Co-workers:

Our laboratory studies DNA methylation, a post-synthetic modification that does not alter the coding potential of DNA, but affects the activity of genes. Methylation is predominantly confined to the short dinucleotide CpG, whose frequency varies widely across the genome. In the great majority of the genome the CpG dinucleotide is under-represented and predominantly methylated. Discrete regions near gene promoters, however, display a CpG frequency that is ~10-fold higher than elsewhere. About 60% of protein-coding genes have “CpG island” (CGI) promoters of this kind. Unlike the bulk genome, CGIs are usually methylation-free. In fact transcription of the associated gene is invariably silenced if they become methylated.
 
In the past few years we developed a biochemical purification method that permits isolation of all human and mouse CGIs from three tissues: blood, cerebellum and sperm. We have now analysed these by high throughput DNA sequencing and found, unexpectedly, that many (about half) are not at annotated promoters, but are either within or between genes. Like CGIs at known promoters, these “orphan” CGIs are often associated with the foci of methylation at lysine 4 of histone H3 and there is evidence for transcripts arising at ~40% of them. Therefore most if not all orphan CGIs appear to contain novel promoters whose functions are unknown. It is likely that many produce non-coding RNAs.

A few CGIs become methylated during development and this is associated with long-term promoter silencing, but the extent of CGI methylation not well described. Using our comprehensive set of CGIs and an affinity purification method based on the methyl-CG binding domain, we have looked for CGI methylation during differentiation of the mouse immune system. The advantage of this
system is that individual cell types can be isolated more or less pure. Also, T-helper lymphocytes can be differentiated in culture to give Th1 or Th2 derivatives.
By analysing CGI methylation in these and other haematopoietic cell types, we found that, despite the very large number of gene expression differences between these cell types, there are surprisingly few CGI methylation events. Between immune cells and a distantly related tissue, brain, however, there are many differences, suggesting that CGI methylation is dynamic in the early stages of development, but much less so during the later stages when cells differentiate. Interestingly, methylation of orphan CGIs is much more dynamic than that at annotated promoter CGIs. As a result, understanding the functional significance of orphan CGIs is now high on our agenda.

Selected publications:

 Deaton, A.M., Webb, S., Kerr, A.R., Illingworth, R.S., Guy, J., Andrews, R., and Bird, A. (2011). Cell type-specific DNA methylation at intragenic CpG islands in the immune system. Genome Res. doi:10.1101/gr.118703.110.

Deaton, A.M., and Bird, A. (2011). CpG islands and the regulation of transcription. Genes Dev 25, 1010-1022.

Illingworth, R.S., Gruenewald-Schneider, U., Webb, S., Kerr, A.R., James, K.D., Turner, D.J., Smith, C., Harrison, D.J., Andrews, R., and Bird, A.P. (2010). Orphan CpG islands identify numerous conserved promoters in the mammalian genome. PLoS Genet 6(9): e1001134. doi:10.1371/journal.pgen.1001134.

 The functional significance of CpG islands

Figure 1. The genomic distribution of CpG islands. Top: CGIs can be located at annotated transcription start sites (TSS), within gene bodies (Intragenic) or between annotated genes (Intergenic). Intra- and intergenic CGIs of unknown function are classed as “orphan” CGIs. Empty circles = unmethylated CpG residues, filled circles = methylated CpG residues. Bottom: The genomic distribution of CGIs in the human and mouse genome. The total number of CGIs is given at the top of each graph.

Figure 2. Alternative chromatin states at CpG islands. A) CGIs usually exist in an unmethylated, transcriptionally permissive state marked by histone acetylation (H3/H4Ac), trimethylation of lysine 4 of histone H3 and depleted trimethylation at lysine 36 of histone H3. Nucleosome deficiency and constitutive binding of RNAPII may also contribute to this transcriptionally permissive state. B) DNA methylation is associated with stable long-term silencing of CGI promoters. This can be mediated by methyl-binding domain (MBD) proteins which recruit co-repressor complexes associated with histone deacetylase (HDAC) activity or through inhibition of transcription factor binding by DNA methylation. C) CGIs are sometimes silenced by polycomb group proteins and may be involved in polycomb recruitment. Interestingly, transcriptionally permissive and polycomb repressed states can co-exist at “bivalent” CGIs in embryonic cells.