Beware of Manipulations on the Genome: Epigenetic Destabilization through (Foreign) DNA Insertions

Keywords::

• biology and the concepts of dark energy and dark matter
• DNA methylation
• epigenetic consequences and evolution
• epigenetic consequences of foreign DNA insertions
• epigenetic mechanisms of oncogenesis
• epigenetic sequelae of genome manipulations
• food ingested DNA
• foreign DNA integration into mammalian genomes
• hypothesis on DNA as recipient for cosmic emissions
• transcriptional profiles

Introduction

Experimental evidence supports the notion that the integration of foreign DNA into a mammalian genome can lead to alterations in cellular methylation and transcription patterns. The mechanisms behind this epigenetic destabilization of the genome are unknown, and the phenomenon may not invariably occur. It is unknown in what way the site of foreign DNA integration and the genomic loci of epigenetic alterations are interrelated. In this article, I propose new ideas about an epigenetic role in oncogenesis and during evolution. As the technique of foreign DNA insertion is widely applied in numerous procedures in biology and medicine, data obtained from the use of genomically manipulated cells and organisms should be interpreted with caution. Results from transgenic organisms, knock-out, knock-in and gene therapeutic procedures, as well as the highly acclaimed CRISPR/Cas-9 methods might require critical re-evaluations.

Review of original observations & research

Our laboratory has demonstrated that the integration of foreign DNA into an established mammalian genome can lead to alterations in the affected cells’ methylation and transcriptional profiles [1–5]. The consequences of these epigenetic destabilizations for the cell have not been systematically investigated. With a compromised genome, cells might activate the apoptotic or the deregulated (oncogenic) pathway, perish or gain evolutionary advantage and survive under the most favorable conditions of positive selection.

Adenovirus DNA as transgenome

Studies on the integrated state of adenovirus type 12 (Ad12) DNA in the genome of Ad12-transformed Syrian hamster cells revealed that the state of methylation of the ~900 copies of intracisternal A particles (IAPs) per genome was markedly increased in Ad12 DNA transgenomic cells as compared with nontransgenomic cells [6]. Single gene sequences also showed altered methylation profiles. BHK21 hamster cells had served as targets for Ad12 transformation. Ad12 virus infection of BHK21 cells did not comparably affect the methylation status of the IAP elements.

Excision of the transgenome: insert & excise, hit-and-run

Surprisingly, IAP hypermethylation proved stable in revertants of the Ad12-transformed cells which had lost all Ad12 DNA copies [6]. This finding suggested a hit-and-run mechanism linking foreign DNA integration to epigenetic destabilization of the genome. Apparently, the insertion of 10–15 copies of the 34 kbp Ad12 genome in the Ad12-transformed T637 cell line [7] had somehow led to IAP hypermethylation by an unknown mechanism. Inserting and subsequently excising foreign genomes can cause and then maintain the epigenetic alterations even after the excision of the foreign genome. Since epigenetic destabilizations are a hallmark of oncogenesis, a hit-and-run scenario gains increased significance also for tumor biology.

DNA of bacteriophage lambda (λ)

The integration of bacteriophage λ DNA into the genomes of BHK21 cells also leads to hypermethylation of the IAP sequences, albeit to a lesser extent than in cells transgenomic for Ad12 DNA [8]. In cells carrying integrated DNA of either Ad12 or of bacteriophage λ, transcriptional profiles of numerous cellular genes were markedly altered [9]. These data support the notion of genome-wide alterations in transcriptional profiles in transgenomic cells.

A 5.6 kbp bacterial plasmid transgenome leads to the epigenetic destabilization of the human genome

In a more structured approach in this project, we rendered five clones of human HCT116 (human colon tumor) cells transgenomic for a 5.6 kbp bacterial plasmid which contained the kanamycin resistance gene under early SV40 promoter control to facilitate selection of transgenomic cells. In five nontransgenomic human HCT116 cell clones, the transcriptional activities in 28,869 genome segments proved identical or very similar. This latter finding was the indispensable precondition to render the analysis of five plasmid-transgenomic cell clones meaningful. A total of 1343, in other words, 4.7%, of the 28,869 genome segments analyzed showed altered transcription levels in the transgenomic cell clones, 907 were upregulated and 436 were downregulated [10]. The most significantly affected genome segments belonged to small nucleolar RNA genes, which are involved in the modification of ribosomal, transfer and small nuclear RNAs. A number of signaling genes was also differentially transcribed. Differential DNA methylation in 3791 of >480,000 CpGs was demonstrated in the same plasmid-transgenomic as compared with the nontransgenomic cell clones, 1504 were hyper- and 2287 were hypomethylated [10]. However, the regulatory sequences in the human endogenous retroviral (HERV-K, -E, -W) or LINE-1.2 (long interspersed nuclear elements) sequences in the same transgenomic cell clones had remained epigenetically unaffected [11]. Epstein–Bar virus- or telomerase gene-transformed cells exhibit altered methylation patterns.

The fragile X syndrome (FXS) is one of the most frequent causes of mental retardation in humans. It is caused by the loss of function of the FMR1 (fragile X mental retardation 1) gene, which is most frequently due to an expansion of a naturally occurring CGG repeat in the first exon of this gene and to hypermethylation of the FMR1 promoter [12] (a recent review). We have identified a distinct DNA methylation boundary upstream of the FMR1 promoter, and this boundary is ubiquitous in all types of human and mouse cells investigated [13]. The boundary is lost in DNA from FXS patients [13]. In cells from normal and from FXS individuals, immortalized by EBV or by transformation with the telomerase gene, the methylation boundary is preserved, however, the hypermethylated region way upstream of the boundary has become massively de-methylated, probably due to the introduction of foreign DNA into the transformed cells [14].

Foreign DNA ingested via the GI tract

The most frequent portal of foreign DNA entry into an organism is the GI tract with the continuous food supply. Food-ingested DNA in mice can survive the digestive tract in small amounts, in highly fragmented form and on a transient time scale [15]. An even lower percentage of the DNA fragments persisting in the gut can reach white cells in peripheral blood and in the spleen of mice. Transcription of the persisting foreign DNA has never been detected. An occasional white cell in the spleen cell carries fragments of the gut-ingested DNA as integrate in the cellular genome [15]. The germ line of the experimental animals appeared to be protected from the penetrance of gut-ingested foreign DNA [16]. It is conceivable that food-ingested DNA might contribute to epigenetic sequelae in affected cells [2] – an eye-opener for work in oncology?

Conclusion & caveat

On the basis of the results summarized above, I have pursued the notion 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 eminent importance for the critical evaluation of results obtained from many lines of research. Surprisingly, the literature is practically silent on this issue. In the ensuing discussion, I will address several generally relevant implications of the consequences of foreign DNA insertion or of genome manipulations in the following areas of biology and medicine:

• Epigenetic factors in (viral) oncogenesis;

• Thoughts on epigenetics and evolution;

• Experimental approaches using genome manipulations.

Epigenetic factors in (viral) oncogenesis

For many years, researchers in tumor biology have opened their eyes wide to epigenetic considerations. There are thousands of publications on topics dealing with altered transcription profiles in cancer, cancer epigenetics, histone modifications and cancer, alterations of DNA methylation patterns in many types of tumors and on mutations in tumor cells. In tumor biology, in particular in malignancies with a presumed viral causation, the integration of foreign DNA could have led to genome-wide epigenetic alterations and would thus have been causative in generating malignant cells with genome-wide alterations of transcription patterns as compared with normal cells. At least, this is one way of attempting to find a common denominator between these widely diverging aspects presented by changes in the transcriptional programs. Such changes have been reported independent of the huge panoply among the different malignancies. Since the regulation of many genes has been compromised in malignant cells, alterations in the activity of repair genes might be responsible for the large number of mutations frequently found in many different types of human malignancies [17]. Moreover, similar epigenetic mechanisms might be at play in interfering with the function of the immune surveillance mechanisms [18,19] which are thought to keep frequently arising tumor cells at bay in the host organism. The loss or modifications in tumor surveillance could then have become an important factor in permitting cells transformed to the deregulated or tumor-like phenotype to develop into tumors, and to metastasize throughout the affected organisms.

At this time, it remains hypothetical to what extent, or whether at all, food-ingested DNA fragments transiently persisting in the GI tract could reach the genomes of somatic human cells, become integrated into the genome and contribute to epigenetic alterations and oncogenesis as outlined in this and previous discussions [1,2,6,15]. Should one feel inclined to argue that in that case tumor cells should carry traces of food-ingested DNA, that is not necessarily so. The hit-and-run mechanism shown to exist in the causation of epigenetic effects would explain that the causative foreign DNA insert could have been lost whereas the epigenetic re-programming elicited by the lost insertions would have persisted and remained stable [6]. Since we are still groping for a rational approach to finding the probably many causes of the different malignancies, in 2017 politicians in the USA are trying to financially lure scientists into a US$1 billion ‘moonshot’ program on cancer research [20]. Such programs have worked well to shoot man to the earth’s moon, but despite all the good intentions behind them may not achieve their goals in cancer research. Nevertheless, this program might encourage similar rather unconventional approaches to cancer research.

Thoughts on epigenetics & evolution

During evolution, both genome stability and flexibility must have been decisive factors which were in constant competition with each other. Since foreign DNA is readily inserted into established genomes, this competition continues to be relevant in today’s biology. The widespread presence of ancient retroviral and retrotransposon elements also in the human genome attests to the long evolutionary history of retroviral insertions into ancient genomes. The ancient, now degenerate retroviral genomes, continue to be transcribed with unknown function; but active viral genomes are not produced. Each insertion in evolutionary times added novel genetic information. However, more importantly, each impact upon insertions of foreign DNA into an ancestral genome had epigenetic consequences in that it altered methylation and transcription profiles and could have led to the generation of completely new cell types. Depending on the environmental conditions then prevalent, the novel cell types were either eliminated or had gained an evolutionary advantage, survived and contributed to the development of present day (human) genomes. Hence, the most significant contribution of the insertion of ancient foreign genomes would have been the generation of new (epi)-genetic profiles and in that way to the rise of new cell types. Thus epigenetic destabilizations of ancient genomes are thought to have been an important driving force in evolution.

Might we have missed 95% of cosmic reality?

In an age when physicists lecture us on unidentified dark energy and dark matter constituting about 95% of cosmic reality, biologists might have to be alerted to consider completely unheard-of mechanisms at work during evolution. Should the physicists’ revolutionary concepts be proven right, we as biologists would have to recognize that so far we have confined our thinking to only 5% of this reality. In that case, it would be likely that 95% of the laws of nature had also remained unrecognized.

Here is an audacious attempt and a reprise of an earlier hypothesis [21] on evolution, “DNA – a molecule in search of additional functions”:

• The entirety (termed pool in the earlier article) of all information, all laws governing all events in the universe, all energy and matter including the ‘dark ones’ has not existed and does not exist in silence but emits cosmic information ‘waves’ of unknown physical properties. Obviously, we lack the sensory antennas to perceive and receive any of this form of informative energy. This transmission system is thought to have originated simultaneously with the big bang at about 13.8 billion years ago. The physicists have postulated the existence of dark energy and dark matter to comprise about 95% of the entirety of the cosmos;

• These cosmic transmissions have been the evolutionary driving force for the generation of all DNA molecules with different, species-specific nucleotide sequences. Over evolutionary time scales, the molecule grew from oligo-nucleotides to the highly complex nucleotide sequences which exist today. Apart from and in addition to the cosmic energy sources, the development of the multitude of these sequences has been influenced by many different factors in the biological environments on the planet earth which DNA has been exposed to;

• Since, according to this hypothesis, DNA arose from interactions of cosmic energy transmissions with matter, DNA might be the only molecule capable of ‘sensing’ the cosmic information waves;

• Human DNA and its sequence serves as one of these receptors and is probably the most sophisticated one in existence. We are completely unaware of these transmissions since we lack a sensory reception system to respond to them. However, un- or rather subconsciously, we are subject to this steady flow of information at all times. Quality and extent of this receptor capacity depends on our DNA sequence. Without our awareness, the DNA receptor acting as antenna constantly expands the received information to our neural system which, under the most fortunate circumstances, might create new ideas. Also, our psychological condition could be modulated by this system.

Experimental approaches using genome manipulations

For many years, researchers in the following areas of experimental biomedicine have closed their eyes to epigenetic considerations, with notable exceptions [22]. Therefore, they might be concerned about their results with respect to epigenetic destabilizations of the genomes of manipulated cells or organisms:

• Transgenic and transgenomic cells and organisms in all biological systems;

• Gene therapeutic regimens;Induced embryonic stem cells;

• Knock-out or knock-in experiments;

• Applications of the CRISPR-Cas9 technology [23].

Technical perfections in molecular biology have rendered genome-wide analyses of transcriptional and methylation patterns a reality. Hence it can be expected that with time there will be active reconsiderations of data obtained with these technologies, even though there might be resistance among those who have placed their interests on the application of these interesting technologies.

Financial & competing interests disclosure

Between 1972 and 2000, the author’s research at the Institute of Genetics in Cologne was supported by the Deutsche Forschungsgemeinschaft (DFG) through SFB74 and SFB274 as well as by the Center for Molecular Medicine Cologne (CMMC, TP13). Research at the Institute for Virology in Erlangen (2002 to the present) has been funded at different times by the Thyssen Stiftung, Köln (Az. 10.07.2.138 and a research fellowship to Anja Naumann, Az. 40.12.0.029), the DFG, Bonn-Bad Godesberg (DO 165/28-1), the Staedtler Stiftung, Nürnberg (WW/eh 01/15) and the Institute for Virology, University Erlangen-Nürnberg. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

Between 1972 and 2000, the author’s research at the Institute of Genetics in Cologne was supported by the Deutsche Forschungsgemeinschaft (DFG) through SFB74 and SFB274 as well as by the Center for Molecular Medicine Cologne (CMMC, TP13). Research at the Institute for Virology in Erlangen (2002 to the present) has been funded at different times by the Thyssen Stiftung, Köln (Az. 10.07.2.138 and a research fellowship to Anja Naumann, Az. 40.12.0.029), the DFG, Bonn-Bad Godesberg (DO 165/28-1), the Staedtler Stiftung, Nürnberg (WW/eh 01/15) and the Institute for Virology, University Erlangen-Nürnberg. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.