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Jonathan D. Turner

Dept. Immunology,
Laboratoire Nationale de Santé / Luxembourg institute of health
20A rue Auguste Lumiere
Grand Duchy of Luxembourg
According to EU and WHO estimates, stress represents the single most important cause of disease, causing costs as high as 3-4 % of the European GNP. Stress is responsible for up to 60% of all days lost to disease. Many of these diseases are related to infections and aberrant immune reactions. In addition, stress affects social behaviour, mood, learning and memory. We are a department of the “Forschungsinstitut für Psychobiologie des Stresses” of the University of Trier. As the leader of the PsychoImmunology group, my interest is in explaining the transcriptional regulation of the glucocorticoid receptor, concentrating on genetic, epigenetic, transcriptional, translation and post-translational mechanisms.

We have extensively studied the regulation of the glucocorticoid receptor (GR) as one of the main mediators of the stress response system, the hypothalamic-pituitary-adrenal (HPA). The GR mediates the HPA feed-back and is responsible for homeostasis of many cell and organ functions including inflammatory and immune processes, in particular during stress. Mechanisms underlying the transcriptional and translational control of the human GR and its activity on a molecular level help to understand the interaction between the central nervous system, the HPA axis and the immune system under stress. The level of the GR is of critical importance in adapting the GC sensitivity of each tissue. Since the initial report of environmentally induced DNA methylation controlling both GR levels and HPA axis reactivity in the rat, we have shown that human GR levels are transcriptionally controlled through a highly variable 5’ gene region that codes for 11 alternative untranslated first exons (Turner 2005, 2006) each with its own promoter region (Cao-Lei et al 2011). Each promoter is susceptible to DNA methylation (Turner 2008). In immune cells, promoter methylation levels and patterns were stochastic and unique among individuals. Within the GR promoters the majority of evolutionarily conserved, and confirmed active transcription factor binding sites contain methylatable CpG sites, suggesting that methylation helps control GR expression in a tissue specific manner (Turner 2008). We have also demonstrated the effects of reduced GR levels, and an altered HPA axis on circulating lymphocytes in stress related disorders (e.g. fibromyalgia patients), on GR target gene expression, cytokine regulation and levels of adhesion molecules (Macedo et al 2007, 2008).

Transcriptional and translational control of the human GR.
After elucidating the GR structure, we showed that environmentally induced DNA methylation in this region controls both GR levels and HPA axis (Cao-Lei et al. 2013, Turner et al in preparation 2014b). These 5’UTR transcript variants influenced mRNA structures, free energy and total mRNA levels, as well as influencing relative levels of the different N-terminal protein isoforms. Levels of the two principal 3’ splice transcripts, GR-α and –β were not dependent upon the 5’UTR. Membrane-GR specific labelling confirmed that it is a product of the classical GR gene, and the alternate 5’ transcripts appear to play a role in its production (Turner et al. 2014a). We confirmed that each 5’UTR has its own functional promoter, and that was silenced by DNA methylation (Cao-Lei et al 2011). Detailed examination demonstrated that methylation occurs in clusters <100bp long, but there is a strong correlation between promoter-wide methylation and GR expression (Cao-Lei et al. 2013, Witzmann et al 2012). We demonstrated that GR promoter methylation levels were constant throughout the human brain (Cao-Lei et al. 2013), and did not vary in major depression (Alt et al. 2010 Klok 2011). Importantly, differences in key HPA axis tissue methylation levels (pituitary and adrenal gland) were reflected in PBMCs, suggesting they may be surrogates for the HPA axis in e.g. EpiPath (Witzmann et al 2012). The advent of massively parallel sequencing has allowed us to repeat our initial examination of the GR 5’UTR structure. With more than 800,000 sequences, (c.f. 20-50 colonies previously), we identified a total of 358 TSSs distributed throughout the promoter region. These TSSs were distributed in 38 loci, with an average of 9.4 adjacent normally distributed TSSs (Leenen et al. in preparation). Additionally we have collaborated with other departments, elucidating the structure of Mlc1 (Henseler et al. 2011) and C15orf53 (Krantz et al. 2012).

Downstream effects of GCs.
We have shown that GC and the HPA axis orchestrate alternating tidal waves of immune cells (Trifonova et al 2013b); GC induced gene expression; and the GC induced proteome (Billing et al. 2007; Billing et al. 2011). In the latter studies we characterized the GC effect on cellular signalling pathways such as interferon signalling. This was extended to the rapid effects of GCs both in vivo (Billing et al. 2012) and in vitro. The membrane GR receptor was shown to be responsible for rapid GC effects that aligned with and paved the way for classical GR effects (Vernocchi et al. 2013). GC induced gene expression was investigated using both the natural circadian rhythm, and inducing cortisol secretion with the TSST (Trifonova et al. In preparation). Our data suggests that GC induced GR-target gene expression is highly gene- and donor-dependent, although, transcription was induced in a window up to 3h post GC exposure.

Epigenetic programming of the GR.
Using chronic restraint and psychosocial stress models we successfully programmed the HPA axis is rats, particularly through increased GR promoter methylation in peripheral HPA axis tissues (Witzmann et al 2012). To assess the effects of epigenetic programming of GR promoters on HPA axis function in man, we have developed a mathematical deconvolution model of the HPA axis, accessing and characterizing cortisol secretion from the adrenal gland using saliva, a sampling technique that has the advantage of not activating the HPA axis (Trifonova et al 2013a). We subsequently applied our deconvolution model in a psychobiological model of image processing (Ferreira de Sá et al. 2014)

Lifelong effects of epigenetic programming.
Two complementary genome-wide DNA methylation analysis techniques, MeDIP-Seq and Methyl-Seq, have been setup. We have further developed Methyl-Seq, targeting specific genomic regions, optimising for example gene- or CpG island- coverage with a series of methylation sensitive and insensitive isoschizomers (Kirschner et al in preparation a). Prior to starting cohort studies, we investigated the influence of the PBMC heterogeneity on inter-individual variability in DNA methylation. Patterns of co-regulated CpG sites that were detectable in cord blood became more prominent in adult blood, although methylation levels were clearly influenced by cellular composition (Jacoby, M. et al. 2012). We are currently focussed on two epigenetic programming events, early life immune challenge (ELIC, Perinatal) and adversity (ELA, Epipath). Both paradigms affect HPA axis reactivity, and share many long term effects. After ELIC, the reaction to subsequent exposure was reduced. This diminished response was accompanied by increased global methylation levels, and was transmitted to subsequent untreated generations (Kirschner et al in preparation b). We are currently in the early phase of EpiPath, recruiting individuals that were adopted during the 1990s from orphanages worldwide, a severe form of ELA. Preliminary data suggests that our subjects have reduced HPA axis and an increased immune response to stress. In this project we are collecting genome wide MeDIP-Seq, environmental, behavioural, and psychological data, as well as functional data on the status of their immune, cardiovascular and stress response systems. Integrating these data will provide a broad and unique insight on the influence of ELA on the development of adult disease.

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