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.