The focus of the research group’s interests has been on the biological function of DNA methylation in genome stability and in the regulation of biological processes which are of interest in genetics (epigenetics) and medicine. In this report, we describe current results and plans for future projects.
The human genome sequence contains about 28 million CpG pairs which are potential targets for the modification of cytidine- to 5-methyldeoxycytidine-residues (5-mC) by DNA methyltransferases. The distribution of 5-mC’s across the human genome can vary with cell type. Depending on environmental conditions, CpG methylation patterns can be subject to change. Lacking a complete map of 5-mC locations in the human genome, how might one visualize these patterns which hold high functional significance for genomic stability and activity? Definite information is not available. The challenges raised by CpG methylation landscapes emerge from the large number of CpG’s, and from the quest to decipher their functional meaning. By selecting the two following examples for more detailed inspection, we are fully aware that there will be many additional ones worth consideration.
(i) The presence of 5-mC residues in specific, functionally decisive positions of the genome is undoubtedly related to genetic activity.
(ii) Perhaps as importantly, the genome-inherent 5-mCpG versus CpG algorithms might be an important guardian of genomic stability, and capable of recognizing any threat against it. Much like innate and acquired immunity respond to the intrusion of foreign, often pathogenic molecules or cells, the CpG arrays are thought to be highly sensitive to the invasion of foreign DNA into the cell. The CpG guardian might already be alerted by the contact of foreign nucleic acids with the cell surface or the mere application of techniques for gene transfer. This CpG alarm clock has probably developed early in evolution and, like other ancient biological defenses, has progressed and evolved over evolutionary times. This system is flexible and permits alterations, not always under strictly controlled conditions. Altered methylation patterns can be transmitted over cell generations, i.e. are at least in part inheritable.
The notion of a guardian for genome stability is caught between two contradicting, equally essential options. (i) Maintaining the inherited genome is the precondition for survival in the real world that abounds with a gamut of competing molecules and organisms. However, will defense of genome maintenance suffice as the major principle for survival? (ii) More realistically, the system requires the genetic and epigenetic potential to exploit competing organisms and their intruding foreign genetic information. Novel genetic and epigenetic information from foreign sources might be convenient to have around and could be constantly scanned for internal usefulness. Ubiquitous non-homologous recombination mechanisms enable the cell to incorporate newly-acquired foreign DNA into its own genome. Subsequently, this acquisition could be screened for internal advantage or might be eliminated from the cell’s indigenous nucleotide sequence if advantageous. Selection in a competitive environment would then determine survival of propitious acquisitions of foreign DNA sequences.
We have set out to study changes in the cellular CpG methylation profiles upon introducing foreign DNA into mammalian cells. As stress factors served the genomic integration of foreign (viral or bacterial plasmid) DNA, virus infections or the immortalization of cells with Epstein Barr Virus (EBV). In several systems studied alterations in cellular CpG methylation and transcription profiles were observed to different degrees. In the case of adenovirus DNA integration in adenovirus type 12 (Ad12)-transformed hamster cells, the extensive changes in cellular CpG methylation persisted even after the loss of the transgenomic Ad12 DNA. Hence, stress-induced alterations in CpG methylation can be inherited independent of the continued presence of the transgenome. Upon adeno-virus infections, changes in cellular CpG methylation have not been observed. In EBV immortalized as compared to control cells, CpG hypermethylation in the far-upstream region of the human FMR1 promoter decreased four-fold. In the wake of cellular stress due to foreign DNA entry, preexisting CpG methylation patterns were altered, possibly at specific CpG dinucleotides. Frequently, transcription patterns were also affected. As a caveat towards manipulations of cells with foreign DNA, such cells can no longer be considered identical to their unmanipulated counterparts.
Aim: We previously reported changes of DNA methylation and transcription patterns in mammalian cells that carry integrated foreign DNA. Experiments were now designed to assess the epigenetic consequences after the insertion of a 5.6 kbp plasmid into the human genome.
Methods: Differential transcription and CpG methylation patterns were compared between plasmid-transgenomic and non-transgenomic cell clones by using gene chip microarray systems.
Results: In 4.7% of the 28,869 gene segments analyzed, the transcriptional activities were up-regulated (907 genes) or down-regulated (436 genes) in plasmid-transgenomic cell clones in comparison to non-transgenomic control clones. Frequent up-regulations were noted in small nucleolar RNA genes which affect RNA metabolism and in genes involved in signaling pathways (see Table below).
|Top canonical pathways for differentially expressed genes|
|Regulation of eIF4 and p70S6K signaling|
|Insulin receptor signaling|
|Small nucleolar RNA's|
Genome-wide methylation profiling was performed for 361,983 CpG sites. In comparisons of methylation levels in five transgenomic versus four non-transgenomic cell clones, 3,791 CpG's were differentially methylated, 1,504 CpG's were hyper- and 2,287 were hypo-methylated (see Table below).
|Top canonical pathways for differentially methylated CpG’s|
|Neuropathic pain signaling in dorsal horn neurons|
|Axonal guidance signaling|
|CREB signaling in neurons|
|Glutamate receptor signaling|
|GABA receptor signaling|
Conclusions: The epigenetic consequences of foreign DNA integration can be considered a general effect also in human cells. We do not understand the role of transgenome size, CG content or copy number. The mechanism(s) underlying the observed epigenetic alterations are still unknown. Extent and location of alterations in genome activities and CpG methylation might depend on the site(s) of foreign DNA insertion. Genome manipulations in general – work with transgenomic or knocked cells and organisms – have assumed a major role in molecular biology and medicine. The consequences of cellular genome manipulations for epigenetic stability have so far received unwarrantedly limited attention. Before drawing far-reaching conclusions from work with cells or organisms with manipulated genomes, critical considerations for and careful analyses of their epigenomic stability could prove prudent.
Aim: DNA methylation and transcriptional profiles were determined in the regulatory sequences of the human endogenous retroviral (HERV-K, -W, -E) and LINE-1.2 elements and were compared between non-transgenomic and plasmid-transgenomic cells (see previous section for plasmid-transgenomic HCT116 cell clones).
Methods: DNA methylation profiles in parts of the HERV (K, W, E) as well as LINE-1.2 sequences were determined by bisulfite genomic sequencing. The transcription of these genome segments was assessed by quantitative real-time PCR.
Results: In HERV-K, HERV-W and LINE-1.2 sequences the levels of DNA methylation ranged between 75 and 98%, while in HERV-E they were around 60%. Nevertheless, the HERV and LINE-1.2 sequences were actively transcribed. No differences were found in comparisons of HERV and LINE-1.2 CpG methylation and transcription patterns between non-transgenomic and the same plasmid-transgenomic HCT116 cell clones which showed altered transcription and CpG methylation profiles in other regions of their genomes (see previous section).
Conclusions: The insertion of a 5.6 kbp plasmid into the HCT116 genome had no effect on the HERV and LINE-1.2 methylation and transcription profiles, although other parts of the HCT116 genome had shown marked changes (Weber et al., 2015). Nevertheless, some of these heavily methylated repetitive sequences proved actively transcribed, probably because the large number of HERV and LINE-1.2 elements harbor copies with non- or hypo-methylated long terminal repeat sequences. These ancient genome constituents are possibly less sensitive to epigenetic alterations in the wake of foreign DNA insertions, since due to their long-term presence in the human genome, they might already have attained a “final” epigenetic mode.
On the basis of the results summarized in Weber et al. 2015, and 2018, the notion has been pursued that manipulations of (mammalian) genomes in cultured cells can elicit genome-wide epigenetic alterations of transcriptional and methylation profiles and thus fundamentally alter the characteristics of the affected cells and organisms. It is unknown whether these events occur generally in all instances of foreign DNA insertions or whether manipulations other than insertions and excisions could have comparable sequelae. So far, we have not yet investigated the mechanisms which recognize and respond to insertions of foreign DNA into the cell nucleus or into the genome. Since the integration of foreign DNA stands at the center of many experimental approaches in biology and medicine, I consider our field of research of importance for the critical evaluation of results obtained from many lines of genome manipulations. The literature has practically kept silent on this issue. Several generally relevant implications of the consequences of foreign DNA insertion or of genome manipulations will have to be discussed in the following areas of biology and medicine: (i) Epigenetic factors in (viral) oncogenesis. (ii) Thoughts on epigenetics and evolution. (iii) Experimental approaches using genome manipulations.
In our current work we have asked the question, whether the infection of mammalian cells with DNA viruses, like adenovirus type 12 (Ad12) or African Swine Fever Virus (ASFV) can lead to alterations in the CpG methylation status of (i) the intruding viral genome or (ii) the genomes of the recipient cells. The data available today are the following.
(i) In the course of the productive infection of human HCT116 cells with Ad12 or of monkey cells with ASFV, the viral genomes do not become de novo methylated. For the Ad12 genome, it had been shown much earlier that the viral DNA inside virions is not CpG methylated.
(ii) Preliminary findings suggest that there are no changes in the analyzed CpG’s of human cellular DNA between 12 and 48 h after Ad12 infection. For ASFV-infected monkey cells, such investigations have not yet been done.
The immortalization of human cells with EBV elicits changes in the cellular DNA methylation profile in a region far-upstream of the human FMR1 gene promoter (Naumann et al., 2014). We have started to investigate this region as a potential indicator site in the human genome which is capable of responding to the entry of foreign DNA or of viruses with alterations in its methylation pattern. Should these earlier data be verified and expanded in more systematic studies, we intend to expose human cells in culture, e.g. cell line HCT116 and others, to various methods of gene transfer by transfection (Ca2+-precipitation, lipofection, nucleofection), to the addition of foreign DNA to the culture medium of the cells, to the transfection with plasmid or viral DNAs, and to the infection with a wider selection of DNA or RNA viruses. In each set of experiments, the kinetics of possible changes in DNA methylation will be followed at various times after the application of the challenge. The far-upstream region of the FMR1 promoter encompasses about 25 CpG dinucleotides and will be analyzed by the bisulfite sequencing technique which has been routinely used in our laboratory.