Research Group of W. Doerfler

Virology

Head of Institute:
Prof. Dr. med. Klaus Überla

Epigenetic Consequences of Foreign DNA Insertions into the Human Genome

The interests of the research group have been focused on the biological functions of DNA methylation in the regulation of biological processes which are of interest in genetics (epigenetics) and medicine. Here we describe current results of the following projects:

  1. In a pilot study, we have documented genome-wide epigenetic destabilization in the genomes of human cells which had been rendered transgenomic for a 5.6 kbp bacterial plasmid. These findings are thought to be of importance for the understanding of viral and general oncogenesis and of evolution.
  2. Infections of mammalian cells with DNA viruses can lead to genome-wide increases in the CpG methylation profiles of the recipient host cell genomes. Adenovirus type 12, African swine fever virus and herpes simplex virus type 1 have been studied in these experiments. Does this increase in genome-wide methylation of cellular DNA reflect the apoptotic process in virus infections?
  3. We have now extended previous studies on methylation patterns in HIV1 proviral genomes to tissue (rectum) samples from HIV1-infected individuals. The data obtained so far have not revealed methylation of proviral DNA in these samples.

Destabilization of the human epigenome: consequences of foreign DNA insertions

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 signalling pathways (see Table below).

Top canonical pathways for differentially expressed genes
EIF2 signalling
Regulation of eIF4 and p70S6K signalling
Glutathione-mediated detoxification
FAK signalling
Insulin receptor signalling
ErbB4 signalling
Small nucleolar RNA's

Genome-wide methylation profiling was performed for >480,000 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 signalling in dorsal horn neurons
Axonal guidance signalling
CREB signalling in neurons
Glutamate receptor signalling
GABA receptor signalling
Netrin signalling

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.

Weber S., Hofmann A., Herms S, Hoffmann P, Doerfler, W. (2015). Destabilization of the human epigenome: consequences of foreign DNA insertions. Epigenomics. 7: 745-55.

DNA methylation and transcription in HERV (K, W, E) and LINE-1.2 sequences remain unchanged upon foreign DNA insertions

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 section 1 for plasmid-transgenomic HCT116 cell clones).

Methods: DNA methylation profiles in the HERV (K, W, E) and 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 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 LINE1.2 CpG methylation and transcription patterns between non-transgenomic and plasmid-transgenomic HCT116 cells.

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). These repetitive sequences are 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.

Weber, S., Jung, S., Doerfler, W. (2016). DNA methylation and transcription in HERV (K, W, E) and LINE sequences remain unchanged upon foreign DNA insertions. Epigenomics. 8: 157-65.

Beware of manipulations on the genome: epigenetic destabilization through (foreign) DNA insertions

On the basis of the results summarized in Weber et al. 2015, 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 this field of research of importance for the critical evaluation of results obtained from many lines of research. The literature has practically kept silent on this issue. Several generally relevant implications of the consequences of foreign DNA insertion or of genome manipulations have been 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.

Doerfler, W. (2016). Beware of manipulations on the genome: epigenetic destabilization through (foreign) DNA insertions. Invited Commentary - Epigenomics. 8: 587-91.

Epigenetic Changes in Host Cellular DNA upon Virus Infections

Sequence-specific CpG methylation of DNA, in particular of promoters, constitutes one of the most important epigenetic signals for the long-term modulation of genetic activities. Starting in the late 1970s, we have discovered changes in methylation patterns in adenovirus 12 (Ad12) DNA which was integrated into Ad12-induced hamster tumor genomes. Virus infections lead to extensive and specific changes in cellular gene expression in time-of-infection dependent, almost algorhythm-like patterns. We have been interested in studying whether these alterations in gene expression profiles are accompanied by changes in cellular DNA methylation profiles in the recipient host cell genomes. In general, methylation patterns are known to be rather stable under certain conditions. Nevertheless, it is conceivable that viral pathogenesis challenges this presumed stability. The literature on viral infections abounds with reports on the effects of viral infections on cellular gene activities. We have asked the question of whether and to what extent the infection of human cells with adenovirus type 12 (Ad12) or of monkey cells with African swine fever virus (ASFV) affects the genome-wide methylation status of the infected cells at various times after infection. In these viral systems, global methylation of host cellular DNA was markedly increased. By using the bisulfite sequencing technique, we demonstrated that the viral DNA in different parts of the ASFV genome at different times after infection of Vero monkey cells remained unmethylated. Methylation analyses of the ASFV genome had not been studied previously. The observed alterations of genome-wide methylation patterns in the wake of virus infections have to be considered a crucial factor in virus-induced cell damage and death. We currently investigate whether global increases in cellular DNA methylation upon virus infections could be part of the apoptotic pathway.

Weber, S., Zakaryan, H., Doerfler, W. (2017). Epigenetic changes in cellular DNA upon virus infections and after the insertion of foreign genomes. Abstract: Euro Virology, London, 2017.

Levels of DNA methylation in HIV1 proviral genomes in rectum samples from HIV1-infected individuals

In collaboration with Rupert Kaul’s group in Toronto, we have extended previous studies on DNA methylation patterns in HIV1 proviral genomes in HIV1-infected individuals to tissue (rectum) samples from individuals at various stages of HIV-1 infection. The results of bisulfite analyses of these DNA samples obtained so far have not revealed methylation of proviral DNA.

Weber, S., Kaul, R., Burger, H., Weiser, B., Doerfler, W. Manuscript in preparation, 2017.

 
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