Mechanism and relevance of active DNA methylation during monocyte differentiation
Biochemistry
Immunology
Final Report Abstract
Aim of this project was to decipher the mechanisms that initiate active DNA demethylation in the differentiation of human blood monocytes (MO), the post-mitotic precursors of macrophages (MAC) and dendritic cells (moDC), and to reveal the functional relevance of this process for monocyte differentiation. As part of this project, we comprehensively profiled and compared epigenetic landscapes during IL-4/GM-CSF–driven MO differentiation across the genome and detected several thousand regions that were actively demethylated during culture, both with or without accompanying changes in chromatin accessibility or transcription factor (TF) binding. We knocked-down Tet2 with a specifically designed siRNA, but only identified few genes affected during moDC differentiation, implying a minor importance for active DNA demethylation during MO differentiation. However, since there is a significant delay between siRNA-mediated mRNA knock-down and loss of protein, we may retain sufficient residual TET activity during the initial culture period, when most demethylation events are initiated. Hence, we cannot say yet, whether the process is important for MO differentiation, or not. Here, more rapid degradation approaches would be required, which are currently not applicable to the postmitotic differentiation system. Using computational and functional approaches, we further identified and characterized TF that are globally associated with DNA demethylation processes in human MO. We designed siRNAs that allowed the efficient knock-down of two candidate factors (IRF4, EGR2) and studied their role in moDC differentiation and active DNA demethylation. Interferon regulatory factor 4 (IRF4) was found to control hallmark dendritic cell functions (IRF4-deficient MO developed into macrophage-like cells in moDC culture conditions) with less impact on DNA methylation. Early growth response 2 (EGR2), however, proved essential for MO differentiation into moDC as well as DNA methylation turnover at its binding sites. We showed that ERG2 interacts with the 5mC hydroxylase TET2, and its consensus binding sequences show a characteristic DNA methylation footprint at demethylated sites with or without detectable protein binding. Hence, our findings reveal an essential role for EGR2 as epigenetic pioneer in human MO and suggest that active DNA demethylation can be initiated by the TET2-recruiting TF both at stable and transient binding sites.
Publications
- (2021). The epigenetic pioneer EGR2 initiates DNA demethylation in differentiating monocytes at both stable and transient binding sites. Nat Commun 12:1556
Mendes K, Schmidhofer S, Minderjahn J, Glatz D, Kiesewetter C, Raithel J, Wimmer J, Gebhard C, Rehli M
(See online at https://doi.org/10.1038/s41467-021-21661-y)
