Cell types are defined by their spatiotemporal gene expression patterns and their differential activity of promoters and enhancers. Enhancers are cis-regulatory elements in the DNA critical for the acquisition and maintenance of cellular identities by regulating the expression of key genes. Enhancers serve as landing pads for transcription factors (TFs) which are DNA-binding proteins that interpret the genomic code and enhance gene expression upon their binding. However, the underlying DNA sequence does not solely convey binding specificity, and therefore it is still largely elusive what additional factors regulate TF binding.
An important regulatory layer in gene expression are dynamic and reversible epigenetic modifications of chromatin including DNA and histone proteins. To date, dozens of histone modifications have been identified that are associated with different genomic contexts and transcriptional states. For instance, histone H3 lysine acetylation has been generally associated with active chromatin as active enhancers and promoters, while histone H3 tri-methylation at lysine 23 (H3K27me3) is coupled to transcription repression. Yet, the causal contribution of such histone modifications to the regulation of enhancer activity and TF binding is still large unknown.
To address this question, I developed a technical approach to analyse TF binding at DNA molecules where a certain histone modification of interest is present. For this, I combined a genomic enrichment technique with a single molecule footprinting (SMF) approach that allows to detect TF binding at single DNA molecule resolution. However, this experimental set-up paired with different optimization approaches did not produce high enough enrichments of DNA molecules harboring certain histone modifications to suffice the required statistical power. Therefore, the focus was laid on investigating the causal role of DNA methylation.
DNA methylation in CpG context is the most common epigenetic modification in the mammalian genome that covers 70-80% of all CpG dinucleotides. Despite its prevalence, DNA methylation can be highly dynamic, especially at enhancer elements that exhibit reduced methylation levels during their activation. Previous studies have identified that the binding of TFs to enhancers is correlated with the partial loss in DNA methylation and it has been suggested that DNA methylation regulates enhancer activity. This hypothesis has remained elusive up to date, which has multiple reasons. First, the relationship between TFs and DNA methylation is bidirectional. Previous studies have identified many methyl-sensitive TFs in vitro whose binding is reduced upon methylation of their DNA binding motif. Some of those have been confirmed by in vivo studies, which showed that DNA methylation prevents the spurious binding of those TFs in the genome. Opposingly, TFs have also been identified to be directly responsible for the demethylation of enhancers. In consequence, the bidirectional regulation between DNA methylation and TF binding has prevented the establishment of a causal relationship between them. Second, the cell-to-cell epigenetic variability observed as intermediate methylation at enhancers elements makes common bulk-cell genomics approaches ineffective to identify a direct correlation between DNA methylation and TF binding and to determine whether DNA methylation generally contributes to the regulation of enhancer activity.
In the here presented PhD project, I overcame these issues and limitation by advancing the single molecule footprinting (SMF) approach to resolve chromatin accessibility, TF binding, and simultaneously quantify the presence of DNA methylation on the same DNA molecules. By applying this technology across the murine genome, I demonstrate that TFs can bind most (>90%) enhancers irrespective of the underlying DNA methylation, suggesting that presence of DNA methylation does not generally impede enhancer activity. Yet, for stem cells and three somatic cell types, I identified active enhancers where TF occupancy is directly repressed by DNA methylation, including enhancers involved in the control of key cell identity genes. Using global perturbation assays and orthogonal enhancer activity measurements, I was able to show that at these active sites, DNA methylation directly controls the occupancy levels of TFs such as Max-Myc, that play a key role in the control of stem cell identity and proliferation. In the end, my data suggest a model where the function of DNA methylation extends beyond protecting the genome from spurious TF binding, by directly regulating the activation of cell-type specific enhancers.
This detailed analysis is an important addition to our general knowledge on gene regulation and suggest that while epigenetic factors may have largely redundant functions, their individual contributions can play important and instructive roles in tuning the quantitative expression of key cell- specific genes. Understanding the regulation of such genes involved in cell identity will have important implications in the comprehension of development and disease
