Question

17-11. Methylation of eukaryotic DNA controls gene expression.

a) Describe in words the control of methylation of DNA in eukaryotes.

b) Describe in words how silencing starts with methylation.

Answer 1

a. The DNA methylation landscape of vertebrates is very particular compared to other organisms. In vertebrates, around 60–80% of CpG are methylated in somatic cells and DNA methylation appears as a default state that has to be specifically excluded from defined locations. By contrast, the genome of most plants, invertebrates, fungi, or protists show “mosaic” methylation patterns, where only specific genomic elements are targeted, and they are characterized by the alternation of methylated and unmethylated domains.

High CpG methylation in mammalian genomes has an evolutionary cost because it increases the frequency of spontaneous mutations. Loss of amino-groups occurs with a high frequency for cytosines, with different consequences depending on their methylation. Methylated C residues spontaneously deaminate to form T residues over time; hence CpG dinucleotides steadily deaminate to TpG dinucleotides, which is evidenced by the under-representation of CpG dinucleotides in the human genome. On the other hand, spontaneous deamination of unmethylated C residues gives rise to U residues, a change that is quickly recognized and repaired by the cell.

In mammals, the only exception for this global CpG depletion resides in a specific category of GC- and CpG-rich sequences termed CpG islands that are generally unmethylated and therefore retained the expected CpG content. CpG islands are usually defined as regions with 1) a length greater than 200bp, 2) a G+C content greater than 50%, 3) a ratio of observed to expected CpG greater than 0.6, although other definitions are sometimes used. Excluding repeated sequences, there are around 25,000 CpG islands in the human genome, 75% of which being less than 850bp long. They are major regulatory units and around 50% of CpG islands are located in gene promoter regions, while another 25% lie in gene bodies, often serving as alternative promoters. Reciprocally, around 60-70% of human genes have a CpG island in their promoter region.The majority of CpG islands are constitutively unmethylated and enriched for permissive chromatin modification such as H3K4 methylation. In somatic tissues, only 10% of CpG islands are methylated, the majority of them being located in intergenic and intragenic regions.

DNA methylation was probably present at some extent in very early eukaryote ancestors. In virtually every organism analyzed, methylation in promoter regions correlates negatively with gene expression. CpG-dense promoters of actively transcribed genes are never methylated, but reciprocally transcriptionally silent genes do not necessarily carry a methylated promoter. In mouse and human, around 60–70% of genes have a CpG island in their promoter region and most of these CpG islands remain unmethylated independently of the transcriptional activity of the gene, in both differentiated and undifferentiated cell types. Of note, whereas DNA methylation of CpG islands is unambiguously linked with transcriptional repression, the function of DNA methylation in CG-poor promoters remains unclear; albeit there is little evidence that it could be functionally relevant.

DNA methylation may affect the transcription of genes in two ways. First, the methylation of DNA itself may physically impede the binding of transcriptional proteins to the gene,and second, and likely more important, methylated DNA may be bound by proteins known as methyl-CpG-binding domain proteins (MBDs). MBD proteins then recruit additional proteins to the locus, such as histone deacetylases and other chromatin remodeling proteins that can modify histones, thereby forming compact, inactive chromatin, termed heterochromatin. This link between DNA methylation and chromatin structure is very important.

b. A function that appears even more conserved than transposon silencing is positively correlated with gene expression. In almost all species where DNA methylation is present, DNA methylation is especially enriched in the body of highly transcribed genes. The function of gene body methylation is not well understood. A body of evidence suggests that it could regulate splicing and suppress the activity of intragenic transcriptional units (cryptic promoters or transposable elements). Gene-body methylation appears closely tied to H3K36 methylation. In yeast and mammals, H3K36 methylation is highly enriched in the body of highly transcribed genes. In yeast at least, H3K36me3 recruits enzymes such as histone deacetylases to condense chromatin and prevent the activation of cryptic start sites.In mammals, DNMT3a and DNMT3b PWWP domain binds to H3K36me3 and the two enzymes are recruited to the body of actively transcribed genes.