Functional analysis of DNA demethylation during IL4/GM-CSF-driven differentiation of human monocytes

Epigenetic processes, such as DNA (de)methylation and chromatin remodeling are fundamental for hematopoietic cell differentiation and lineage specification. Hence, their dysregulation is associated with many hematological malignancies. In this thesis, the analyses were focused on DNA demethylation processes during monocyte (MO) to immature dendritic cell (iDC) differentiation in the presence of the cytokines IL4 and GM-CSF. Due to the absence of cell proliferation in this system, we were able to investigate TET-mediated active demethylation events occurring in a replication-independent context. In hematopoietic cells, comprising MO and MO-derived cells, the TET2 enzyme is the main hydroxylase catalysing the initial oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and likely the remaining oxidative steps as well. In this thesis, the TET2 gene was transiently knocked-down in MO using a well-established siRNA-mediated approach and its functional consequences during iDC differentiation were assessed. Overall, TET2 depletion was associated with very mild effects on iDC differentiation, transcriptional programs and chromatin remodeling processes. Nevertheless, the data was reproducible among donors and corroborated the importance of TET2-mediated active DNA demethylation processes during MO differentiation. Based on preliminary genome-wide methylation analyses in MO and iDCs, 7610 iDC-specific differentially methylated regions (DMRs) were identified. These regions were characterized by a progressive active DNA demethylation (5hmC enrichment) and an increase in chromatin accessibility. Since we were mostly interested on differentiation-associated processes, the analyses were centered on transcription factors (TFs) enriched across DMRs and strongly induced in iDCs, such as IRF4 and EGR2. Consequently, both TFs were individually and transiently knocked-down in MO and their effects on iDC differentiation and associated epigenetic processes were determined. IRF4 depletion prevented the differentiation of iDC, and instead cells acquired a macrophage-like morphology confirming previous mouse work244. Interestingly, we found that IRF4 might be involved in open chromatin at its binding sites regardless of changes in DNA demethylation, as reflected by the reduced chromatin accessibility and the minor effects on DNA methylation upon its depletion. Remarkably, this work is the first to describe an essential role of EGR2 in IL4/GM-CSF-mediated MO differentiation. Indeed, data showed a strong impact of EGR2 depletion on cell morphology and viability as well as profound changes in iDC transcriptional programs. Besides its importance on MO biology, EGR2 is implicated in active DNA demethylation and chromatin remodeling processes as indicated by iDC DMRs that remained methylated and failed to gain accessibility upon EGR2-silencing. Notably, the presence of a methylation footprint in the EGR2 motif associated with its ability to bind methylated DNA, suggest that the epigenetic pioneer EGR2 can target de novo demethylation processes at its transient and stable binding sites. In fact, co-immunoprecipitation data showed an interaction between EGR2 and TET2, emphasizing the EGR2 role in targeting the DNA demethylation machinery. In summary, this thesis provides new insights into dynamic epigenetic changes through IL4/GM-CSF-mediated MO differentiation. Accordingly, we identified key regulators on MO biology and differentiation-associated epigenetic processes, specifically IRF4 and EGR2. Importantly, EGR2 was found to recruit TET2 to its binding sites even before chromatin opening or TF binding detection, suggesting that at certain iDC DMRs active DNA demethylation and chromatin accessibility changes are uncoupled. In addition, this work demonstrated that DNA methylation-spikes (identified in EGR2 as well as other consensus motifs of TFs) are cell type-specific and protected from demethylation by bound key epigenetic pioneer factors.