Establishment of DNA methylation patterns during mouse development

Abstract

Methylation is the only known modification of DNA and in animals it mainly occurs at cytosines in a CpG context. The pattern of DNA methylation varies among organisms; some invertebrates are totally devoid of it, while others have densely methylated regions embedded in an otherwise unmethylated genome. The genome of mammals on the other hand, is very rich in DNA methylation with the exception of regions with high CpG frequency, known as CpG islands, that are often found devoid of methylation. Little is known about the factors that determine the genome-wide pattern of DNA methylation. Moreover, although there appears to be a specific developmental program for the establishment of methylation in specific genomic regions, the molecular events that lead to methylation establishment remain unknown. The establishment of methylation in the regulatory region of the murine Oct4 gene as well as the occurrence and establishment of methylation in mouse CpG islands are investigated in this study. The promoter of Oct4, which encodes an important developmental regulator, is known to gain methylation as the gene becomes silenced during early development. An in vitro model of murine early development has been used to recapitulate the events that lead to the gene’s silencing. In accordance to other reports, detailed methylation analysis of the gene’s entire upstream region and expression analysis showed that DNA methylation establishment follows the gene’s downregulation. Moreover, establishment of methylation at the Oct4 locus seems to start from the gene’s proximal enhancer and then spread towards the distal enhancer and the promoter. Although the initial establishment of methylation in the distal enhancer was not impaired in G9a -/- cells, methylation in these cells was unable to spread and accumulate. These findings demonstrate that the promoter of the gene is not the primary target for methylation as previously assumed and give rise to two possible mechanisms for DNA methylation establishment at this gene; one possibility is that methylation is actively targeted to the proximal enhancer, while the other is that the promoter and the distal enhancer are resistant to methylation, perhaps because of transcription factors bound to them. Moreover, the finding that G9a is not necessary for DNA methylation establishment but appears to have a role in methylation spreading, together with observations on the kinetics of the downregulation and the timing of methylation establishment, allowed the formation of a possible model for the role of DNA methylation in this gene’s downregulation. According to this model, DNA methylation acts to accelerate the gene’s downregulation ensuring its coordinated repression in the developing organism. For the study of methylation in CpG islands, first a novel algorithm was applied for the identification of CpG islands in the mouse genome. Approximately 21,000 CpG islands were identified in the mouse genome, half of which localised at the 5’ of genes, while the majority of the remaining was equally distributed in intragenic and intergenic regions. Only a very small proportion of the CpG islands localised at the 3’ of genes. When the gene ontology terms related with the CpG island-associated genes where interrogated, two main gene functions emerged as being preferentially associated with CpG islands, development and cell maintenance. Then, an affinity purification method, together with microarray hybridisation was applied for the identification of methylated CpG islands from mouse brain. Approximately 18% of all CpG islands were methylated in brain, with the big majority localised at 5’ and intragenic regions. When the gene ontology of the methylated CpG island-associated genes was analysed, developmental but not housekeeping genes were overrepresented in the methylated fraction. In order to further investigate the relationship of CpG islands with developmental genes, the same methodology was applied for the identification of CpG islands that become methylated after the in vitro induction of differentiation of ES cells. Although this approach failed to produce genome-wide data, it enforced the idea of a developmental program for CpG island methylation.