DNA demethylation processes have been studied for many years and entered the focus of extensive research with the discovery of active demethylation mechanisms (He et al., 2011; Ito et al., 2011; Iyer et al., 2009; Kriaucionis and Heintz, 2009; Tahiliani et al., 2009). These processes contribute to the regulation of cell type-specific gene expression patterns and the dynamics of other epigenetic mechanisms (Wu and Zhang, 2014). The investigation of the different types of mechanisms and their role in different cell types or developmental stages is an important challenge to understand the complex regulatory processes in mammals. The data presented in this work allowed further insights into the active demethylation processes and contributed to the understanding of regulatory mechanisms in different hematopoietic cell types. Using an in vitro model system, representing the human mononuclear phagocyte system, we were able to characterize the active DNA demethylation mechanism in the absence of passive demethylation events. The data revealed that the targeted, locus-specific active DNA demethylation process is initiated by the modification of 5mC to 5hmC. Further experiments based on the knockdown of candidate enzymes identified TET2 as the initiator of the active DNA demethylation process and as being responsible for the conversion of 5mC to 5hmC. Investigation of further possible players like TDG, MBD4, OGT, and HELLS gave first insights into a possible contribution to the process and so far the data indicated that none of the enzymes is involved in the first conversion step. Functional investigation of the demethylated regions in reporter gene assays linked the local binding of TFs like PU.1 and synchronous demethylation events to the activation of potential enhancer elements. The data demonstrated that their activation depended on the methylation level and that demethylation led to enhancer activation in a cell type-specific manner. Moreover the results indicated that the activation of cell type-specific enhancer elements requires a corresponding set of TF to open the regions, which may include the removal of 5mC in this process.
The validation and adaption of a 5hmC-enrichment method to next generation sequencing allowed us to investigate the active demethylation processes on a genome-wide level. Using the Hydroxymethyl CollectorTM kit we assessed the global dynamics of DNA demethylation and its association with the key hematopoietic transcription factor PU.1 in differentiating monocytes. The global screen illustrated dynamic patterns of 5hmC and confirmed its role as an intermediate of active demethylation events accompanying the transition into another cell type. Local binding of PU.1 at demethylated sites further supported the theory of a correlation between demethylation events and the recruitment of PU.1. However, active DNA demethylation events were not altogether dependent on PU.1 binding, since several regions accumulated 5hmC in the absence of this TF, indicating the involvement of other factors and thus a site-specific recruitment of PU.1. The data further hinted at a possible regulatory role of 5hmC as an epigenetic mark, actively recruiting or passively impeding other factors. Further gene ontology analyses confirmed the immunological background of the cells and presented genes involved in the immune response and inflammation to be associated with active demethylation processes and the local appearance of PU.1. Corresponding expression changes suggested an involvement of PU.1 in the regulation of transcriptional changes during monocyte differentiation. However, regions with increasing or stable 5hmC levels displayed transcriptional changes independent of demethylation or PU.1 and supported the involvement of other factors in their regulation as well as possible regulatory functions of 5hmC.
A global screen of PU.1 distribution in differentiating monocytes illustrated dynamic PU.1 binding patterns upon the transition into another cell type and confirmed the association with demethylation events at subsets of PU.1 target regions. Comparing the 5hmC and PU.1 dynamics during monocyte differentiation we presented first evidence for a distinct chronology of PU.1 and demethylation events. In a subset of PU.1 target regions demethylation was present in monocytes but recruited PU.1 primarily on the transition into a new cell type. It is still unclear, if PU.1 generally profits from the opening of demethylated regions or if it administrates various functions at different target regions. The localization of the PU.1 patterns to active, cell type-specific regulatory elements revealed distinct distribution dynamics during cell differentiation. PU.1 is mainly targeted to promoter and promoter-distal regulatory regions that are activated in a cell type-specific manner. The active nature of the regions supported an involvement of PU.1 in hematopoietic cell differentiation. PU.1 binding was associated with marginally dynamic, demethylated states and indicated a role for PU.1 in the maintenance of an active transcriptional state or its independent recruitment to demethylated regulatory regions.
In summary the data presented in this work contributed to the understanding of the active DNA demethylation mechanism and revealed dynamic global association of demethylation events and PU.1 binding accompanying cell fate decisions in hematopoietic cells
