Turnover and function of DNA methylation at transcription factor binding sites

Cell type identity is largely determined by regulatory networks consistent of various transcription factors. Transcription factor activity requires interaction with DNA and thus critically depends on the accessibility of binding motifs. Growing evidence suggests that interactions between transcription factors and DNA are modulated by distinct chromatin modifications which in turn are influenced by transcription factors. Thus, ultimately transcriptional output is a product of intimate interactions between DNA, transcription factors and chromatin modifications. While recent studies support a model in which DNA sequence in collaboration with transcription factors can autonomously determine chromatin states, exact relationship between all these components is not well understood. Full genome single basepair resolution mammalian methylomes (Hodges et al, 2011; Stadler et al, 2011) demonstrated a correlation between transcription factor occupancy and hypomethylation at distal regulatory regions. Importantly, these low methylated states critically depend on the presence of transcription factors. Here we analyzed how DNA binding factors impact DNA methylation. Using chromatin immunoprecipitation followed by bisulfite sequencing, we show that CTCF bound molecules can vary in their methylation levels at such low methylated regions (LMRs). This observation suggests that no tight link exists between DNA binding of transcription factors and unmethylated state. While cytosines which are highly occupied by CTCF indeed are fully devoid of methylation, cytosines within sites of low occupancy display heterogeneous methylation levels. Moreover, at these sites CTCF occupancy correlates with the likelihood of being demethylated. 5-hydroxymethylcytosine (5hmC) is a putative intermediate of active demethylation. In support of a dynamic model of interaction between transcription factors and DNA methylation, we found that 5hmC is highly enriched at cell type specific and constitutive LMRs in embryonic stem cells and upon their neuronal differentiation. Furthermore, regions with hydroxymethylation changes between these cell types are enriched for cell type specific LMRs. This suggests a participation of transcription factor mediated oxidative demethylation in reprogramming of distal regulatory elements. Knockout of CTCF is lethal for embryonic stem cells. Therefore, in order to test the relationship between transcription factor binding and hydroxymethylation we chose an embryonic stem (ES) cell line with genetic deletion of REST, another factor previously shown to be involved in formation of low methylated states. Indeed, deletion of REST decreased 5-hydroxymethylcytosine levels while concomitantly increasing methylation levels at its binding sites within the analyzed LMRs. These results indicate that transcription factor mediated turnover of DNA methylation acts in maintenance and reprogramming of distal regulatory regions. To test whether the observed turnover is selective for active regulatory regions, we decided to delete the two de novo DNA methyltransferases DNMT3A and DNMT3B in embryonic stem cells. Surprisingly, using this approach we detected loss of methylation at both, low and fully methylated regions. In order to compare the turnover kinetics between different segment subtypes, we collected DNA from ES cells at various time points after DNMT3A/B deletion. This indeed revealed an accelerated turnover at low methylated regions. On average full demethylation was achieved after eight days, suggesting that binding of transcription factors can induce rapid changes in DNA methylation. In summary, this study supports a model in which methylation at distal regulatory regions is maintained and reprogrammed by a transcription factor mediated turnover. We furthermore provide evidence that this turnover depends on TET proteins for demethylation and on DNMT3A/B for remethylation. Quantification suggests that while DNA methylation turnover is present throughout the genome it is accelerated at active distal regulatory elements.