The Mysterious Role of non-CpG Methylation in the Brain
DNA methylation is a key mechanism of epigenetic regulation of gene expression and contributes to a multitude of process such as embryonic development, cancer, aging and learning and memory. In the metazoan genome, CpG methylation is the primary form of DNA methylation and is therefore extensively studied. However, key questions regarding non-CpG methylation, including its prevalence, regulation and epigenetic function, remain poorly understood.
In 2000, it was reported that in embryonic stem cells, non-CpG methylation constituted about 25% of total DNA methylation, while in somatic cells, non-CpG methylation prevalence diminished significantly to about 7%1. Results from other groups corroborated these findings – revealing a significant higher frequency of non-CpG methylation in pluripotent cell lines compared to several somatic cell lines investigated, including smooth muscle, skeletal muscle, blood cells and pancreatic islet cells2. Non-CpG methylation is preferentially located in genomic regions with low CpG density, suggesting CpG methylation-independent functions. Indeed, it has been shown that non-CpG methylation is unaffected by null mutation of DNMT1, the methyltransferase required for CpG methylation maintenance1. More recently, it was shown that in type 2 diabetic individuals, a particular promoter region, PGC-1α, is hypermethylated in skeletal muscles – primarily in a non-CpG context. Hypermethylation of this promoter was found to be coupled with decreased levels of mitochondrial respiratory chain proteins, as well as reduced mitochondrial number3, suggesting a possible role of non-CpG methylation in metabolic diseases!
Interestingly, in the postnatal mammalian brain, non-CpG methylation accumulates from juvenile to adult, correlating well with both synaptogenesis and adolescent synaptic pruning4. However, whether or not non-CpG methylation regulates neuronal gene expression, as well as how non-CpG methylation patterns are established, maintained and recognized, remain elusive. The recent work of Guo et al. attempts to address these questions. In their study, the methylome of adult mouse dentate gyrus (a region believed to contribute to the formation of new episodic memories and spontaneous exploration of novel environments) was profiled at a single-nucleotide resolution. They found that roughly 25% of total neuronal DNA methylation was found at non-CpG loci! Additionally, the non-CpG methylation patterns were found to be somewhat heterogeneous between individuals from a homogenous population – contrasting the conserved nature of conventional CpG methylation. They also found that the particular loci at which the non-CpG methylation occurs is relatively conserved across species – as 83% of genes with non-CpG methylation in the human brain have non-CpG methylated ortholog genes in the mouse brain.
The group also found that both CpG and non-CpG methylation marks were diminished at neuronal transcription factor binding regions, as well as at the transcription start sites of genes. Consistent with this, mRNA-seq analysis reveals a negative correlation between the presence of neuronal non-CpG methylation and the expression levels of associated genes. Using a GFP expressing plasmids, they showed that methylated at non-CpG sites resulted in repressed GFP expression when transfected into hippocampal neurons. The degree of repression was similar between plasmids carrying non-CpG methylation and those carrying CpG methylation. These results provide evidence that non-CpG methylation is capable of gene expression repression in neurons!
The authors then investigated how neuronal non-CpG methylation is established, maintained and recognized. They identified MeCP2 as a non-CpG methylation binding/recognizing protein and found that it is able to bind to synthetically methylated oligonucleotides containing non-CpG methylation sites in vitro (however, the presence of CpG methylation greatly aiding the binding affinity). DNA obtained from chromatin immunoprecipitation (ChIP) for MeCP2 of adult mouse hippocampus (famously involved in learning and memory) show enrichment of non-CpG as well as CpG methylated sequences.
Consistent with other studies, time course experiments demonstrated an increase in neuronal non-CpG methylation during postnatal neuronal maturation4,5. The authors suggest that the low levels of non-CpG methylation in fetal brains indicate that non-CpG methylation patterns are established postnatally.
The distinct temporal dynamics and inter-individual variability of non-CpG methylation raise interesting questions regarding its role in the neuronal epigenetic regulation. Is it an important part of learning and memory and experienced induced plasticity? Are the patterns of non-CpG methylation unique for each neuronal type? Are aging and neurological diseases coupled with a change in non-CpG patterns, or are they preceded by it? Studies addressing these issues will greatly expand our knowledge of the epigenetics of the brain.
1. Ramsahoye BH, Biniszkiewicz D, Lyko F, Clark V, Bird AP, & Jaenisch R (2000). Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proceedings of the National Academy of Sciences of the United States of America, 97 (10), 5237-42 PMID: 10805783
2. Ziller MJ, Müller F, Liao J, Zhang Y, Gu H, Bock C, Boyle P, Epstein CB, Bernstein BE, Lengauer T, Gnirke A, & Meissner A (2011). Genomic distribution and inter-sample variation of non-CpG methylation across human cell types. PLoS genetics, 7 (12) PMID: 22174693
3. Barrès R, Osler ME, Yan J, Rune A, Fritz T, Caidahl K, Krook A, & Zierath JR (2009). Non-CpG methylation of the PGC-1alpha promoter through DNMT3B controls mitochondrial density. Cell metabolism, 10 (3), 189-98 PMID: 19723495
4. Lister R, Mukamel EA, Nery JR, Urich M, Puddifoot CA, Johnson ND, Lucero J, Huang Y, Dwork AJ, Schultz MD, Yu M, Tonti-Filippini J, Heyn H, Hu S, Wu JC, Rao A, Esteller M, He C, Haghighi FG, Sejnowski TJ, Behrens MM, & Ecker JR (2013). Global epigenomic reconfiguration during mammalian brain development. Science (New York, N.Y.), 341 (6146) PMID: 23828890
5. Guo JU, Su Y, Shin JH, Shin J, Li H, Xie B, Zhong C, Hu S, Le T, Fan G, Zhu H, Chang Q, Gao Y, Ming GL, & Song H (2014). Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain. Nature neuroscience, 17 (2), 215-22 PMID: 24362762