Methylation of cytosines is an essential epigenetic modification in mammalian genomes, yet the rules that govern methylation patterns remain largely elusive. To gain insights into this process, we generated base-pair-resolution mouse methylomes in stem cells and neuronal progenitors. Advanced quantitative analysis identified low-methylated regions (LMRs) with an average methylation of 30%. These represent CpG-poor distal regulatory regions as evidenced by location, DNase I hypersensitivity, presence of enhancer chromatin marks and enhancer activity in reporter assays. LMRs are occupied by DNA-binding factors and their binding is necessary and sufficient to create LMRs. A comparison of neuronal and stem-cell methylomes confirms this dependency, as cell-type-specific LMRs are occupied by cell-type-specific transcription factors. This study provides methylome references for the mouse and shows that DNA-binding factors locally influence DNA methylation, enabling the identification of active regulatory regions.

Cytosine methylation is a repressive, epigenetically propagated DNA modification. Although patterns of DNA methylation seem tightly regulated in mammals, it is unclear how these are specified and to what extent this process entails genetic or epigenetic regulation. To dissect the role of the underlying DNA sequence, we sequentially inserted over 50 different DNA elements into the same genomic locus in mouse stem cells. Promoter sequences of approximately 1,000 bp autonomously recapitulated correct DNA methylation in pluripotent cells. Moreover, they supported proper de novo methylation during differentiation. Truncation analysis revealed that this regulatory potential is contained within small methylation-determining regions (MDRs). MDRs can mediate both hypomethylation and de novo methylation in cis, and their activity depends on developmental state, motifs for DNA-binding factors and a critical CpG density. These results demonstrate that proximal sequence elements are both necessary and sufficient for regulating DNA methylation and reveal basic constraints of this regulation.

To gain insight into the cellular readout of DNA methylation, we established a strategy for systematically profiling the genome-wide distribution of chromatin-interacting factors. This enabled us to create genomic maps for the methyl-CpG-binding domain (MBD) family of proteins, including disease-relevant mutants, deletions, and isoforms. In vivo binding of MBD proteins occurs predominantly as a linear function of local methylation density, requiring functional MBD domains and methyl-CPGs. This interaction directs specificity of MBD proteins to methylated, CpG-dense, and inactive regulatory regions. In contrast, binding to unmethylated sites varies between MBD proteins and is mediated via alternative domains or protein-protein interactions. Such targeting is exemplified by NuRD-complex-mediated tethering of MBD2 to a subset of unmethylated, active regulatory regions. Interestingly, MBD3 also occupies these sites, but like MBD2, binding is independent of the presence of hydroxymethylation. These functional binding maps reveal methylation-dependent and -independent binding modes and revise current models of DNA methylation readout through MBD proteins.