Resumen de Molecular mechanisms for active DNA demethylation in cellular immune models
Epigenetic factors, such as histone post-translational modifications and DNA methylation, are critical determinants of gene expression, which ultimately defines cell identity and function. Despite intense research in DNA methylation over the past twenty years, there are still many open questions in relation to the mechanisms underlying its targeting to different genomic sites, its specific functional roles and the identity of enzymes involved in its establishment and removal. Immune cells are extraordinarily diverse and require of many differentiation and activation steps in response to cytokines, growth factors and many other extracellular signals, including exposure to pathogenic molecules. The complexity of immune differentiation and responses provides excellent models to investigate questions about epigenetic mechanisms, including DNA methylation. In this thesis, we aimed at investigating the molecular mechanisms of active DNA demethylation and its genomic targeting through the use of two different immune models. Firstly, we have explored the potential role of activation-induced cytidine deaminase (AID) in mediating active DNA demethylation through its deaminase activity. To this end, we performed whole genome bisulfite sequencing of naïve and memory B cells isolated from healthy controls and patients with hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency characterized by loss-of-function of AID. Comparison between the methylomes of B cell subsets from controls and HIGM2 patients, revealed global DNA methylation alterations associated with AID loss. In fact, the observed alterations include a blockage of demethylation during the transition from naïve to memory B cells. In addition, AID deficiency also results in alterations in naïve B cells. The analysis of the HIGM2-associated alterations occurring in the transition from naïve to memory B cell rules out the direct involvement of AID in active demethylation and suggests the participation of TET enzymes. Finally, the exploration of the DNA methylation alterations in HIGM2 naïve B cells revealed a role for AID in early stages of B cell development removing autoreactive B cells with a DNA methylation signature characteristic of BCR- stimulated cells. Secondly, we also explored the role of DNA demethylation in the regulation of immunosuppressive phenotype of monocyte- (MO) derived dendritic cells (DC) differentiated in presence of vitamin D, focusing on the interplay among different signal-dependent transcription factors (TF) and the DNA demethylation machinery. In this study, we performed high-throughput DNA methylation screening of both inflammatory DCs and DCs differentiated in presence of vitamin D (TL). DNA methylation analysis revealed extensive condition-specific DNA demethylation events associated with differential immune properties. As expected, we observed that demethylation occurs in enhancer regions and displays an inverse correlation with gene expression. We proved that, vitamin D receptor (VDR) can bind to closed chromatin and correlates with TL-specific demethylation. Interestingly, we observed that tolerogenic properties in DCs are acquired together with activation of the IL- 6/JAK/STAT3 pathway. In fact, VDR directly binds the IL-6 gene and JAK2- mediated STAT3 phosphorylation is specific to vitamin D-stimulation. In addition, we also observed that VDR interacts with both STAT3 and TET2. Finally, we reported that pharmacological inhibition of STAT3 phosphorylation reverts the vitamin–induced tolerogenic properties of DCs.
