A distinct DNA-methylation boundary in the 5'- upstream sequence of the FMR1 promoter binds nuclear proteins and is lost in fragile X syndrome.

We have discovered a distinct DNA-methylation boundary at a site between 650 and 800 nucleotides upstream of the CGG repeat in the first exon of the human FMR1 gene. This boundary, identified by bisulfite sequencing, is present in all human cell lines and cell types, irrespective of age, gender, and developmental stage. The same boundary is found also in different mouse tissues, although sequence homology between human and mouse in this region is only 46.7%. This boundary sequence, in both the unmethylated and the CpG-methylated modes, binds specifically to nuclear proteins from human cells. We interpret this boundary as carrying a specific chromatin structure that delineates a hypermethylated area in the genome from the unmethylated FMR1 promoter and protecting it from the spreading of DNA methylation. In individuals with the fragile X syndrome (FRAXA), the methylation boundary is lost; methylation has penetrated into the FMR1 promoter and inactivated the FMR1 gene. In one FRAXA genome, the upstream terminus of the methylation boundary region exhibits decreased methylation as compared to that of healthy individuals. This finding suggests changes in nucleotide sequence and chromatin structure in the boundary region of this FRAXA individual. In the completely de novo methylated FMR1 promoter, there are isolated unmethylated CpG dinucleotides that are, however, not found when the FMR1 promoter and upstream sequences are methylated in vitro with the bacterial M-SssI DNA methyltransferase. They may arise during de novo methylation only in DNA that is organized in chromatin and be due to the binding of specific proteins.

Epigenetic consequences of foreign DNA insertions: de novo methylation and global alterations of methylation patterns in recipient genomes.

The insertion of foreign DNA into mammalian or plant genomes is a frequent event in biology. My laboratory has pursued a long-standing interest in the structure of integrated adenovirus genomes and in the mechanism of foreign DNA insertions in mammalian cells. The long-term consequences of the integration of alien DNA are only partly known, and even less well understood are the mechanisms that bring them about. Evidence from viral systems has contributed to the realization that foreign DNA insertions entail a complex of sequelae that have also become apparent in non-viral systems: (i) The de novo methylation of integrated foreign DNA sequences has frequently been observed. (ii) Alterations of DNA methylation patterns in the recipient genome at and remote from the site of foreign DNA insertion have been demonstrated but it remains to be investigated how generally this phenomenon occurs. Many viral genomes find and have found entry into the genomes of present-day organisms. A major portion of mammalian genomes represents incomplete retroviral genomes that frequently have become permanently silenced by DNA methylation. It is still unknown how and to what extent the insertion of retroviral or retrotransposon sequences into established genomes has altered and shaped the methylation and transcription profiles of present day genomes. An additional reason for concern about the effects of foreign DNA integration is the fact that in all fields of molecular biology and medicine, the generation of transgenic or transgenomic cells and organisms has become a ubiquitously applied experimental technique.

Ubiquitous Micro-Modular Homologies among Genomes from Viruses to Bacteria to Human Mitochondrial DNA: Platforms for Recombination during Evolution?

The emerging Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and its variants have raised tantalizing questions about evolutionary mechanisms that continue to shape biology today. We have compared the nucleotide sequence of SARS-CoV-2 RNA to that of genomes of many different viruses, of endosymbiotic proteobacterial and bacterial DNAs, and of human mitochondrial DNA. The entire 4,641,652 nt DNA sequence of Escherichia coli K12 has been computer-matched to SARS-CoV-2 RNA. Numerous, very similar micro-modular clusters of 3 to 13 nucleotides lengths were detected with sequence identities of 40 to >50% in specific genome segments between SARS-CoV-2 and the investigated genomes. These clusters were part of patch-type homologies. Control sequence comparisons between 1000 randomly computer-composed sequences of 29.9 kb and with the A, C, G, T base composition of SARS-CoV-2 genome versus the reference Wuhan SARS-CoV-2 sequence showed similar patterns of sequence homologies. The universal A, C, G, T genetic coding mode might have succeeded in evolution due in part to its built-in capacity to select for a substantial reservoir of micro-modular domains and employ them as platforms for integrative recombination. Their role in SARS-CoV-2 interspecies transition and the generation of variants appears likely, but their actual involvement will require detailed investigations.

Epigenetic mechanisms in human adenovirus type 12 oncogenesis.

For the past 30 years, my laboratory has concentrated its work on demonstrating that the epigenetic consequences of foreign DNA insertion into established mammalian genomes -de novo DNA methylation of the integrate and alterations of methylation patterns across the recipient genome - are essential elements in setting the stage towards oncogenic transformation. We have primarily studied human adenovirus type 12 (Ad12) which induces undifferentiated tumors in Syrian hamsters (Mesocricetus auratus) either at the site of subcutaneous Ad12 injection or intraperitoneally upon intramuscular injection. Up to 90% of the hamsters injected with Ad12 develop tumors within 3-6 weeks. Integration of foreign DNA, its de novo methylation, and the consequences of insertion on the cellular methylation and transcription profiles have been studied in detail. While viral infections are a frequent source of foreign genomes entering mammalian and other hosts and often their genomes, we have also pursued the fate of food-ingested foreign DNA in the mouse organism. The persistence of this DNA in the animals is transient and there is no evidence for the expression or germ line fixation of foreign DNA. Nevertheless, the occasional cell that carries integrated genomes from that foreign source deserves the oncologist's sustained interest.

Adenoviral Vector DNA- and SARS-CoV-2 mRNA-Based Covid-19 Vaccines: Possible Integration into the Human Genome - Are Adenoviral Genes Expressed in Vector-based Vaccines?

Vigorous vaccination programs against SARS-CoV-2-causing Covid-19 are the major chance to fight this dreadful pandemic. The currently administered vaccines depend on adenovirus DNA vectors or on SARS-CoV-2 mRNA that might become reverse transcribed into DNA, however infrequently. In some societies, people have become sensitized against the potential short- or long-term side effects of foreign DNA being injected into humans. In my laboratory, the fate of foreign DNA in mammalian (human) cells and organisms has been investigated for many years. In this review, a summary of the results obtained will be presented. This synopsis has been put in the evolutionary context of retrotransposon insertions into pre-human genomes millions of years ago. In addition, studies on adenovirus vector-based DNA, on the fate of food-ingested DNA as well as the long-term persistence of SARS-CoV-2 RNA/DNA will be described. Actual integration of viral DNA molecules and of adenovirus vector DNA will likely be chance events whose frequency and epigenetic consequences cannot with certainty be assessed. The review also addresses problems of remaining adenoviral gene expression in adenoviral-based vectors and their role in side effects of vaccines. Eventually, it will come down to weighing the possible risks of genomic insertions of vaccine-associated foreign DNA and unknown levels of vector-carried adenoviral gene expression versus protection against the dangers of Covid-19. A decision in favor of vaccination against life-threatening disease appears prudent. Informing the public about the complexities of biology will be a reliable guide when having to reach personal decisions about vaccinations.

SARS-CoV-2 worldwide replication drives rapid rise and selection of mutations across the viral genome: a time-course study - potential challenge for vaccines and therapies.

Scientists and the public were alarmed at the first large viral variant of SARS-CoV-2 reported in December 2020. We have followed the time course of emerging viral mutants and variants during the SARS-CoV-2 pandemic in ten countries on four continents. We examined > 383,500 complete SARS-CoV-2 nucleotide sequences in GISAID (Global Initiative of Sharing All Influenza Data) with sampling dates extending until April 05, 2021. These sequences originated from ten different countries: United Kingdom, South Africa, Brazil, United States, India, Russia, France, Spain, Germany, and China. Among the 77 to 100 novel mutations, some previously reported mutations waned and some of them increased in prevalence over time. VUI2012/01 (B.1.1.7) and 501Y.V2 (B.1.351), the so-called UK and South Africa variants, respectively, and two variants from Brazil, 484K.V2, now called P.1 and P.2, increased in prevalence. Despite lockdowns, worldwide active replication in genetically and socio-economically diverse populations facilitated selection of new mutations. The data on mutant and variant SARS-CoV-2 strains provided here comprise a global resource for easy access to the myriad mutations and variants detected to date globally. Rapidly evolving new variant and mutant strains might give rise to escape variants, capable of limiting the efficacy of vaccines, therapies, and diagnostic tests.

An origin of DNA replication in the promoter region of the human fragile X mental retardation (FMR1) gene.

Fragile X syndrome, the most common form of inherited mental retardation in males, arises when the normally stable 5 to 50 CGG repeats in the 5' untranslated region of the fragile X mental retardation protein 1 (FMR1) gene expand to over 200, leading to DNA methylation and silencing of the FMR1 promoter. Although the events that trigger local CGG expansion remain unknown, the stability of trinucleotide repeat tracts is affected by their position relative to an origin of DNA replication in model systems. Origins of DNA replication in the FMR1 locus have not yet been described. Here, we report an origin of replication adjacent to the FMR1 promoter and CGG repeats that was identified by scanning a 35-kb region. Prereplication proteins Orc3p and Mcm4p bind to chromatin in the FMR1 initiation region in vivo. The position of the FMR1 origin relative to the CGG repeats is consistent with a role in repeat maintenance. The FMR1 origin is active in transformed cell lines, fibroblasts from healthy individuals, fibroblasts from patients with fragile X syndrome, and fetal cells as early as 8 weeks old. The potential role of the FMR1 origin in CGG tract instability is discussed.

Essential concepts are missing: Foreign DNA in food invades the organisms' cells and can lead to stochastic epigenetic alterations with a wide range of possible pathogenetic consequences.

In this article, a new concept for general pathogenesis has been proposed. Advances in molecular genetics have led to the realization that essential concepts in the framework of molecular biology are still missing. Clinical medicine is plagued by similar shortcomings: The questioning of current paradigms could open new vistas and invite challenging approaches. This article presents an unconventional idea. Foreign DNA which is regularly ingested with the essential food supply is not completely degraded. Small quantities of fragmented DNA rather persist transiently in the gastro-intestinal tract of mice and can be traced to various organ systems, except for cells in the germ line. Foreign DNA entering and persisting in mammalian cells can stochastically lead to genome-wide alterations of transcriptional and CpG DNA methylation profiles. In the course of food-ingested DNA invading somatic cells, completely new cell types can be generated which might be involved in the causation of common ailments. Projects emanating from this perception merit critical analysis and rigorous pursuit.

Signal hotspot mutations in SARS-CoV-2 genomes evolve as the virus spreads and actively replicates in different parts of the world.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was first identified in Wuhan, China late in 2019. Nine months later (Sept. 23, 2020), the virus has infected > 31.6 million people around the world and caused > 971.000 (3.07 %) fatalities in 220 countries and territories. Research on the genetics of the SARS-CoV-2 genome, its mutants and their penetrance can aid future defense strategies. By analyzing sequence data deposited between December 2019 and end of May 2020, we have compared nucleotide sequences of 570 SARS-CoV-2 genomes from China, Europe, the US, and India to the sequence of the Wuhan isolate. During worldwide spreading among human populations, at least 10 distinct hotspot mutations had been selected and found in up to > 80 % of viral genomes. Many of these mutations led to amino acid exchanges in replication-relevant viral proteins. Mutations in the SARS-CoV-2 genome would also impinge upon the secondary structure of the viral RNA molecule and its repertoire of interactions with essential cellular and viral proteins. The increasing frequency of SARS-CoV-2 mutation hotspots might select for dangerous viral pathogens. Alternatively, in a 29.900 nucleotide-genome, there might be a limit to the number of mutable and selectable sites which, when exhausted, could prove disadvantageous to viral survival. The speed, at which novel SARS-CoV-2 mutants are selected and dispersed around the world, could pose problems for the development of vaccines and therapeutics.

Human CAR gene expression in nonpermissive hamster cells boosts entry of type 12 adenovirions and nuclear import of viral DNA.

Adenovirus type 12 (Ad12) propagation in hamster BHK21 cells is blocked prior to viral DNA replication. The amounts of Ad12 DNA in the nuclei or cytoplasm of hamster cells are about 2 orders of magnitude (2 h postinfection [p.i.]) and 4 to 5 orders of magnitude (48 h p.i.) lower than in permissive human cells. Cell line BHK21-hCAR is transgenic for and expresses the human coxsackie- and adenovirus receptor (hCAR) gene. Nuclear uptake of Ad12 DNA in BHK21-hCAR cells is markedly increased compared to that in naïve BHK21 cells. Ad12 elicits a cytopathic effect in BHK21-hCAR cells but not in BHK21 cells. Quantitative PCR or [(3)H]thymidine labeling followed by zone velocity sedimentation fails to detect Ad12 DNA replication in BHK21 or BHK21-hCAR cells. Newly assembled Ad12 virions cannot be detected. Thus, the block in Ad12 DNA replication in hamster cells is not released by enhanced nuclear import of Ad12 DNA.

Inheritable epigenetic response towards foreign DNA entry by mammalian host cells: a guardian of genomic stability.

Apart from its well-documented role in long-term promoter silencing, the genome-wide distribution patterns of ~ 28 million methylated or unmethylated CpG dinucleotides, e. g. in the human genome, is in search of genetic functions. We have set out to study changes in the cellular CpG methylation profile 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 all instances investigated, 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 complete loss of all transgenomic Ad12 DNA. Hence, stress-induced alterations in CpG methylation can be inherited independent of the continued presence of the transgenome. Upon virus infections, changes in cellular CpG methylation appear early after infection. In EBV immortalized as compared to control cells, CpG hypermethylation in the far-upstream region of the human FMR1 promoter decreased four-fold. We conclude that 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 working concept, we view CpG methylation profiles in mammalian genomes as a guarding sensor for genomic stability under epigenetic control. As a caveat towards manipulations of cells with foreign DNA, such cells can no longer be considered identical to their un-manipulated counterparts.

Intracellular African swine fever virus DNA remains unmethylated in infected Vero cells.

AIM: Sequence-specific CpG methylation of eukaryotic promoters is an important epigenetic signal for long-term gene silencing. We have now studied the methylation status of African swine fever virus (ASFV) DNA at various times after infection of Vero cells in culture. METHODS & RESULTS: ASFV DNA was detectable throughout the infection cycle and was found unmethylated in productively infected Vero cells as documented by bisulfite sequencing of 13 viral DNA segments. CONCLUSION: ASFV DNA does not become de novo methylated in the course of infection in selected segments spread across the entire genome. Thus DNA methylation does not interfere with ASFV genome transcription. Lack of de novo methylation has previously been observed for free intracellular viral DNA in cells permissively infected with human adenoviruses, with human papillomaviruses and others.

Human adenovirus type 12: crossing species barriers to immortalize the viral genome.

When viruses cross species barriers, they often change their biological and pathogenetic properties. In the author's laboratory the nonproductive interaction of Syrian hamster cells with human adenovirus type 12 (Ad12) has been studied. Ad12 induces undifferentiated tumors in newborn hamsters (Mesocricetus auratus) at high frequency. Ad12 inefficiently enters hamster (BHK21) cells, and only small amounts of viral DNA reach the nucleus. Viral DNA replication and late transcription are blocked. In Ad12-induced tumor cells, multiple copies of viral DNA are chromosomally integrated. The integrated viral DNA becomes de novo methylated. Cellular DNA methylation and transcription patterns in Ad12-transformed cells and in Ad12-induced tumor cells are altered. These changes may be related to the oncogenic potential of Ad12 in hamsters. In this chapter, concepts and techniques for the study of the Ad12-hamster cell system are summarized.

Influence of in vitro manipulation on the stability of methylation patterns in the Snurf/Snrpn-imprinting region in mouse embryonic stem cells.

Recent work on embryonic stem (ES) cells showed that stem cell-derived tissues and embryos, cloned from ES cell nuclei, often fail to maintain epigenetic states of imprinted genes. This deregulation is frequently associated with in vitro manipulations and culture conditions which might affect the cells potential to develop into normal fetuses. Usually, epigenetic instability is reported in differentially methylated regions of mostly growth-related imprinted genes. However, little is known about the epigenetic stability of genes that function late in organogenesis. Hence, we set out to investigate the epigenetic stability of neuronal genes and analyzed DNA methylation patterns in the Snurf/Snrpn imprinted cluster in several cultured mouse ES cell lines. We also determined the effects of in vitro stress factors such as consecutive passaging, trypsination, mechanical handling, single cell cloning, centrifugation, staurosporine-induced neurogenesis and the insertion of viral (foreign) DNA into the host genome. Intriguingly, none of these in vitro manipulations interfered with the stability of the methylation patterns in the analyzed neuronal genes. These data imply that, in contrast to growth-related genes like Igf2, H19, Igf2r or Grb10, the methylation imprints of the analyzed neuronal genes in the Snurf/Snrpn cluster may be particularly stable in manipulated ES cells.

DNA methylation and transcription in HERV (K, W, E) and LINE 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. METHODS: DNA methylation profiles in the HERV (K, W, E) and LINE 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 LINE-1.2 CpG methylation and transcription patterns between non-transgenomic and plasmid-transgenomic HCT116 cells. CONCLUSION: 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. 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.

DNA methylation in promoter regions of red cell membrane protein genes in healthy individuals and patients with hereditary membrane disorders.

The methylation state of 5'-CG-3' sites is known to be linked to the regulation of promoter function by modulating DNA-protein interactions and to the structure of chromatin. As part of a project to determine methylation patterns in the human genome, we examined the methylation profiles of several genes for human erythroid membrane proteins: ELB42 (protein 4.2), EPB3 (band 3), SPTB gene (beta-spectrin), and ANK1 (ankyrin). The bisulfite protocol of the genomic sequencing method was applied. The number of 5'-CG-3' dinucleotides was the most abundant in SPTB and ANK1, much less in EPB3, and the least in ELB42. In the DNA of peripheral blood mononuclear cells from healthy individuals, the promoter regions of EPB3 and ELB42 were extensively methylated, but the SPTB and ANK1 promoters were totally unmethylated. We also investigated methylation profiles in peripheral blood mononuclear cells from patients with red cell membrane diseases, such as complete protein 4.2 deficiency due to ELB42 mutations, hereditary spherocytosis with EPB3 mutations, and hereditary elliptocytosis with SPTB mutations. The DNA methylation states in these genes of erythroid cells, which we obtained at the second phase of the 2-phase liquid culture of erythroid precursor cells in the peripheral blood, were essentially identical or very similar to those of peripheral blood mononuclear cells. In disease states, the DNA methylation profiles of these red cell membrane protein genes were essentially not different from those in healthy individuals (statistically not significant).

Relationships between DNA methylation and expression in erythrocyte membrane protein (band 3, protein 4.2, and beta-spectrin) genes during human erythroid development and differentiation.

Red cell membrane proteins are sequentially expressed during erythroid development and differentiation. Spectrins have already been synthesized in early erythroid precursors such as pronormoblasts, and band 3 (B3) appears at nearly the same stage. Protein 4.1 appears next, followed by protein 4.2 (P4.2) at the very late erythroblast stage. The methylation states of the promoter 5'-CG-3' sites are known to be linked to the regulation of promoter function by modulating DNA-protein interactions and the structure of chromatin. Hence, the genes for B3, P4.2, and beta-spectrin (beta-SP) appear to be suitable models to study the relationship between methylation of promoter 5'-CG-3' sites and the sequential expression of genes during human erythroid development and differentiation. We have examined methylation profiles in the promoter regions of the genes (ELB42, EPB3, and SPTB) for the human erythroid membrane proteins P4.2, B3, and beta-SP by applying the bisulfite genomic sequencing method. Our results demon strate the following: (1) The promoter regions of EPB3 and ELB42 are extensively methylated in DNA from human peripheral blood mononuclear cells, but the SPTB promoter is totally unmethylated. (2) During erythroid differentiation, DNA methylation patterns change as follows: (a) ELB42 is unmethylated in DNA from erythroid-committed blastic cells, such as the human cell lin UT-7/EPO, but is methylated in erythroblasts from peripheral blood burst-forming unit erythroid (BFU-E) in the second phase of the liquid-culture method. Messenger RNA (mRNA) from ELB42 is first detected in early erythroblasts, and P4.2 is expressed in late erythroblasts. (b) In contrast, EPB3 is consistently methylated in UT-7/EPO cells and in cultured erythroblasts from BFU-E from human peripheral blood. B3 mRNA and protein are already expressed in early erythroblasts. (c) SPTB remains unmethylated in human DNA from UT-7/EPO cells and from cultured erythroblasts. These results document the diversity of the reactions of human promoter sequences to the modulating influence of DNA methylation. Whereas the human SPTB promoter conforms to expectations in that it is unmethylated and fully active throughout erythroid development, high levels of promoter methylation correlate with promoter activity for the EPB3 and ELB42 genes during their sequential activation in erythrocyte differentiation.

High specificity of quantitative excision repair cross-complementing 1 messenger RNA expression for prediction of minor histopathological response to neoadjuvant radiochemotherapy in esophageal cancer.

PURPOSE: The excision repair cross-complementing 1 (ERCC1) gene is coding for a nucleotide excision repair protein involved in the repair of radiation- and chemotherapy-induced DNA damage. We examined the potential of quantitative ERCC1 mRNA expression to predict minor or major histopathological response to neoadjuvant radiochemotherapy (cisplatin, 5-fluorouracil, and 36 Gy of radiation) followed by transthoracic en bloc esophagectomy in patients with locally advanced esophageal cancer (cT(2-4), N(x), M(0)). EXPERIMENTAL DESIGN: Tissue samples were collected by endoscopic biopsy before treatment. RNA was isolated from biopsies, and quantitative real-time reverse transcriptase PCR assays were performed to determine ERCC1 mRNA expression. Relative mRNA levels (tumor/normal ratios) were calculated as (ERCC1/beta-actin in tumor)/(ERCC1/beta-actin in paired normal tissue). ERCC1 expression levels were correlated with the objective histopathological response in resected specimens. Histomorphological regression was defined as major response when resected specimens contained <10% of residual vital tumor cells or in case a pathologically complete response was achieved. RESULTS: Twelve of 36 tumors showed a major histopathological response, and 24 of 36 showed a minor histopathological response. Relative expression levels of ERCC1 of >1.09 were not associated with a major histopathological response (sensitivity, 62.5%; specificity, 100%) and 15 of 24 patients with minor histopathological response to the delivered neoadjuvant radiochemotherapy could be unequivocally identified. This association of dichotomized relative ERCC1 mRNA expression and histopathological response was statistically significant (P < 0.001). CONCLUSIONS: Relative expression levels of ERCC1 mRNA determined by quantitative real-time reverse transcriptase-PCR appear highly specific to predict minor response to our neoadjuvant radiochemotherapy protocol in patients with locally advanced esophageal cancer and could be applied to prevent expensive, noneffective, and potentially harmful therapies in a substantial number (42%) of patients.

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 of inserting a 5.6 kbp plasmid into the human genome. METHODS: Differential transcription and CpG methylation patterns were compared between transgenomic and nontransgenomic cell clones by using gene chip microarray systems. RESULTS: In 4.7% of the 28.869 gene segments analyzed, transcriptional activities were up- or downregulated in the transgenomic cell clones. Genome-wide profiling revealed differential methylation in 3791 of > 480,000 CpG's examined in transgenomic versus nontransgenomic clones. CONCLUSION: The data document genome-wide effects of foreign DNA insertions on the epigenetic stability of human cells. Many fields in experimental biology and medicine employ transgenomic or otherwise genome-manipulated cells or organisms without considering the epigenetic consequences for the recipient genomes.