Does defective binding of 5-hmC by MeCP2 contribute to Rett syndrome?
DNA methylation is an epigenetic modification that plays an important role in gene expression. Aberrant changes of DNA methylation in the genome are associated with many epigenetic-related neurological disorders. However, the mechanism by which DNA methylation affects chromatin structure and gene expression is not completely understood. Methyl-CpG-binding protein 2, (MeCP2), was first identified by its affinity for DNA containing 5-methycytosine (5-mC), and mutations in MeCP2 are associated with Rett Syndrome, a disorder that results in impaired development of the nervous system. In an article recently published in Cell, Mellen et al. discovered that in addition to 5-mC, MeCP2 also binds with high affinity to DNA containing 5-Hydroxymethycytosine (5-hmC), a more recently discovered epigenetic mark with highest levels in the brain. Furthermore, they found that this binding facilitates gene expression through its effects on chromatin organization in cells of the cerebellum.
The authors analyzed the expression of mRNAs that were actively translated in three types of cells in the cerebellum including glial cells and two types of neuronal cells, Purkinje cells and granule cells, using a method called the Translating Ribosome Affinity Purification and sequencing (TRAP-seq). They found that a large fraction of the actively translated mRNAs are cell type specific and enriched for genes required for specific functions such as neuronal support, synapse formation, and axonal maintenance, respectively. To determine whether the cell type specific gene expression signatures were a consequence of the cytosine modification status of each cell type, the authors next performed genome-wide 5-mC and mapping analysis using methylated DNA immunoprecipitation (MeDIP) and a selective 5-hmC chemical labeling and enrichment strategy. Consistent with previously published studies on brain tissues, 5-hmC was found preferentially enriched over the gene bodies of expressed genes while 5-mC was depleted over these regions. Interestingly, the depletion of 5-mC and enrichment of 5-hmC varied between cell types. For example, in the granule cells and the glial cells, genes that are most highly expressed tended to have more 5-hmC and less 5-mC modifications. However in the Purkinje cells, the loss of cytosine modifications (both 5-mC and 5-hmC) at specific CpG sites, rather than elevated 5-hmC levels at gene bodies, correlated better with gene expression. These findings indicate that both the accumulation of 5-hmC and loss of cytosine modifications in the gene body can contribute to expression. To investigate whether there was a protein that decodes 5-hmC in the central nervous system, the authors employed a method combining affinity purification using beads coated with DNA containing either unmodified Cytosine, 5-mC, or 5-hmC and mass spectroscopy. They identified that MeCP2, which was previously identified to bind 5-mC, was a major 5-hmC binding protein. The binding of MeCP2 to 5-hmC increased the accessibility of 5-hmC-containing chromatin DNA to nuclease digestion, suggesting that MeCP2 can alter chromatin structure. Remarkably, a mutant form of MeCP2, R133C, which causes Rett syndrome, was specifically defective for 5-hmC binding without affecting 5-mC binding, potentially giving a molecular explanation of Rett syndrome pathology.
This report was the first to provide evidence for an interaction between MeCP2 and 5-hmC modifications in DNA, and also suggested that MeCP2 may play a role in establishing a dynamic state of chromatin that is responsible for cell specific gene expression pattern in the CNS. Furthermore these results improved our understanding of the role 5-hmC plays in the pathophysiology of Rett syndrome.
Mellén M, Ayata P, Dewell S, Kriaucionis S, & Heintz N (2012). MeCP2 Binds to 5hmC Enriched within Active Genes and Accessible Chromatin in the Nervous System. Cell, 151 (7), 1417-30 PMID: 23260135