Influence of extremely-low-frequency magnetic fields on epigenetic programming and cellular differentiation

Cellular homeostasis and function, including the controlled interaction with the environment, not only depends on the genetic code but also and primarily on the epigenetic information contained in histone tail modifications and DNA methylation. Histone modifications are most prominently acetylation or methylation of specific residues in the histone tails whereas cytosine on the DNA can be methylated to form 5-methylcytosine (5mC). Virtually all cells from a multicellular organism contain the same genetic code; the epigenetic system shapes the nuclear organisation of chromatin and DNA accessibility, determining gene expression programs in development to facilitate lineage commitment into specific tissue types. Thereby, patterns of epigenetic modifications change dramatically as cells undergo functional and morphological determination during differentiation and de-differentiation, including cancerous. Genetic mutations as well as aberrations of epigenetic modifications are hallmarks of cancer, but may also be the driving force of cancerogenesis. Acute leukaemia is characterised by high abundance of progenitor cells and they often have alterations in genes encoding epigenetic modifiers. Hence, mutations in epigenetic modifiers in leukaemia and the blocked differentiation of progenitor cells may indicate a defect in the epigenetic control of differentiation. Additionally, epigenetic programming in differentiating cells is sensitive to disturbance by environmental factors. Extremely-low-frequency electromagnetic fields (ELF MFs) have been considered as one of these environmental factors, caused by modern life style. The ever-increasing use of electronic appliances generating electromagnetic fields in the ELF MF range of 50 Hz has raised concerns regarding potential risks for human health. Due to epidemiological studies, indicating a correlation of ELF MF exposure with an increased risk for childhood leukaemia, ELF MF was evaluated as being possibly carcinogenic to humans (group 2B) by the International Agency for Research on Cancer (IARC). However, the molecular mechanisms underlying this correlation have remained elusive. The energy transmitted by ELF MF is not sufficient to directly damage DNA and thereby unlikely to induce cancer-promoting mutations, but whether or not it has the potential to influence the epigenetic program of genomes has not been systematically addressed. The aim of my PhD thesis was to evaluate the influence of ELF MF on the epigenetic code as a potential molecular explanation of the implicated leukaemogenic activity. Therefore, we analysed ELF MF effects on the epigenetic stability in leukaemic cells and on the epigenetic reprogramming during an in vitro haematopoietic differentiation into the neutrophilic lineage. Additionally, I investigated the epigenetic dynamics of histone modifications and DNA methylation during neutrophilic granulopoiesis in a second part.
Previous studies indicated a certain genotoxic potential of ELF MF exposure, although the energy content of ELF MF is not high enough to induce structural damage to DNA. It is possible, however, that secondary effects such as changes in cell proliferation, cell cycle progression or apoptosis can account for the low levels of DNA breaks observed. On the other hand, the second layer of information superposed to the basic DNA sequence, the epigenetic code, has been hardly assessed with respect to ELF MF exposure. There are a few studies indicating that the epigenome can be modified by ELF MF exposure, but the potential of ELF-MF exposure to destabilize epigenetic modifications in general and in a cancer-relevant manner in particular has not been addressed systematically. We examined the influence of ELF MF exposure on the chromatin landscape of the leukaemic Jurkat cell line by analysing the alterations of histone modifications. Furthermore, we studied the impact of ELF MF exposure on the dynamics of histone modifications and DNA methylation during the epigenetic programming of human cord blood stem cells, differentiating into the neutrophilic lineage. We generated genomic profiles of the activating histone mark H3K4me2 and the repressive histone mark H3K27me3 as well as of DNA cytosine methylation. We report that ELF MF exposure has no significant and consistent influence on epigenetic modifications in differentiated leukaemic cells as well as during haematopoietic differentiation. However, our data showed a consistent effect of ELF-MF exposure on the reproducibility of these histone and DNA modification profiles, indicating that ELF MF may influence the robustness of histone modifications and DNA methylation most pronounced in the course of global reorganization of chromatin in the neutrophilic differentiation process. Moreover, our results indicate that ELF MF exposure may stabilize the epigenetic features that are associated with open chromatin during differentiation as regions marked by H3K4me2, losing H3K27me3 or CpG demethylation. Our data suggest a stochastic effect of ELF MF exposure on the chromatin landscape of individual cells.
Acute myeloid leukaemia (AML) is the most common leukaemia in adults (80%). It is characterized by high accumulation of progenitor stages mainly neutrophilic progenitors and alterations in modifiers of epigenetic modifications. To understand the potential contribution of aberrant epigenetic programming in differentiation to carcinogenesis, it is important to understand the physiological epigenetic pattern established during the differentiation process. As the dynamic epigenetic programming during human neutrophilic granulopoiesis has not been addressed systematically, we investigated the reorganization of histone modifications (H3K4me2 and H3K27me3) and DNA cytosine methylation during in vitro neutrophilic granulopoiesis at genome scale. The results show a fundamental reorganisation of the chromatin landscape most pronounced at lineage commitment at the transition of human cord blood stem cells into the neutrophilic lineage, shaping an overall more compact chromatin. We observed that epigenetic repression of pluripotency and developmental genes with a poised chromatin state in human cord blood stem cells occurred through a gain of H3K27me3 alone or with concomitant de novo DNA methylation, but rarely with DNA methylation alone. By contrast, our data indicate that epigenetic activation of neutrophil-specific genes preferentially appeared through DNA demethylation without simultaneous alterations in histone modifications. Our data suggest a specific regulatory role of DNA demethylation in lineage restriction of neutrophils.
This work was part of the European Commission funded project “Advanced Research on Interaction Mechanisms of electromagnetic exposure with Organisms for Risk Assessment” (ARIMMORA), evaluating possible pathways to explain the association between ELF MF exposure and childhood leukaemia. I contributed to the final risk assessment applying an IARC Monograph evaluation scheme to hazard identification of ELF MF. The ARIMMORA consortium concluded that the relationship between ELF MF and childhood leukaemia remains consistent with the classification by IARC that ELF MFs are possibly carcinogenic to humans (Group 2B). The risk assessment was published in a peer-reviewed comment.
Taken together, in collaboration with others, I was able to elucidate and describe alterations of epigenetic modifications during an in vitro differentiation and induced by an environmental factor. As a proof-of-concept, I showed that ELF MF, as an environmental factor, is able to influence the epigenetic code of histone modifications and DNA methylation. ELF MF effects are subtle and stochastic rather than deterministic; the analysis of epigenetic patterns in cell populations showed altered reproducibility between replicate samples. These effects suggest that ELF MF affects the robustness of epigenetic reprogramming by stabilizing epigenetic modifications associated with open chromatin. These observations will support further mechanistic studies analysing the contribution of ELF MF exposure to cancerogenesis. Additionally, my data of the epigenetic programming during neutrophilic granulopoiesis provided novel insights into the regulatory role of DNA methylation and histone modifications in lineage restriction and cell plasticity, revealing a specific epigenetic pattern for activated and repressed genes during differentiation. These data may support further studies investigating chromatin epigenetic regulators as targets for leukaemia induced differentiation therapy.