The data presented here identify a role for MeCP2 in the regulation of chromatin structure, and support a model for the organization of chromatin and gene expression that is of particular importance for CNS. This model depends on three major factors: depletion of 5mC within the bodies of expressed genes, accumulation of high levels of 5hmC within these gene bodies, and occupation of 5hmC binding sites by the abundant and CNS-enriched protein MeCP2. The contributions of each of these factors to gene expression vary between cell types, suggesting that each of them can be regulated independently. Based on our data, and the fact that both 5hmC and MeCP2 are at least an order of magnitude more abundant in CNS than in the periphery (Kriaucionis and Heintz, 2009
; Skene et al., 2010
), we propose that binding of 5hmC by MeCP2 plays a central role in the epigenetic regulation of neural chromatin and gene expression. Advances in our understanding of the pathophysiology of RTT will require further investigation of this new role for MeCP2 in facilitating gene expression when bound to 5hmC in the context of the traditional repressive functions it elicits upon its binding to 5mC (Guy et al, 2011).
Although a mechanism by which MeCP2 binding to 5hmC could regulate chromatin accessibility remains to be determined, several inferences can be drawn from the existing data. First, the distribution of 5hmC throughout the transcription unit of highly expressed genes distinguishes this mechanism from the established roles of MeCP2 and other MBD family proteins in the organization of repressive chromatin complexes at promoters and enhancers (Guy et al,2011; Yildirim et al., 2011
). Our data support the idea that the action of MeCP2 is more akin to a linker histone (Skene et al., 2010
), occupying expressed genes through its binding to 5hmC. They are also consistent with the observations that MeCP2 stably associates with nucleosomes (Chandler et al., 1999
), that it can compete with histone H1 for nucleosome binding sites (Ghosh et al., 2010
), and that the levels of MeCP2 and histone H1 are inversely correlated in neurons (Skene et al., 2010
). However, our observations that MeCP2 binds with high affinity to 5hmC and that 5hmC is enriched in expressed genes that are nuclease sensitive forces a reevaluation of the role of MeCP2 binding to chromatin in neural cell types. We propose that binding of MeCP2 to 5hmC in expressed genes facilitates transcription through organization of dynamic chromatin domains. This model provides a mechanistic explanation for the recent demonstration that MeCP2 can also activate gene expression, as some genes are both downregulated upon loss of MeCP2 and upregulated in mice with increased MeCP2 gene dosage (Ben-Shachar et al., 2009
Second, our data suggest that both depletion of gene body 5mC and MeCP2 binding to 5hmC are important to establish chromatin domains that facilitate transcription. Thus, there is a strong inverse correlation between gene expression and gene body 5mC. It seems probable that this reflects both the biochemical nature of 5mC binding by MBD proteins, and the consequences of their action. For example, it has recently been shown that in the brain two populations of MeCP2 are present: one in chromatin regions that are enriched in nucleosomes and the other that is loosely bound to highly accessible chromatin domains (Thambirajah et al., 2012
). Given our demonstration that genes enriched in 5hmC are also preferentially present in these MNase sensitive domains, it seems likely that this loosely bound MeCP2 is associated with 5hmC rather than 5mC. This suggests that the interaction of MeCP2 with 5hmC establishes a dynamic state of chromatin that would be quite sensitive over time to the presence of much more stable complexes established within that domain by binding of MeCP2 or other less abundant MBD family proteins to 5mC (Lopez-Serra et al., 2006
). A cell-specific and dynamically regulated gene expression pattern might be explained by a three-dimensional chromatin structure established by regulating levels of 5mC, 5hmC, MeCP2 and other MBD proteins. Changes in the level or activity of MeCP2 would disrupt this balance, resulting alterations in chromatin structure and, consequently, gene expression. Since in each cell type the levels of 5hmC, 5mC and the proteins that bind them vary, the phenotypic consequences of changes in the function of MeCP2, whether as a result of mutation (Adkins et al., 2011
; Tao et al., 2009
; Amir et al., 1999
) or postranslational modification (Rutlin et al., 2011
; Gonzales et al., 2012
), will be cell type and circuit specific.
Third, our understanding of the pathophysiology of RTT must now encompass both the role of MeCP2 binding to 5mC in the repression of gene expression (Chahrour and Zoghbi, 2008
), and present results supporting a model in which MeCP2 binds to 5hmC within active transcription units. For example, the observations that the distribution of 5hmC, 5mC and their relationship to gene expression vary depending on cell type, and that disease causing mutations of MeCP2 can impact 5hmC binding preferentially (e.g. R133C), could lead to important insights into the specific phenotypes associated with altered MeCP2 function. Our data both support previous genetic studies demonstrating that the consequences of MeCP2 loss in different neural cell types differ both quantitatively and qualitatively (Ben-Shachar et al., 2009
), and suggest that the specific biochemical properties of mutant MeCP2 proteins may inform our understanding of their clinical consequences. For example, it is well documented that patients carrying the R133C mutation have a milder form of RTT that is characterized by delayed onset regression, with improved speech and motor skills (Bebbington et al., 2008
). However, for many other characteristics, including breathing abnormalities, sleep problems, mood disturbances, and epilepsy prevalence, no significant differences are evident between patients bearing R133C or other mutations (Bebbington et al., 2008
). Does this mean that these latter clinical features of RTT are associated with loss of its 5hmC binding capacity, and that they reflect differences in the relative importance of 5hmC versus 5mC binding in different cell types? Is it possible that 5hmC plays a role in the phenotypes that result in categorization of RTT as an Autism Spectrum Disorder? We cannot presently answer these questions, although generation of mouse models with “improved” MeCP2 mutations that continue to strongly impact 5hmC binding yet retain WT 5mC interaction offers an important avenue toward investigation of these issues.
Finally, while we believe binding of MeCP2 to 5hmC is a major step in decoding 5hmC in the CNS, many issues remain to be addressed. We have not, for example, assessed the influence of activity dependent mechanisms (Cohen et al., 2011
) on the interactions of MeCP2 with 5mC or 5hmC containing DNA. We have not yet had the opportunity to analyze the relationships between gene expression, 5mC and 5hmC in other glial cell types that have been shown recently to play important roles in mouse models of RTT (Derecki et al., 2012
; Lioy et al., 2012
). We do not understand the relative importance of the mechanism described here and the recent observation that MBD3 can bind to 5hmC containing DNA (confirmed here), and that it is co-localized with Tet1 at 5hmC containing promoters in ES cells (Yildirim et al., 2011
). And we do not know if 5hmC mediated demethylation plays a role in the dynamic control of epigenetic regulation of specific CNS cell types (Cortellini et al. 2011; Ito et al., 2011
). Investigation of these and other issues in specific neuronal and glial cell types will be essential if we are to decipher the role of 5hmC in CNS, and understand its contributions to the pathophysiology of RTT.