction of viral US3 RNA was also evident at low MOI. Whether lower production of IE1 p72 and IE2 p86 causes a reduction in expression of the immediate-early (IE) class US3 gene remains to be determined. We conclude that the MIE distal enhancer region possesses a mechanism for augmenting viral IE gene expression and genome replication at low MOI, but this regulatory function is unnecessary at high MOI.     

The IE2 promoter/enhancer region from mouse CMV provides high levels of therapeutic protein expression in mammalian cells

Protein expression in mammalian cells is key for the production and manufacturing of bio-therapeutics with human-like properties and activities. As a molecular basis for reaching high protein expression levels, efficient promoter/enhancer systems are a prerequisite. Here we identify a novel enhancer from the mouse cytomegalovirus (CMV) immediate early 2 (IE2) region as a strong expression-promoting element. We further demonstrate its activity in bi-directional promoter architecture and apply it to generate production clones for IL-18BP, a protein with therapeutic indications in autoimmune diseases. These data show that the IE region from mouse CMV, and the IE2 enhancer/promoter in particular, have a broad potential for application in novel gene expression systems for research, development, and manufacturing of protein drugs.     

The EF‐1α promoter maintains high‐level transgene expression from episomal vectors in transfected CHO‐K1 cells

In our previous study, we demonstrated that episomal vectors based on the characteristic sequence of matrix attachment regions (MARs) and containing the cytomegalovirus (CMV) promoter allow transgenes to be maintained episomally in Chinese hamster ovary (CHO) cells. However, the transgene expression was unstable and the number of copies was low. In this study, we focused on enhancers, various promoters and promoter variants that could improve the transgene expression stability, expression magnitude (level) and the copy number of a MAR‐based episomal vector in CHO‐K1 cells. In comparison with the CMV promoter, the eukaryotic translation elongation factor 1 α (EF‐1α, gene symbol EEF1A1) promoter increased the transfection efficiency, the transgene expression, the proportion of expression‐positive clones and the copy number of the episomal vector in long‐term culture. By contrast, no significant positive effects were observed with an enhancer, CMV promoter variants or CAG promoter in the episomal vector in long‐term culture. Moreover, the high‐expression clones harbouring the EF‐1α promoter tended to be more stable in long‐term culture, even in the absence of selection pressure. According to these findings, we concluded that the EF‐1α promoter is a potent regulatory sequence for episomal vectors because it maintains high transgene expression, transgene stability and copy number. These results provide valuable information on improvement of transgene stability and the copy number of episomal vectors.     

De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase.

Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.     

In vitro DNA cytosine methylation of cis-regulatory elements modulates c-Ha-ras promoter activity in vivo.

The effect of DNA cytosine methylation on promoter activity was assessed using a transient expression system employing pHrasCAT. This 551 bp Ha-ras-1 gene promoter region is enriched with 84 CpG dinucleotides, six functional GC boxes, and is prototypic of many genes possessing CpG islands in their promoter regions. Bacterial modification enzymes HhaI methyl transferase (MTase) and HpaII MTase, alone or in combination with a human placental DNA methyltransferase (HP MTase) that methylates CpG sites in a generalized manner, including asymmetric elements such as GC box CpG's, were used to methylate at different types of sites in the promoter. Methylation of HhaI and HpaII sites reduced CAT expression by approximately 70%-80%, whereas methylation at generalized CpG sites with HP MTase inactivated the promoter by greater than 95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in non-promoter regions.     

Methylation of CpG dinucleotides in the open reading frame of a testicular germ cell-specific intronless gene,Tact1/Actl7b,represses its expression in somatic cells

Methylation of CpG islands spanning promoter regions is associated with control of gene expression. However, it is considered that methylation of exonic CpG islands without promoter is not related to gene expression, because such exonic CpG islands are usually distant from the promoter. Whether methylation of exonic CpG islands near the promoter, as in the case of a CpG-rich intronless gene, causes repression of the promoter remains unknown. To gain insight into this issue, we investigated the distribution and methylation status of CpG dinucleotides in the mouse Tact1/Actl7b gene, which is intronless and expressed exclusively in testicular germ cells. The region upstream to the gene was poor in CpG, with CpG dinucleotides absent from the core promoter. However, a CpG island was found inside the open reading frame (ORF). Analysis of the methylation status of the Tact1/Actl7b gene including the 5′-flanking area demonstrated that all CpG sites were methylated in somatic cells, whereas these sites were unmethylated in the Tact1/Actl7b-positive testis. Trans fection experiments with in vitro-methylated constructs indicated that methylation of the ORF but not 5′ upstream repressed Tact1/Actl7b promoter activity in somatic cells. Similar effects of ORF methylation on the promoter activity were observed in testicular germ cells. These are the first results indicating that methylation of the CpG island in the ORF represses its promoter in somatic cells and demethylation is necessary for gene expression in spermatogenic cells.     

Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes

Previously, we established an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese hamster ovary (CHO) cell line. With a gradual increase in methotrexate (MTX) concentration, gene-amplified cell pools had high and stable specific growth and production rates. Moreover, the phenotype of gene-amplified cells seemed to be affected by the location of the amplified gene in chromosomal DNA. We suspected that various kinds of gene-amplified cells might appear during the long-term selection to construct gene-amplified cell pools. To clarify the behavior of gene-amplified cell pools during a stepwise increase of MTX concentration, we isolated gene-amplified clones derived from gene-amplified cell pools. We compared the characteristics of isolated clones, such as the productivity of recombinant protein, stability of amplified genes, and the location of amplified genes. As a result, telomere-type clones, in which the amplified gene was located near the telomeric region, were found to be more stable and productive than other types of clones. Telomere-type clones had over 100 copies of amplified genes in the chromosomal DNA. In contrast, a large number of other types of clones had less than 10 copies of amplified genes. During long-term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.     

Orthology between genomes of Brachypodium, wheat and rice

Background

Differential expression of perforin (PRF1), a gene with a pivotal role in immune surveillance, can be attributed to differential methylation of CpG sites in its promoter region. A reproducible method for quantitative and CpG site-specific determination of perforin methylation is required for molecular epidemiologic studies of chronic diseases with immune dysfunction.

Findings

We developed a pyrosequencing based method to quantify site-specific methylation levels in 32 out of 34 CpG sites in the PRF1 promoter, and also compared methylation pattern in DNAs extracted from whole blood drawn into PAXgene blood DNA tubes (whole blood DNA) or DNA extracted from peripheral blood mononuclear cells (PBMC DNA) from the same normal subjects. Sodium bisulfite treatment of DNA and touchdown PCR were highly reproducible (coefficient of variation 1.63 to 2.18%) to preserve methylation information. Application of optimized pyrosequencing protocol to whole blood DNA revealed that methylation level varied along the promoter in normal subjects with extremely high methylation (mean 86%; range 82–92%) in the distal enhancer region (CpG sites 1–10), a variable methylation (range 49%–83%) in the methylation sensitive region (CpG sites 11–17), and a progressively declining methylation level (range 12%–80%) in the proximal promoter region (CpG sites 18–32) of PRF1. This pattern of methylation remained the same between whole blood and PBMC DNAs, but the absolute values of methylation in 30 out of 32 CpG sites differed significantly, with higher values for all CpG sites in the whole blood DNA.

Conclusion

This reproducible, site-specific and quantitative method for methylation determination of PRF1 based on pyrosequencing without cloning is well suited for large-scale molecular epidemiologic studies of diseases with immune dysfunction. PBMC DNA may be better suited than whole blood DNA for examining methylation levels in genes associated with immune function.     

染色质调节元件与不同启动子的相互作用对基因表达调控的影响

为探究DNA序列元件对不同启动子调节转基因稳定表达的影响,利用遍在染色质开放元件 (Ubiquitous chromatin opening elements,UCOE) 和基质黏附序列 (Scaffold/matrix-attachment regions,MAR) 分别与含增强子的oct4基因启动子、含CpG岛的sox2基因启动子和不含调控元件的nanog基因启动子以及同时包含增强子和CpG岛的CMV启动子组合构建pOCT4-MAR、pOCT4-UCOE、pSOX2-MAR、pSOX2-UCOE、pNANOG-MAR、pNANOG-UCOE、pCMV-UCOE、pCMV-MAR等质粒,分析这些质粒稳定转染后的表达量和嵌合表达差异。结果发现,UCOE与含增强子元件的oct4启动子组合能较稳定高效表达,而MAR与含CpG岛的sox2启动子组合能较稳定高效表达。利用排除位置效应原因的嵌合表达对染色质高级结构调控基因表达的稳定性分析表明：(1) 通常情况下UCOE比MAR调节的表达载体的表达更高效和更稳定;UCOE连接含CpG岛的启动子形成开放染色质调节的高表达更稳定;(2) MAR与启动子上TATA盒或增强子可能通过染色质环产生高表达,但相对不稳定。结论：染色质调节元件UCOE和MAR与启动子调控元件之间能通过染色质开放状态或染色质环调控基因稳定表达。