In an attempt to understand DNA methylation in various contexts, we have examined chromatin modification at enhancers and CpG islands. At both DNA features, we find the binding of transcription factors is the major determinant of methylation status.
At the enhancer for the tissue-specific inflammatory gene Il12b, we attempted to isolate the DNA sequence necessary for the establishment of a low methylation window usually present in most cell types. We cloned Il12b enhancer deletions into a bacterial artificial chromosome that could recapitulate native chromatin when stably transfected into murine ES cells, but were unable to remove the low methylation window without deleting the full enhancer sequence. The Il12b enhancer is uniquely methylated in embryonic stem cells compared to all other cell types; it has higher methylation than usual and responds to certain changes in growth conditions. DNA methylation increases globally during stem cell differentiation, but DNA methylation at the Il12b enhancer remains constant in successfully differentating cells. Finally, we take advantage of variable Il12b enhancer methylation in embryonic stem cells to demonstrate that, following differentiation to a macrophage fate, moderate enhancer methylation does not prevent Il12b expression.
To understand what factors influence the well studied low DNA methylation, histone 3 lysine 4 trimethylation (H3K4me3), and low nucleosome occupancy at CpG islands, we cloned CpG rich DNA into bacterial artificial chromosomes which were stably transfected into ES cells. Analysis of the integrated BACs revealed that CpG island features are each controlled through separate mechanisms. We determined several properties of CpG island features based on experimental deletions and fusions of a small CpG island and the 601 positioning sequence. Protection from DNA methylation at CpG islands can occur either by binding of a specific transcription factor, or by a size threshold mechanism in murine ES cells. H3K4me3 marking requires low DNA methylation, but unmethylated CpGs are not sufficient to recruit high levels. Nucleosome density is influenced by transcription factor binding and sequence positioning determinants, but is unaffected by low DNA methylation and moderate H3K4me3 levels.
We expanded our analysis of CpG islands to include all CpG rich regions in the human genome, which were computationally determined based on our own criteria. Using available chromatin datasets, we assayed the effect of nucleotide content on CpG island features. We found that CpG density and island size correlated with high levels of CpG island features. However, by far the strongest determinant of CpG island features was association with a promoter. Promoter CpG rich regions were strongly biased to accumulate high levels of all CpG island features, which could not be explained by nucleotide content. Instead, we showed that promoter CpG islands have much higher transcription factor binding then other CpG islands in the genome, and high binding is correlated with lower DNA methylation and higher H3K4me3. Finally, we observed a difference in the DNA methylation at human and mouse CpG islands. High CpG density mouse CpG islands are much more susceptible to demethylation.
This multifaceted study elucidates many previously undefined relationships between transcription factors and chromatin properties. These findings will be beneficial to describing the complex mechanisms that drive regulation of cell fate and gene expression.