Linking active DNA demethylation by Thymine DNA Glycosylase with epigenetic regulation of gene expression

The correct regulation of epigenetic modifications is crucial for cell plasticity and the establishment of cell identity. The underlying molecular mechanisms are not clear, but a role for DNA repair proteins has been implicated in this context, the investigation of which was the overall aim of my PhD thesis.
The Thymine DNA Glycosylase (TDG) was described to excise the deamination products of cytosine (C) and 5-methylcytosine (5-mC), thereby initiating base excision repair (BER; Neddermann and Jiricny, 1994; Wiebauer and Jiricny, 1990). Furthermore, TDG was implicated in transcriptional regulation both physically and functionally (Leger et al., 2014; Tini et al., 2002; Um et al., 1998). Also, TDG was suggested to directly demethylate 5-mC (Zhu et al., 2000; Jost, 1993), however, this finding was highly controversial. It turned out only recently, that TDG indeed is critically involved in DNA demethylation by excising the oxidation products of the ten-eleven translocation (TET) proteins. The current view of active DNA demethylation is that the TET proteins iteratively oxidize 5-mC to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) and TDG then excises 5-fC and 5-caC, thereby initiating a repair process that integrates non-methylated C (He et al., 2011; Maiti and Drohat, 2011). However, the exact mechanisms for the recruitment, targeting and regulation of TET and TDG are still unclear.
I was involved in the characterization of the phenotype of the first Tdg knockout mouse to be reported, which unexpectedly showed an embryonic lethal phenotype. Analyses of TDG-proficient and TDG-deficient cells did not reveal a defect in DNA repair, but instead misregulation of developmental genes in differentiating mouse embryonic stem cells (ESCs). This correlated with an imbalance of histone marks and stochastic accumulation of DNA methylation in CpG island (CGI) promoters of developmental genes in TDG-deficient cells, linking TDG-dependent DNA repair to the establishment and maintenance of an active chromatin state at gene promoters during lineage commitment (Appendix III).
We generated genome-wide DNA methylation profiles of TDG-proficient and TDG-deficient ESCs and in vitro differentiated neuronal progenitor cells (NPs). In differentiated cells only, we identified a large number of differentially methylated regions (DMRs). Surprisingly, DMRs overlapping with CGIs were almost exclusively hypomethylated in TDG-deficient NPs. Since these CGIs were prone to acquire DNA methylation in TDG-proficient NPs, this indicates that TDG is required to establish de novo methylation upon differentiation. Global levels of 5-mC, and particularly its oxidized derivatives 5-fC and 5-caC, were elevated in TDG-deficient ESCs. We observed this also at the very CGI DMRs, implicating an engagement of TDG activity in cyclic DNA methylation and demethylation at CGIs undergoing epigenetic transitions during cell differentiation (Appendix I).
I then characterized the genomic sites, where TET proteins and TDG engage to effect cyclic DNA methylation and demethylation. I established procedures for chromatin-immunoprecipitation-coupled next generation sequencing (ChIP-seq) for TET1, TET2 and TDG and generated the respective datasets. Bioinformatical analyses revealed clear but differential correlation of TET and TDG association with gene regulatory elements, especially gene promoters and active enhancers. Notably, sites of 5-fC enrichment in TDG-deficient cells, CGIs and bivalent chromatin domains showed a preferential co-occupancy with all three proteins, indicating that highly dynamic active DNA demethylation may involve TET1, TET2 and TDG. Contrarily, sites where uniquely TDG shows chromatin association were more often located in active enhancers. There further was a strong correlation particularly of TET1-TET2-TDG co-occupancy with the occurrence of the dynamic histone variants H3.3 and H2A.Z, suggesting that the targeted oxidation and excision of 5-mC by TET-TDG at gene regulatory elements may serve the generation of single-stranded DNA breaks to trigger nucleosomal dynamics and facilitate epigenetic plasticity and transcription at developmental genes (Appendix II). Preliminary evidence further supports a role for TDG in the regulation of transcription by directly affecting the assembly and progression of the transcription machinery (Supplementary Results).
Taken together, my PhD thesis contributes to the understanding of the epigenetic function of TDG-mediated active DNA demethylation with respect to time (during differentiation) and space (at gene regulatory elements). My model depicts an attractive concept where TDG maintains chromatin plasticity at critical genomic regions destined to undergo epigenetic regulation in response to developmental or environmental cues. We propose that the targeted demethylation of DNA by TDG finally facilitates transcription.